JP2004215649A - Anti-C-reactive protein monoclonal antibody, cell line producing the same, method for producing anti-C-reactive protein monoclonal antibody, and C-reactive protein detection kit - Google Patents

Abstract

An object of the present invention is to provide an anti-CRP monoclonal antibody and a CRP detection kit capable of accurately and easily detecting and measuring CRP.
As shown in FIG. 1, the method for producing an anti-CRP monoclonal antibody of the present invention comprises the steps of: fusing mammalian spleen cells immunized with CRP with cells derived from mammalian myeloma; A step St1 of obtaining a cell line producing a monoclonal antibody; and the monoclonal antibody produced from the cell line is assayed for its ability to bind to CRP, complement C1q, and serum amyloid P by an immunoassay, thereby being specific for CRP. Selecting a cell line that produces a monoclonal antibody that binds specifically.
[Selection diagram] Fig. 1

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an anti-C-reactive protein monoclonal antibody, a cell line producing the same, a method for producing the anti-C-reactive protein monoclonal antibody, and a C-reactive protein detection kit.
[0002]
[Prior art]
C-reactive protein (abbreviated CRP) contains pneumococcal capsular C-polysaccharide and Ca in the blood of pneumonia patients. ²⁺ It was found as a protein showing a sedimentation reaction in the presence. CRP is a pentamer with a molecular weight of about 130,000, is a typical acute phase reactant found in the β globulin fraction by electrophoresis, and significantly increases when there is an inflammatory disease, necrosis of body tissue, etc. Is a plasma protein. CRP can be used, for example, for bacterial or viral infections, collagen diseases such as rheumatoid arthritis, malignant tumors, myocardial infarction, hepatitis, gastrointestinal diseases such as cirrhosis, or a wide range of tissue disintegrating diseases such as major trauma. Affected subjects show high detection values.
[0003]
Thus, a CRP test, although not enough to identify a disease, is very useful for detecting the presence of a histolytic disease in a subject. For example, using a CRP antibody, a pre-test for a person with poor physical condition (if the antigen-antibody reaction with CRP is positive, a further test is performed to identify the disease), a screening of the disease state by quantifying the amount of CRP, and confirmation of the therapeutic effect , Prognosis and the like can be determined.
[0004]
Conventionally, anti-human CRP serum, which is a polyclonal antibody, has been used to specifically detect CRP by an immunological technique. Most conventional CRP testing methods test for CRP by the presence or absence of a sedimentation line or an increase in turbidity due to the agglutination reaction between antiserum and CRP. However, unpurified antiserum often contains various antibodies against various antigens in serum, and may detect antigens other than CRP. Therefore, in order to prevent an antigen other than CRP from being erroneously detected in the test, an antibody that binds to the protein in the human standard serum is removed from the antiserum using a technique such as affinity chromatography. By this, it is common to prevent a reaction derived from an antigen other than CRP in human standard serum.
[0005]
Further, it has been disclosed that CRP is measured by an immunoturbidimetric method using an anti-CRP monoclonal antibody (Patent Document 1).
[0006]
[Patent Document 1]
JP-A-62-192661.
[0007]
[Problems to be solved by the invention]
However, in the anti-human CRP serum, the group of anti-human CRP antibodies obtained differs depending on the serum produced. For this reason, the reactivity with CRP may fluctuate. Furthermore, detection of human CRP using anti-human CRP serum is complicated in operation, and quantification of CRP is not easy.
[0008]
Patent Document 1 discloses that CRP is measured by an immunoturbidimetric method using an anti-CRP monoclonal antibody. However, in actuality, when the anti-CRP monoclonal antibody and CRP aggregate, When there is an influence of steric hindrance, it may be difficult to quantify CRP.
[0009]
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an anti-CRP monoclonal antibody and a CRP detection kit that can accurately and easily detect and measure CRP. .
[0010]
[Means for Solving the Problems]
The method for producing an anti-C-reactive protein monoclonal antibody of the present invention comprises producing a monoclonal antibody by cell fusion between a mammalian spleen cell immunized with a C-reactive protein and a cell derived from a mammalian myeloma. (A) obtaining a cell line, and assaying the monoclonal antibody produced from the cell line for its ability to bind to C-reactive protein, complement C1q, and serum amyloid P by immunoassay. (B) selecting a cell line that produces a monoclonal antibody that specifically binds to the protein.
[0011]
According to the present invention, monoclonal antibodies produced from each cell line are assayed for their ability to bind to CRP, complement C1q, and serum amyloid P by an immunoassay, whereby monoclonal antibodies that specifically bind to CRP are determined. Select the cell line to produce. Therefore, an anti-CRP monoclonal antibody having extremely low cross-reactivity to complement C1q and serum amyloid P can be selected. Therefore, it is possible to provide an anti-CRP monoclonal antibody and a CRP detection kit that can accurately and simply detect and quantify CRP.
[0012]
In the above step (b), it is preferable to select a cell line that produces a monoclonal antibody that has a binding ability to C-reactive protein and does not bind to complement C1q and serum amyloid P.
[0013]
The cell lines of the present invention produce monoclonal antibodies that have the ability to bind to C-reactive protein and do not bind to complement C1q and serum amyloid P.
[0014]
The cell line may be Accession Number FERM BP-7941 of the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
[0015]
The cell line may be Accession No. FERM BP-7942, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
[0016]
The cell line may be Accession Number FERM BP-7939, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
[0017]
The cell line may be Accession Number FERM BP-7940, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
[0018]
The cell line may be Accession No. FERM BP-8185, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
[0019]
The cell line may be Accession Number FERM BP-8186, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
[0020]
The above cell line is a deposit number of FERM BP.
The above cell line is a deposit number of FERM BP.
The above cell line is a deposit number of FERM BP.
The anti-C-reactive protein monoclonal antibody of the present invention has a binding ability to C-reactive protein and does not bind to complement C1q and serum amyloid P.
[0021]
The C-reactive protein detection kit of the present invention is a C-reactive protein detection kit for detecting a C-reactive protein in a sample solution, comprising a first antibody and a second antibody, The antibody is an anti-C-reactive protein antibody, the second antibody is a labeled anti-C-reactive protein antibody, and at least one of the first antibody or the second antibody is a complement C1q And anti-C-reactive protein monoclonal antibodies that do not bind to serum amyloid P.
[0022]
In the C-reactive protein detection kit of the present invention, since at least one of the first antibody and the second antibody is an anti-CRP monoclonal antibody that does not bind to complement C1q and serum amyloid P, complement C1q and serum amyloid P , The amount of the antibody having binding ability is greatly reduced. Therefore, the first antibody and the second antibody almost certainly bind in a sandwich manner with the CRP interposed therebetween, and form a complex by an antigen-antibody reaction. Therefore, for example, CRP can be measured with high sensitivity and high accuracy by applying the kit of the present invention to an immunoassay using conventional spectroscopic means (immunopathy, immunoassay, etc.). .
[0023]
The first antibody is immobilized on a solid phase, and the second antibody is an anti-C-reactive protein monoclonal antibody that does not bind to complement C1q and serum amyloid P, and is contained in a mobile phase. I have.
[0024]
Since the second antibody is an anti-C-reactive protein monoclonal antibody that does not bind complement C1q and serum amyloid P, no label indicating the presence of complement C1q and serum amyloid P is detected. Therefore, CRP can be accurately detected and measured with higher sensitivity.
[0025]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described.
[0026]
In order to quantify CRP by a simple method, sandwich-type immunochromatography using a monoclonal antibody is often used.
[0027]
In sandwich immunochromatography, two types of antibodies are used, and one of them is first immobilized on a solid phase. Next, the other labeled antibody is allowed to react with CRP, and this is allowed to flow as a mobile phase against a solid phase portion. As a result, the two antibodies bind in a sandwich via CRP. Therefore, the label is confirmed on the solid phase only when CRP is present.
[0028]
CRP is a pentamer in which five peptides are repeated (pentamer), and has a molecular weight of about 130,000. For this reason, it is relatively easy to obtain a pair of antibodies that can bind in a sandwich across the CRP. However, the present inventors have noticed that when a monoclonal antibody is used, since the antibody is homogeneous, cross-reactivity of the antibody is a much larger problem than when only a polyclonal antibody is used.
[0029]
The amino acid sequence of CRP has 51% homology with the amino acid sequence of serum amyloid P, and CRP is known to have homology with a part of the amino acid sequence of complement C1q. Therefore, among the antibodies against CRP, there may be antibodies having binding ability not only to CRP but also to complement C1q and serum amyloid P. When such an antibody is used in the sandwich method, accurate quantification of CRP may be hindered. Therefore, the present inventors considered that it was necessary to use an anti-CRP monoclonal antibody having the lowest possible cross-reactivity to complement C1q and serum amyloid P.
[0030]
Therefore, in the present embodiment, an anti-CRP monoclonal antibody and a cell line producing the same are prepared by the following method. A method for producing the anti-CRP monoclonal antibody of the present embodiment will be described with reference to FIG.
[0031]
As shown in FIG. 1, the method for producing an anti-CRP monoclonal antibody of the present embodiment comprises the steps of: fusing a spleen cell of a mammal immunized with CRP with a cell derived from a mammalian myeloma; A step of obtaining a cell line to be produced St1, and a monoclonal antibody produced from the cell line is specifically bound to CRP by assaying the binding ability to CRP, complement C1q, and serum amyloid P by immunoassay. Selecting a cell line that produces a monoclonal antibody to St2.
[0032]
According to the method of the present embodiment, monoclonal antibodies produced from each cell line are specifically bound to CRP by assaying the binding ability to CRP, complement C1q, and serum amyloid P by immunoassay. A cell line producing a monoclonal antibody is selected. Therefore, an anti-CRP monoclonal antibody having extremely low cross-reactivity to complement C1q and serum amyloid P is obtained. Therefore, it is possible to provide an anti-CRP monoclonal antibody and a CRP detection kit that can accurately and simply detect and quantify CRP.
[0033]
In the above-mentioned step St2, it is preferable to select a cell line that has a binding ability to CRP and produces a monoclonal antibody that does not bind to complement C1q and serum amyloid P.
[0034]
Next, details of each step will be described in order. In the present embodiment, unless otherwise specified, protein separation and analysis methods and immunological techniques known in the art may be employed. These techniques can be performed using commercially available enzymes, kits, antibodies, labeling substances and the like. Further, CRP, serum amyloid P, and complement C1q described in the following description are all derived from humans. In this embodiment, a human CRP and an anti-human CRP monoclonal antibody that specifically binds to the CRP will be described. However, the present invention is directed to a CRP of a mammal other than a human (dog, cat, cow, horse, etc.). The same applies to anti-CRP monoclonal antibodies that bind specifically.
[0035]
First, the details of the step St1 will be described.
[0036]
(Immunity)
By immunizing a mammal with CRP, antibody-producing cells are prepared in the animal.
[0037]
Examples of “mammals” include mice, rats, cows, rabbits, goats. Mammals are preferably mice and rats, and more preferably mice. When the mammal to be immunized is a mouse or a rat, it is convenient in terms of handling of the animal and immunization. Examples of mice include A / J strain, BALB / C strain, DBA / 2 strain, C57BL / 6 strain, C3H / He strain, SJL strain, NZB strain, and CBA / JNCrj strain mice. Since BALB / C strain mice show high antibody titers in serum after immunization, it is possible to obtain monoclonal antibodies having extremely high affinity for CRP. It is known that blood antibody titers are associated with the likelihood of specific hybridomas. In addition, BALB / C strain mice are generally used in large-scale production of antibodies by ascites after the establishment of a cell line. Based on the above, BALB / C strain mice are a preferred example for immunization with CRP. Although the age of the experimental animal is not particularly limited, it is typically a mouse or rat of about 4 weeks to about 12 weeks, preferably a mouse or rat of about 6 to about 10 weeks, more preferably Is a mouse or rat about 7 weeks old.
[0038]
As CRP used for immunization, human CRP, a monomer subunit thereof, or a fragment thereof is used, and may be purified from human serum or a commercially available product. CRP is typically used in a purity of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and most preferably about 98% or more.
[0039]
Prior to immunization, CRP may be mixed with an adjuvant to enhance the immune response. Examples of adjuvants include water-in-oil emulsions (eg, incomplete Freund's adjuvant), water-in-oil-in-water emulsions, oil-in-water emulsions, liposomes, aluminum hydroxide gels, silica adjuvants, powdered bentonite, and tapioca adjuvants. BCG, bacterial cells such as Propionibacterium acnes, cell walls and bacterial cell components such as trehalose dicolate (TDM); lipopolysaccharide (LPS) and lipid A fractions, endotoxins of Gram-negative bacteria; β-glucan (polysaccharide Body); muramyl dipeptide (MDP); bestatin; synthetic compounds such as levamisole; proteins or peptide substances derived from biological components such as thymic hormone, thymic hormone humoral factor and tuftsin; and mixtures thereof (eg, complete Freund's adjuvant) ), And the like.
[0040]
These adjuvants are effective in enhancing or suppressing the immune response depending on the administration route, dosage, administration timing and the like. Furthermore, depending on the type of adjuvant, differences are found in the production of blood antibodies to antigens, induction of cellular immunity, classes of immunoglobulins, and the like. Therefore, it is preferable to select an adjuvant appropriately according to the intended immune response. Handling of selected adjuvants, such as methods for mixing with CRP, is well known in the art.
[0041]
Immunization of a mammal is performed according to methods known in the art. For example, the antigen CRP can be injected subcutaneously, intradermally, intravenously, or intraperitoneally in a mammal. Since the immune response depends on the type and strain of mammal being immunized, the immunization schedule can be modified appropriately for the animal used. The challenge may be repeated several times after the first immunization. Such antigen administration is referred to as boosting. Booster immunizations can be performed, for example, two weeks, four weeks, and six weeks after the first immunization.
[0042]
(Confirmation of antibody production)
Following immunization, blood is drawn from the mammal and the resulting blood is assayed for the presence of CRP binding activity, thereby producing antibodies against CRP in the body of the mammal and, at the end of immunization, a switch from IgM to IgG. Check that a class switch has occurred (see, for example, Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, COLD SPRING HARBOR LABORATORY, New York (1988)).
[0043]
Examples of suitable assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent antibody method. In the present invention, it is desirable to obtain an anti-CRP monoclonal antibody having high affinity for CRP. In order to obtain a high-affinity monoclonal antibody-producing cell, it is necessary to show a high antibody titer at the time of antiserum.
[0044]
(Boost)
After confirming the production of CRP-binding antibodies, additional administration of the immunogen can be performed to enlarge the spleen. The amount of CRP administered in the booster of the immunogen is desirably about 4 to 5 times the amount of CRP initially immunized, but is not limited thereto.
[0045]
Boosting is typically performed using an emulsion of CRP and incomplete Freund's adjuvant. However, as the CRP to be administered in the final immunization (additional administration of the immunogen several days before the cell fusion), it is preferable to use a pure product to which no adjuvant is added. The administration route is appropriately determined in consideration of the possibility that an antibody recognizing a different site of CRP may be obtained by subcutaneous, intradermal, intravenous, or intraperitoneal administration.
[0046]
(Cell fusion)
After the final immunization, spleen cells are removed from the immunized mammal and fused with a myeloma-derived cell line.
[0047]
Since the growth ability of the fused cells depends on the type of myeloma-derived cell line used at the time of cell fusion, it is preferable to use a cell line having excellent proliferation ability for cell fusion. Preferably, the cell line derived from myeloma is compatible with the mammal from which the spleen cells to be fused are derived. The cell line derived from myeloma may be newly prepared or a commercially available cell line may be used. Cell lines derived from mouse myeloma include P3X63-Ag8.653, Sp2 / O-Ag14, FO · 1, S194 / 5. XX0. BU. 1, P3 / NS1 / 1-Ag4-1 and the like. The use of P3X63-Ag8.653 is preferred because it does not produce antibody fragments and the fusion cells have excellent growth ability. Therefore, when the cell line derived from mammalian myeloma is mouse myeloma-derived P3X63-Ag8.653, many cells can be assayed in a short time. As a cell line derived from rat myeloma, 210. RCY3. Ag. 1.2.3, YB2 / 0 and the like.
[0048]
Cell fusion is performed according to methods known in the art (Koehler and Milstein, Nature 256: 495 [1975], Kosbor et al., 1983, Immunol. Today 4:72, Cote et al., 1983, Proc. Natl. Acad. Sci. USA, 80: 2026, Cole et al., MONOCLONAL ANTIBODYES AND CANCERHERAPY, Alan R Liss Inc., New York, NY, pp. 77-96 [1985], etc.). Examples of the cell fusion method include a polyethylene glycol method, a method using Sendai virus, a method using electric current, and the like. The polyethylene glycol method is preferred because of relatively low cytotoxicity, easy fusion operation and high reproducibility.
[0049]
The resulting fused cells can be grown according to conditions known in the art. Desired fusion cells can be selected based on the binding ability of the produced antibody.
[0050]
The above is the details of the process St1. Next, the details of the step St2 will be described.
[0051]
(Cell sorting and cloning)
The binding ability of the antibody produced from the fusion cell can be assayed based on a method known in the art. In the present invention, in order to obtain a fused cell that produces an antibody that specifically binds to CRP, the ability to bind to CRP and complement C1q and serum amyloid P, which are proteins highly homologous to CRP as described above, The target cell line is cloned using the selection based on the above. As used herein, “an antibody that specifically binds to CRP” refers to an antibody that has the ability to bind to CRP. Such "specifically binding antibodies" react best with CRP and are distinguishable from reactions with substances other than CRP, which are usually considered in the general context in which the antibodies are used.
[0052]
The evaluation of the binding ability can be performed by an inhibition ELISA method. As used herein, the term “has binding ability” means that an inhibitor is applied in a measurement under substantially the same conditions as in the inhibition ELISA method described in the Examples below, and the half value of the inhibitor is About 10 ^-6 M or less. Preferably, the half value of the inhibition is about 10 ^-7 M or less, more preferably about 10 ^-8 M or less, even more preferably about 10 ^-9 M or less. Still more preferably, about 10 ^-10 M or less. As used herein, the term "not capable of binding" refers to no inhibition in the same measurement. The term "inhibited" refers to a decrease in the amount of antibody that binds to CRP immobilized on a solid phase in the presence of a competitor (inhibitor) as compared to the absence of the inhibitor. The term "inhibition free" means that the amount of antibody that binds to CRP immobilized on a solid phase is substantially equivalent in the presence and absence of the inhibitor. "Half value of the inhibitor" refers to the concentration of the inhibitor at which the absorbance at half the absorbance (reflecting the amount of antibody bound) in the absence of the inhibitor is measured.
[0053]
The binding ability of the antibody is assayed using a method such as an ELISA method, an RIA method, or a fluorescent antibody method as described above for confirming the production of the antibody. The ELISA method is preferred because the antibody can be easily detected with high sensitivity.
[0054]
For cloning of the fused cells, a method known in the art can be used. Examples of the cloning method include a limiting dilution method and a soft agar method. The limiting dilution method is preferred because the operation is easy, there are many results, and the reproducibility is high.
[0055]
In order to select useful cells efficiently from many fusion cells obtained by cell fusion, cell selection is preferably performed from an early stage of cloning.
[0056]
In this way, a fusion cell line that produces an antibody having the desired binding ability is finally selected. Sorted cell lines can be stored semi-permanently in liquid nitrogen. According to the monoclonal antibody-producing cell line of the present invention, by culturing it, an anti-CRP monoclonal antibody having high affinity for CRP and high specificity can be semipermanently provided.
[0057]
The above is the details of the step St2. Further, it is preferable to perform the following steps after the step St2.
[0058]
(Purification of antibody)
By culturing the monoclonal antibody-producing cell line selected as described above in a large amount, a monoclonal antibody that specifically binds to CRP can be produced in a large amount. Methods for large scale culturing of monoclonal antibody producing cell lines include in vivo and in vitro cultivation. Examples of large-scale in vivo culture include a method in which a fused cell is injected into a peritoneal cavity of a mammal to grow the same, and an antibody is produced in ascites. In in vitro culture, the fused cells are cultured in a medium and antibodies are produced in the medium.
[0059]
The monoclonal antibody of the present invention can be purified from ascites or culture supernatant obtained by mass culture using a method known in the art. For purification, for example, DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation and the like are used in an appropriate combination. The antibodies of the present invention are generally purified to a purity of about 90%, preferably about 95%, more preferably about 98%.
[0060]
(Evaluation of antibody)
In order to evaluate the binding ability of the purified monoclonal antibody, CRP is adsorbed to an ELISA plate, several obtained antibodies are reacted, and an antibody with high binding ability is selected by performing inhibition with CRP. did. Antibodies with high binding ability are useful for highly sensitive detection of CRP.
[0061]
As described above, the cell line and the antibody of the present invention can be produced. The anti-CRP monoclonal antibody produced by the cell line of the present invention is a monoclonal antibody that specifically binds to CRP, and is a monoclonal antibody capable of binding to CRP. The binding ability of the anti-CRP monoclonal antibody produced by the cell line of the present invention to CRP was determined by the inhibition in the measurement under substantially the same conditions as in the inhibition ELISA method described in the Examples below. Half value of inhibition is about 10 ^-6 M or less, preferably about 10 ^-7 M or less, more preferably about 10 ^-8 M or less, even more preferably about 10 ^-9 M or less, and even more preferably about 10 ^-10 M or less. Such anti-CRP monoclonal antibodies are typically represented by accession number FERM BP-7941 (MH-IIC1) and accession number FERM BP-7942 (MH-IIC) of the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. IIC3), accession number FERM BP-7939 (MH-C3), accession number FERM BP-7940 (MH-C4), accession number FERM BP-8185 (MH-C5), accession number FERMBP- No. 8186 (MH-C6), accession number FERM BP · ERM BP · · ·
This anti-CRP monoclonal antibody is preferably a monoclonal antibody that has binding ability to CRP and has no binding ability to complement 1Cq and serum amyloid P. That is, CRP is inhibited in the measurement under substantially the same conditions as the inhibition ELISA method described in the following example, and the half value of the inhibition is about 10%. ^-6 M or less, preferably the half-value of this inhibition is about 10 ^-7 M or less, more preferably about 10 ^-8 M or less, even more preferably about 10 ^-9 M or less, even more preferably about 10 ^-10 M or less, but no inhibition is applied to complement 1Cq and serum amyloid P in the measurement under substantially the same conditions as the inhibition ELISA method described in the following example. Such an anti-CRP monoclonal antibody is available under the deposit number of FERM BP-7941 (MH-IIC1), the deposit number of FERM BP-7942 (MH-IIC3), and the deposit number of National Institute of Advanced Industrial Science and Technology. No. FERM BP-7940 (MH-C4), accession number FERM BP-8185 (MH-C5), accession number FERM BP ...
"Anti-CRP monoclonal antibodies" according to the present invention also include functional fragments that retain their binding properties. These fragments can compete with the intact antibody from which they are derived for specific binding to CRP and have at least 10 ⁷ , 10 ⁸ , 10 ⁹ M ^-1 Or 10 ¹⁰ M ^-1 With an affinity of Antibody fragments include immunoglobulin heavy and light chains, Fab, Fab ', F (ab'). ₂ , Fabc and Fv. Antibody fragments can be generated by enzymatic or chemical separation of intact immunoglobulins. For example, F (ab ') ₂ The fragments are protein digested with pepsin at pH 3.0-3.5 using standard methods as described in Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, COLD SPRING HARBOR LABORATORY, New York (1988). Can be obtained from IgG molecules. Fab fragments are converted to F (ab ') by limited reduction. ₂ It can be obtained from fragments or from whole antibodies by papain digestion in the presence of a reducing agent (see Paul, W. Ed., FUNDAMENTAL IMMUNOLOGY 2nd Edition Ravan Press, NY, 1989, Chapter 7).
[0062]
The present invention also provides a kit for detecting CRP. The kit of the present invention can detect CRP in a sample solution with high sensitivity based on an antigen-antibody binding reaction. The kit of the present invention comprises a first antibody and a second antibody, wherein the first antibody is an anti-CRP antibody and the second antibody is a labeled anti-CRP antibody. In particular, at least one of the first antibody or the second antibody is an anti-CRP monoclonal antibody that does not bind to complement C1q and serum amyloid P.
[0063]
That is, a monoclonal antibody having the binding ability to CRP as described above and not having the ability to bind to complement C1q and serum amyloid P was used as one antibody, and the other antibody was used to bind to CRP. Any antibody can be used as long as it has binding ability (this may be a polyclonal antibody or a monoclonal antibody). As such another antibody, a conventional antibody which is commercially available can also be used. Preferably, a monoclonal antibody can be used.
[0064]
In the kit of the present invention, since at least one of the first antibody and the second antibody is an anti-CRP monoclonal antibody that does not bind to complement C1q and serum amyloid P, it also binds to complement C1q and serum amyloid P. The abundance of active antibodies is greatly reduced. Therefore, the first antibody and the second antibody almost certainly bind in a sandwich manner with the CRP interposed therebetween, and form a complex by an antigen-antibody reaction. Therefore, for example, CRP can be measured with high sensitivity and high accuracy by applying the kit of the present invention to an immunoassay using conventional spectroscopic means (immunopathy, immunoassay, etc.). .
[0065]
In this embodiment, the “anti-CRP monoclonal antibody” produced by the above method can be used as the first antibody and the second antibody of the kit of the present invention. Examples of such antibodies include accession number FERM BP-7941 (MH-IIC1), accession number FERM BP-7942 (MH-IIC3), accession number FERM BP-7939 (MH-C3), accession number FERM BP-7940 (MH-C4), accession number FERM BP-8185 (MH-C5), accession number FERM BP-8186 (MH-C6) ), It is particularly desirable that only the accession number FERM BP... Therefore, a monoclonal antibody having binding ability to CRP and not binding ability to complement 1Cq and serum amyloid P is preferable. Accession No. FERM BP-7941 (MH-IIC1), Accession No. FERM BP-7942 (MH-IIC3), Accession No. FERM BP-7940 (MH-IIC) C4), accession number FERM BP-8185 (MH-C5), accession number FERM
No. BP No. 8339 (MH-IIC4) or accession number FERM BP No.
In the kit of the present invention, the first antibody and the second antibody are capable of recognizing and binding to an epitope on an antigen to which the first antibody did not bind in a sandwich reaction. Are included in any combination. The second antibody is preferably an antibody having no binding ability to an epitope to which the first antibody binds. Since CRP is a pentamer, the first antibody and the second antibody may be the same type of antibody.
[0066]
Further, in the kit of the present invention, the first antibody is immobilized on a solid phase, and the second antibody is an anti-C-reactive protein monoclonal antibody that does not bind to complement C1q and serum amyloid P; It may be a kit contained in the mobile phase.
[0067]
In a sandwich reaction, the immobilized antibody and the labeled antibody in the mobile phase are on separate constituent monomers for one CRP antigen, since CRP may have an epitope on each monomer that makes up the pentamer Binding to an epitope. In particular, since the second antibody is an anti-C-reactive protein monoclonal antibody that does not bind to complement C1q and serum amyloid P, a label indicating the presence of complement C1q and serum amyloid P is not detected. Therefore, CRP can be accurately detected and measured with higher sensitivity.
[0068]
In the kit of the present invention, for example, as a combination of the first antibody and the second antibody, accession number FERM BP-7941 (MH-IIC1) of the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and accession number FERM BP-7942 (MH-IIC3), accession number FERM BP-7939 (MH-C3), accession number FERM BP-7940 (MH-C4), accession number FERM BP-8185 (MH-C5) ), Accession number FERM BP-8186 (MH-C6), accession number FERM BP...
[0069]
The above description also applies to the case where the “first antibody” is used in exchange for the “second antibody”.
[0070]
In the kit of the present invention, both the first antibody and the second antibody are produced by the cell line of Accession No. FERM BP-7941 (MH-IIC1) of the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology. Anti-CRP monoclonal antibody.
[0071]
Similarly, in the kit of the present invention, both the first antibody and the second antibody are cell lines of the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology, Accession No. FERM BP-7942 (MH-IIC3). May be an anti-CRP monoclonal antibody produced by the cell line of Accession No. FERM BP-7940 (MH-C4), and an accession number FERM BP- No. 8185 (MH-C5), accession number FERM
BP ...
The second antibody can be labeled with any label by methods known in the art. Examples of the label include an enzyme label, a dye label, a magnetic label, a radioactive label, a label with colored particles (colloidal gold, latex, etc.), and the like.
[0072]
Usually, the first antibody can be immobilized on a solid phase of the kit (eg, an ELISA plate), and the second antibody can be used as a mobile phase.
[0073]
The kit of the present invention can be appropriately prepared by a method known in the art. A kit of the invention may include the first antibody and the second antibody in one or more containers. The kit may also include instructional materials that teach the use of the antibody in a CRP sandwich assay. Kits may include appropriate reagents, wash solutions, dilution buffers, and the like, for detecting the label or for detecting the positive and negative controls. The kit may include a solid phase antigen solution for adsorption to a solid phase, a solid phase blocking solution, and a standard CRP solution for preparing a calibration curve. It is preferable to use PBS-Az (phosphate-buffered saline-sodium azide solution) as a solvent since all solutions create an environment similar to that in a living body. The solid phase blocking solution is preferably a protein solution such as casein, skim milk, bovine serum albumin. In particular, casein is a protein obtained by purifying milk, and is preferable because it has stable quality and is inexpensive. The concentration is preferably about 1% because the solid phase can be efficiently blocked. It is preferable to use the same protein solution as the solid phase blocking solution also for the standard albumin solution, and it is preferable to use the same protein concentration as the solid phase blocking solution because the equilibrium relationship with the solid phase can be efficiently maintained.
[0074]
As described above, according to the method for producing a monoclonal antibody-producing cell line of the present invention, in the cloning of the cell line, the binding ability of the antibody produced from the fused cells to CRP, complement C1q, and serum amyloid P is assayed, The cells of interest are sorted. Therefore, after cell fusion, useful cells can be efficiently selected from many cells existing at the beginning. In addition, cell lines that produce highly specific antibodies while achieving high affinity for CRP can be created.
[0075]
When the immunoassay is an enzyme immunoassay (ELISA), the binding ability of the antibody can be assayed simply and with high sensitivity.
[0076]
When the mammalian myeloma-derived cell line is mouse myeloma-derived P3X63-Ag8.653, it does not produce antibody fragments, and the obtained fused cells have particularly excellent proliferation ability. Many cells can be assayed.
[0077]
When the mammal to be immunized is a mouse or a rat, it is convenient in terms of handling of the animal and immunization. When the mammal to be immunized is a BALB / C strain mouse, it becomes possible to obtain a monoclonal antibody having extremely high affinity for CRP.
[0078]
According to the monoclonal antibody-producing cell line of the present invention, an anti-CRP monoclonal antibody having high affinity for CRP and high specificity can be semipermanently provided by culturing it.
[0079]
When the monoclonal antibody of the present invention is used in the sandwich method, it is possible to provide a highly specific CRP detection kit that does not cross-react with complement C1q and serum amyloid P.
[0080]
In addition, the anti-human CRP monoclonal antibodies of the present invention are also expected to bind to CRPs of other closely related mammals almost similarly specifically. Therefore, it is considered that it can be used for the detection and measurement of CRP of other closely related mammals.
[0081]
【Example】
Hereinafter, the production of the monoclonal antibody-producing cell line of the present invention will be described more specifically. The present invention is not limited by the following examples. Materials, reagents, etc., used in the examples are available from commercial sources unless otherwise specified.
[0082]
<Enzyme immunoassay (ELISA)>
In the following examples, the evaluation of the obtained antiserum, culture supernatant, and monoclonal antibody was performed by ELISA. The operation method is described below.
[0083]
(A) Coating of antigen (CRP)
CRP is 0.1 mg / mL BSA-PBS-Az (bovine serum albumin (hereinafter referred to as BSA) dissolved in 0.04% by weight sodium azide-phosphate buffered saline solution at a concentration of 0.1 mg / mL). ) To a concentration of 5 μg / mL. The antigen solution was injected at 100 μl / well into a microplate (polystyrene high binding flat bottom # 2580, manufactured by Costar) and stored overnight in saturated steam at room temperature. Immediately before the experiment, the antigen solution was removed with an aspirator.
[0084]
(B) Blocking
200 μl / well of 1% by weight BSA-PBS-Az was injected and left at room temperature for 30 minutes. Thereafter, 1% by weight of BSA-PBS-Az was removed with an aspirator. When the subsequent experiments were not carried out on the same day, they were stored in saturated steam at 4 ° C. in this state.
[0085]
(C) Antibody reaction
Antibody solutions (antiserum, culture supernatant, purified antibody, etc.) diluted to various concentrations with 1% by weight BSA-PBS-Az were injected at 50 μl / well, and 1% by weight BSA-PBS-Az at 50 μl / well. . When performing an inhibition (inhibition) experiment for the purpose of measuring relative affinity, 50 μl / well of an inhibitor solution (a solution containing either CRP, complement C1q, or serum amyloid P) was injected, An antibody solution (50 μl / well) was further added and shaken. After standing at room temperature for one and a half hours, it was washed three times with PBS, and the remaining PBS was removed with an aspirator.
[0086]
(D) Reaction of secondary antibody
0.2 μg / mL peroxidase-labeled goat-derived anti-mouse IgG antibody (manufactured by KPL) dissolved in 1% by weight BSA in PBS solution, or 0.2 μg / mL peroxidase-labeled goat-derived anti-mouse IgM A solution prepared by dissolving an antibody (manufactured by KPL) in a 1% by weight BSA solution in PBS was injected at 100 μl / well, and allowed to stand at room temperature for 30 minutes. After washing three times with PBS, the remaining PBS was removed with an aspirator.
[0087]
(E) Substrate reaction and termination
A solution (substrate solution) obtained by dissolving 40 mg of O-phenylenediamine (for biochemistry) in 10 mL of a citric acid-phosphate buffer (pH 5) and adding 4 μL of a 30% by weight hydrogen peroxide solution immediately before use is injected at 100 μl / well. And left at room temperature. After about 3 minutes, the reaction was stopped by injecting 25 μl / well of 4N sulfuric acid.
[0088]
(F) Measurement
The absorbance at 492 nm was measured using a microplate reader (manufactured by Toyo Soda Co., Ltd.).
[0089]
In this example, an enzyme immunoassay was used as the immunoassay, but an RIA method, a fluorescent antibody method, or the like may be used instead.
[0090]
<Example 1>
In this example, taking into account the fact that the present inventors have a track record in the laboratory, and that BALB / C strain mice are most often used in mass culturing of antibodies by ascites after establishing a monoclonal antibody-producing cell line. BALB / C strain mice were used for immunization.
[0091]
(Immunity)
C-reactive protein (CRP) (manufactured by Chemicon International Inc.), which is an immunogen, was adjusted to 2 mg / mL using a phosphate buffer solution (PBS) at a physiological saline concentration. The same volume of a complete adjuvant (ADJUVANT COMPLETE FREUND, manufactured by DIFCO LABORATORIES) was added to the PBS solution of CRP, and the mixture was sufficiently emulsified with a homogenizer at 1,000 rpm to obtain an adjuvant emulsion containing an immunogen.
[0092]
Four 7-week-old female mice (BALB / C) were injected subcutaneously with 100 μl of an adjuvant emulsion containing an immunogen. Two weeks later, a CRP solution adjusted to 2 mg / mL using PBS and an incomplete Freund's adjuvant (ADJUVANT INCOMPLETE FREEUND, manufactured by DIFCO LABORATORIES) of the same volume were emulsified with a homogenizer, and the emulsion was applied to BALB / C mice. 100 μl was injected into the same site (subcutaneous) as the previous time.
[0093]
Thereafter, 4 and 6 weeks after the start of the immunization, 100 μl of the incomplete Freund's adjuvant emulsion containing the same composition and concentration of CRP as that of the immunization 2 weeks later was subcutaneously injected to each mouse. Blood was collected one week after the second injection and one week after the fourth injection, and antibody production was confirmed as described below.
[0094]
(Confirmation of antibody production)
Serum was separated from the collected blood, and antibody production was confirmed using the obtained serum by enzyme-linked immunosorbent assay (ELISA). As a solid phase, 5 μg / mL CRP prepared with 0.1 mg / mL BSA-PBS-Az was dispensed at 100 μl / well, and a microplate coated overnight at room temperature was used. A peroxidase-labeled anti-mouse IgG antibody or a peroxidase-labeled anti-mouse IgM antibody was used as the second antibody. Color development in the wells confirms the presence of antibody binding to CRP in the antibody sample.
[0095]
As a result, production of anti-CRP antibody was observed in all four mice. Furthermore, in all mice, it was confirmed that the antibody production shifted from IgG to IgM after the second injection, and the IgG / IgM ratio was 100 or more after the fourth injection, and class switching occurred sufficiently. I confirmed that.
[0096]
(Cell fusion)
A final immunization was performed to enlarge one spleen, which had the highest titer among the immunized mice. Ten weeks after the start of immunization, the CRP of the immunogen was adjusted to a concentration of 1 mg / mL using PBS, and the mice were injected in an amount of 100 μl without adding an adjuvant.
[0097]
Three days after the last immunization, spleen cells of the mouse were removed. Using a polyethylene glycol having an average molecular weight of 1,500, spleen cells were fused with a mouse myeloma-derived cell line (P3X63-Ag8.653) by a conventional method to obtain fused cells.
[0098]
The fused cells were suspended in hypoxanthine / aminopterin / thymidine (HAT) medium prepared in Ishikov medium containing 15% by weight of fetal calf serum (hereinafter, FCS), and spread on three 96-well plates (200 μl). / Well). At this time, spleen cells of the same mouse individual were used as feeder cells (cells supplying a growth factor at the start of culture). CO ₂ Incubator (CO ₂ The culture was started within a concentration of 5% by volume, a temperature of 37 ° C. and a humidity of 95%. In the following cultures, cultures were performed under the same conditions unless otherwise indicated.
[0099]
(Cell sorting and cloning)
On the 5th and 7th days after the cell fusion, 100 μl of the culture supernatant of the fused cells was collected from each well, and whether or not the antibody was produced was confirmed by the ELISA method as described above. When the supernatant was collected, 100 μl of each HAT medium prepared with Ishikov medium containing 15% by weight of FCS was added, and the culture was continued.
[0100]
On day 9 after the cell fusion, 100 μl of the culture supernatant of the fusion cells was collected in each well, and the remaining culture solution containing the fusion cells was subcultured into 12 24-well plates, and 1 ml of 15% by weight was added to each well. HAT medium prepared with Ishikov medium containing FCS was added. The collected culture supernatant was measured for its binding ability to CRP by a binding ELISA method. The binding ELISA method measures binding ability to an antigen (in this example, CRP), and was performed without adding an inhibitor according to the description in the section <Enzyme-linked immunosorbent assay (ELISA method) described above>. .
[0101]
Five days after the passage of the fused cells into a 24-well plate, cell culture supernatants were collected at 200 μl / well. The binding ability of this culture supernatant to CRP was measured by binding ELISA.
[0102]
By combining the results of the ELISA method of the culture supernatant collected twice, 93 wells having a binding ability to CRP and having a good growth state were selected from 244 wells. Cells in these wells were all passaged into 16 six-well plates for the first stage selection. To each well, 4 ml of hypoxanthine / thymidine (HT) medium containing 15% by weight of FCS was added.
[0103]
These collected culture supernatants were measured for their binding ability to CRP by using an inhibition ELISA method as a first-stage selection. Inhibition ELISA measures the relative affinity of a test substance between a solid phase-bound antigen and an inhibitor in a solution phase, and is described in the above section <Enzyme immunoassay (ELISA)>. Was performed with the addition of an inhibitor as described below.
[0104]
100 μl / well of CRP adjusted to a concentration of 5 μg / mL with 0.1 mg / mL · BSA-PBS-Az was used as a solid phase, and CRP was used as an inhibitor. The cell culture supernatant was used as the antibody solution. As a secondary antibody, a peroxidase-labeled anti-mouse IgG antibody was used. If an antibody that binds to CRP is present in the cell culture supernatant, it binds to soluble CRP added as an inhibitor and inhibits the binding (inhibition of binding to solid-phase CRP). Is not confirmed.
[0105]
As a result, for the second stage of selection, 8 wells that exhibited binding ability to CRP and were inhibited in the inhibition ELISA were selected. The cells in these wells were each passaged into medium flasks (volume 50 ml) three days after the first stage selection. To each well, 45 ml of HT medium containing 15% by weight of FCS was added.
[0106]
Three days after the passage of the first stage of cell sorting, the culture supernatant was collected, and the binding ability to CRP was measured using the inhibition ELISA method as the second stage of selection. The inhibition ELISA method used for the second stage selection is as follows.
[0107]
As a solid phase, CRP prepared at a concentration of 5 μg / mL with 0.1 mg / mL · BSA-PBS-Az was used at 100 μl / well, and CRP and complement C1q (HUMAN C1q ANTIGEN, Cat #: AG704) were used as inhibitors. And Amyloid P (AMYLOID P, Cat #: 0490-2752, manufactured by Biogenesis). The cell culture supernatant was used as the antibody solution. As a secondary antibody, a peroxidase-labeled anti-mouse IgG antibody was used. If an antibody that binds to the inhibitor is present in the cell culture supernatant, it binds to the inhibitor that has been solubilized in the supernatant and the inhibition is applied, so that color development in the well is not confirmed.
[0108]
Two wells were selected that were inhibited only by CRP, but not complement C1q or serum amyloid P.
[0109]
The cells in the two wells are diluted (limit dilution) to a concentration containing two cells per well using an HT medium containing 15% by weight of FCS, and divided into three 96-well microplates. Noted. Thymocytes of 4-week-old female mice (BALB / C) were used as feeders to promote initial proliferation. The culture was advanced while increasing the size of the plate, and the above-mentioned screening of the cell culture supernatant by the inhibition ELISA method in the second step was repeated as appropriate.
[0110]
Cell lines that show high titers against CRP, are not inhibited by complement C1q or serum amyloid P, and show good growth are finally selected and 5% in 200 mL of medium. × 10 ⁵ Culture was advanced to a concentration of cells / mL.
[0111]
Finally, two strains showing binding ability to CRP and not causing a cross-reaction with complement C1q or serum amyloid P were selected.
[0112]
One of the strains that show binding ability to CRP and does not cause a cross-reaction with complement C1q or serum amyloid P is named cell line name: MH-IIC1, and is patented by the National Institute of Advanced Industrial Science and Technology. It was deposited on March 7, 2002 at the Organism Depositary (Accession No. FERM BP-7941).
[0113]
Another strain that shows binding ability to CRP and does not cause a cross-reaction with complement C1q or serum amyloid P is named cell line name: MH-IIC3, and is patented by the National Institute of Advanced Industrial Science and Technology. Deposited with the Biological Depositary on March 7, 2002 (Accession No. FERM BP-7942).
[0114]
The antibodies produced by the MH-IIC1 strain and the MH-IIC3 strain are referred to as MH-IIC1 antibody and MH-IIC3 antibody, respectively.
[0115]
(Preservation of cells)
The finally selected cell line was centrifuged to remove the supernatant, and 1 × 10 ⁷ The cells were suspended in 1 mL of a solution of FCS: dimethylsulfoxide = 9: 1 (volume ratio) at a concentration of cells / mL, preliminarily frozen at -80 ° C, and then transferred to liquid nitrogen for long-term storage.
[0116]
(Purification of antibody)
The selected strain was mass-cultured in Ishikov medium containing 15% by weight of FCS and centrifuged to obtain a culture supernatant. The culture supernatant was subjected to affinity chromatography using a protein A binding gel (Protein A Sepharose 4FF, manufactured by Pharmacia) to purify each monoclonal antibody (MH-IIC1 antibody and MH-IIC3 antibody) under the following conditions.
[0117]
The column packed with protein A binding gel was equilibrated with binding buffer (1.5 M glycine / 3 M NaCl, pH 8.9). The culture supernatant or ascites was diluted about 3 times with a binding buffer, and then applied to an equilibrated column. The column was washed with binding buffer while monitoring the eluate from the column at 280 nm until the elution of the impurities was complete. After washing, an elution buffer (100 mM citric acid, pH 4) was applied to the column (linear flow rate: about 20 cm / hour), and an IgG-containing eluate was collected. The concentration of the antibody was determined by measuring the absorbance of the collected IgG-containing eluate at 280 nm with an absorptiometer and converting the measured absorbance by an extinction coefficient.
[0118]
From comparison with the standard protein by SDS polyacrylamide gel electrophoresis, the purified fractions of these monoclonal antibodies (MH-IIC1 antibody and MH-IIC3 antibody) were all found to have a heavy chain of about 50,000 and a molecular weight of about 25,000. It was confirmed to be an IgG consisting of an L chain. In addition, contamination on the electrophoresis was below the detection limit.
[0119]
(Evaluation of antibody)
The monoclonal antibody purified by the affinity chromatography was subjected to antibody evaluation using the CRP dilution series under the same conditions as the inhibition ELISA method in the selection in the second step.
[0120]
FIGS. 2A-C show CRP (FIGS. 2A and 3A), complement C1q (FIGS. 2B and 3B), and serum amyloid for MH-IIC1 antibody, and FIGS. It is a graph which shows the result of having measured binding ability with respect to P (FIG. 2C and FIG. 3C) by competition reaction (inhibition reaction). In each figure, the vertical axis represents absorbance (wavelength: 492 nm), and the horizontal axis represents CRP concentration (mol / L, hereinafter referred to as M), complement C1q concentration (mg / ml), and serum amyloid P concentration (mg / ml). dl) is shown.
[0121]
As shown in FIGS. 2A-C, for the MH-IIC1 antibody, the half-value of the inhibition to CRP was about 7 × 10 ^-7 M and 2-3 × 10 ^-7 The possibility of detecting M CRP was shown. No reaction was observed with complement C1q and serum amyloid P, confirming that no cross-reaction occurred.
[0122]
As shown in FIGS. 3A-C, for the MH-IIC3 antibody, the half-value of the inhibition to CRP was about 5 × 10 ^-7 M and 2-3 × 10 ^-7 The possibility of detecting M CRP was shown. No reaction was observed with complement C1q and serum amyloid P, confirming that no cross-reaction occurred.
[0123]
<Example 2>
Also in this example, taking into account the fact that the present inventors have a track record in the laboratory, and that BALB / C strain mice are most frequently used in the mass cultivation of antibodies by ascites after the establishment of a monoclonal antibody-producing cell line. BALB / C strain mice were used for immunization.
[0124]
(Immunity)
C-reactive protein (CRP) (manufactured by Chemicon International Inc.), which is an immunogen, was adjusted to 2 mg / mL using a phosphate buffer solution (PBS) at a physiological saline concentration. The same volume of an adjuvant (complete Freund's adjuvant containing killed Mycobacterium tuberculosis (ADJUVANT COMPLETE FREEUND, manufactured by DIFCO LABORATORIES)) was added to the PBS solution of CRP, and the immunogen was sufficiently emulsified with a homogenizer at 1,000 rpm. A combined adjuvant emulsion was obtained.
[0125]
Fifteen female mice (BALB / C) about seven weeks old were injected intraperitoneally or subcutaneously with 100 μl of an adjuvant emulsion containing an immunogen. Two weeks later, a CRP solution adjusted to 2 mg / mL using PBS and an incomplete Freund's adjuvant (ADJUVANT INCOMPLETE FREEUND, manufactured by DIFCO LABORATORIES) of the same volume were emulsified with a homogenizer, and the emulsion was applied to BALB / C mice. 100 μl was injected into the same site as the previous time.
[0126]
Thereafter, 4 weeks, 6 weeks, and 6 months after the start of the immunization, the mice were injected with an incomplete Freund's adjuvant emulsion containing the same composition and concentration of CRP as the immunizations 2 weeks after the immunization, by 100 μl each at the same site as the previous time. . Blood was collected one week after the second injection and one week after the fourth injection, and the following antibody production was confirmed.
[0127]
(Confirmation of antibody production)
Serum was separated from the collected blood, and antibody production was confirmed using the obtained serum by enzyme-linked immunosorbent assay (ELISA). As a solid phase, 5 μg / mL CRP prepared with 0.1 mg / mL BSA-PBS-Az was dispensed at 100 μl / well, and a microplate coated overnight at room temperature was used. As the secondary antibody, a peroxidase-labeled anti-mouse IgG antibody or a peroxidase-labeled anti-mouse IgM antibody was used. Color development in the wells confirms the presence of antibody binding to CRP in the antibody sample.
[0128]
As a result, production of anti-CRP antibody was observed in all 15 mice. Furthermore, in all the mice, it was confirmed that the antibody production shifted from IgM to IgG after the second injection, and the IgG / IgM ratio was 100 or more after the fourth injection, and class switching occurred sufficiently. It was confirmed.
[0129]
(Cell fusion)
A final immunization was performed to enlarge the spleen of the three titers that were particularly high in the immunized mice. Six months after the start of immunization, the CRP of the immunogen was adjusted to a concentration of 5 mg / mL using PBS, and the mice were injected in an amount of 100 μl without adding an adjuvant.
[0130]
One mouse spleen cell was removed from the mouse three days after the final immunization. The spleen cells were fused with a mouse myeloma-derived cell line (P3X63-Ag8.653) by a conventional method using polyethylene glycol having an average molecular weight of 1,500 to obtain fused cells.
[0131]
The fused cells were suspended in hypoxanthine / aminopterin / thymidine (HAT) medium prepared with Ishikov medium containing 15% by weight of fetal calf serum (hereinafter, FCS), and then spread on one 96-well plate ( 200 μl / well). At this time, spleen cells of the same mouse individual were used as feeder cells (cells supplying a growth factor at the start of culture). CO ₂ Incubator (CO ₂ The culture was started within a concentration of 5% by volume, a temperature of 37 ° C. and a humidity of 95%. In the following cultures, cultures were performed under the same conditions unless otherwise indicated.
[0132]
(Cell sorting and cloning)
One week later, 100 µl of the culture supernatant of the fused cells was collected. On the other hand, the remaining culture solution containing the fused cells was subcultured into four 24-well plates, and 1 ml of hypoxanthine / thymidine (HT) medium containing 15% by weight of FCS was added to each well.
[0133]
Four days after the fused cells were passaged into a 24-well plate, cell culture supernatants were collected at 150 μl / well. Using this culture supernatant and the culture supernatant collected one week after the start of the culture, the binding ability to CRP was measured by the binding ELISA method described below.
[0134]
As a solid phase, CRP adjusted to a concentration of 5 μg / mL with 0.1 mg / mL · BSA-PBS-Az was used at 100 μl / well. The cell culture supernatant was used as the antibody solution. As a secondary antibody, a peroxidase-labeled anti-mouse IgG antibody was used.
[0135]
By combining the ELISA results of the culture supernatants collected twice, 53 wells having a high binding ability to CRP and having a good growth state were confirmed. As a first step selection, cells from these wells were all passaged into nine 6-well plates, and 4 ml of HT medium containing 15% by weight of FCS was added to each well.
[0136]
Two days after the cell sorting for the first step, the culture supernatant was collected and the binding ability to CRP was measured by the inhibition ELISA method. The inhibition ELISA method used for the first step selection is as follows.
[0137]
CRP prepared at a concentration of 5 μg / mL with 0.1 mg / mL · BSA-PBS-Az was used as a solid phase, 100 μl / well, and CRP was used as an inhibitor. The cell culture supernatant was used as the antibody solution. As a secondary antibody, a peroxidase-labeled anti-mouse IgG antibody was used. If an antibody that binds to CRP is present in the cell culture supernatant, it binds to soluble CRP added as an inhibitor and inhibits the binding (inhibition of binding to solid-phase CRP). Is not confirmed.
[0138]
As a result, for the second-stage selection, 22 wells that exhibited high binding ability to CRP and were inhibited by the inhibition ELISA were selected. The cells in these wells were each passaged into medium flasks (50 ml volume). As the medium, 45 ml of an HT medium containing 15% by weight of FCS was added.
[0139]
Three days after the passage of the cells subjected to the first-stage selection, the culture supernatant was collected, and as a second-stage selection, the binding ability to CRP was measured using an inhibition ELISA method. The inhibition ELISA method used for the second stage selection is as follows.
[0140]
100 μl / well of CRP prepared at a concentration of 5 μg / mL with 0.1 mg / mL · BSA-PBS-Az is used as a solid phase, and any of CRP, complement C1q or serum amyloid P is used as an inhibitor did. The cell culture supernatant was used as the antibody solution. As a secondary antibody, a peroxidase-labeled anti-mouse IgG antibody was used. If an antibody that binds to the inhibitor is present in the cell culture supernatant, the inhibition occurs, and no color development in the wells is confirmed.
[0141]
Two wells were selected that were inhibited only by CRP and were not inhibited by complement C1q and serum amyloid P. In addition, two wells that were inhibited by CRP and serum amyloid P and not inhibited by complement C1q were selected.
[0142]
The cells in the 4 wells are diluted (limited dilution) to a concentration containing 2 cells per well using HT medium containing 15% by weight of FCS, and dispensed into two 96-well microplates. did. Thorax cells of 4-week-old female mice (BALB / C) were used as feeder cells to promote initial proliferation. The culture was advanced while increasing the size of the plate, and the above-described ELISA screening was repeated for the cell culture supernatant at appropriate times.
[0143]
Cell lines showing high titers against CRP and showing good growth were finally selected and 5 × 10 5 in 200 mL of medium. ⁵ Culture was advanced to a concentration of cells / mL.
[0144]
Finally, one strain showing a binding ability to CRP and not causing a cross-reaction with complement C1q was selected. In addition, one strain showing a binding ability to CRP and not causing a cross-reaction with complement C1q and serum amyloid P was selected.
[0145]
A cell line that exhibits binding ability to CRP and does not cause a cross-reaction with complement C1q was named cell line name: MH-C3, and was assigned to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, 2002. Deposited on March 7 (Accession No. FERM BP-7939).
[0146]
A cell line that exhibits binding ability to CRP and does not cause a cross-reaction with complement C1q and serum amyloid P is designated as cell line name: MH-C4, and is a Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. Was deposited on March 7, 2002 (Accession No. FERM BP-7940).
[0147]
The antibodies produced by the MH-C3 strain and the MH-C4 strain are referred to as MH-C3 antibody and MH-C4 antibody, respectively.
[0148]
(Preservation of cells)
The finally selected cell line was centrifuged to remove the supernatant, and 1 × 10 ⁷ The cells were suspended in 1 mL of a solution of FCS: dimethylsulfoxide = 9: 1 (volume ratio) at a concentration of cells / mL, preliminarily frozen at -80 ° C, and then transferred to liquid nitrogen for long-term storage.
[0149]
(Purification of antibody)
Each of the selected two strains was cultured in large amounts in an Ishikov medium containing 15% by weight of FCS, and the supernatant was centrifuged. In addition, each of the selected two strains was injected intraperitoneally into female BALB / C mice to proliferate and accumulate ascites. The accumulated ascites was collected. The culture supernatant or ascites of each strain is subjected to affinity chromatography using a protein A binding gel (Protein A Sepharose 4FF, manufactured by Pharmacia), and each monoclonal antibody (MH-C3 antibody and MH-C4 antibody) is obtained under the following conditions. Purified.
[0150]
The column packed with protein A binding gel was equilibrated with binding buffer (1.5 M glycine / 3 M NaCl, pH 8.9). The culture supernatant or ascites was diluted about 3 times with a binding buffer, and then applied to an equilibrated column. The column was washed with binding buffer while monitoring the eluate from the column at 280 nm until the elution of the impurities was complete. After washing, an elution buffer (100 mM citric acid, pH 4) was applied to the column (linear flow rate: about 20 cm / h), and an IgG-containing eluate was collected. The concentration of the antibody was determined by measuring the absorbance of the collected IgG-containing eluate at 280 nm with an absorptiometer and converting the measured absorbance by an extinction coefficient.
[0151]
From comparison with the standard protein by SDS polyacrylamide gel electrophoresis, the purified fractions of these monoclonal antibodies (MH-C3 and MH-C4) were all found to have an H chain having a molecular weight of about 50,000 and an L chain of about 25,000. It was confirmed to be an IgG consisting of chains. In addition, contamination on the electrophoresis was below the detection limit.
[0152]
(Evaluation of antibody)
The two types of monoclonal antibodies purified by the affinity chromatography were evaluated using the CRP dilution series under the same conditions as the inhibition ELISA method in the selection in the second step.
[0153]
4A-C show CRP (FIGS. 4A and 5A), complement C1q (FIGS. 4B and 5B), and serum amyloid for MH-C3 antibody and FIGS. It is a graph which shows the result of having measured the binding ability with respect to P (FIG. 4C and FIG. 5C). In each figure, the vertical axis represents the absorbance (wavelength: 492 nm), the horizontal axis represents the CRP concentration (mol / L, hereinafter referred to as M), the serum amyloid P concentration (mol / L, hereinafter referred to as M), and the complement. The logarithmic value of the body C1q concentration (mg / ml) is shown.
[0154]
As shown in FIG. 4A, for the MH-C3 antibody, the half value of the inhibition was about 8 × 10 ^-9 M and 2-3 × 10 ^-9 The possibility of detecting M or more CRP was shown. As shown in FIG. 4B, no inhibition was observed for complement C1q, and it was confirmed that no cross-reaction occurred. On the other hand, as shown in FIG. 4C, the half value of the inhibition was about 5 × 10 5 against serum amyloid P. ^-8 M, indicating cross-reaction. Therefore, this indicates that the MH-C3 antibody has no binding ability to complement C1q.
[0155]
As shown in FIG. 5A, for the MH-C4 antibody, the half value of the inhibition was about 5 × 10 ^-10 M and 2 × 10 ^-10 The possibility of having a CRP detection sensitivity of about M or more was shown. As shown in FIGS. 5B and 5C, no inhibition was observed for complement C1q and serum amyloid P, and it was confirmed that cross-reaction did not occur. This indicates that the MH-C4 antibody has no binding ability to serum amyloid P and complement C1q.
[0156]
<Example 3>
Also in this example, taking into account the fact that the present inventors have a track record in the laboratory, and that BALB / C strain mice are most frequently used in the mass cultivation of antibodies by ascites after the establishment of a monoclonal antibody-producing cell line. BALB / C strain mice were used for immunization.
[0157]
(Immunity)
C-reactive protein (CRP) (manufactured by Chemicon International Inc.), which is an immunogen, was adjusted to 2 mg / mL using a phosphate buffer solution (PBS) at a physiological saline concentration. The same volume of a complete adjuvant (ADJUVANT COMPLETE FREUND, manufactured by DIFCO LABORATORIES) was added to the PBS solution of CRP, and the mixture was sufficiently emulsified with a homogenizer at 1,000 rpm to obtain an adjuvant emulsion containing an immunogen.
[0158]
Four 7-week-old female mice (BALB / C) were injected subcutaneously with 100 μl of an adjuvant emulsion containing an immunogen. Two weeks later, a CRP solution adjusted to 2 mg / mL using PBS and an incomplete Freund's adjuvant (ADJUVANT INCOMPLETE FREEUND, manufactured by DIFCO LABORATORIES) of the same volume were emulsified with a homogenizer, and the emulsion was applied to BALB / C mice. 100 μl was injected into the same site (subcutaneous) as the previous time.
[0159]
Thereafter, 4 and 6 weeks after the start of the immunization, 100 μl of the incomplete Freund's adjuvant emulsion containing the same composition and concentration of CRP as that of the immunization 2 weeks later was subcutaneously injected to each mouse. Blood was collected one week after the second injection and one week after the fourth injection, and antibody production was confirmed as described below.
[0160]
(Confirmation of antibody production)
Serum was separated from the collected blood, and antibody production was confirmed using the obtained serum by enzyme-linked immunosorbent assay (ELISA). As a solid phase, 5 μg / mL CRP prepared with 0.1 mg / mL BSA-PBS-Az was dispensed at 100 μl / well, and a microplate coated overnight at room temperature was used. A peroxidase-labeled anti-mouse IgG antibody or a peroxidase-labeled anti-mouse IgM antibody was used as the second antibody. Color development in the wells confirms the presence of antibody binding to CRP in the antibody sample.
[0161]
As a result, production of anti-CRP antibody was observed in all four mice. Furthermore, in all mice, it was confirmed that the antibody production shifted from IgG to IgM after the second injection, and the IgG / IgM ratio was 100 or more after the fourth injection, and class switching occurred sufficiently. I confirmed that.
[0162]
(Cell fusion)
A final immunization was performed to enlarge one spleen, which had the highest titer among the immunized mice. Ten weeks after the start of immunization, the CRP of the immunogen was adjusted to a concentration of 1 mg / mL using PBS, and the mice were injected in an amount of 100 μl without adding an adjuvant.
[0163]
Three days after the last immunization, spleen cells of the mouse were removed. Using a polyethylene glycol having an average molecular weight of 1,500, spleen cells were fused with a mouse myeloma-derived cell line (P3X63-Ag8.653) by a conventional method to obtain fused cells.
[0164]
The fused cells were suspended in hypoxanthine / aminopterin / thymidine (HAT) medium prepared in Ishikov medium containing 15% by weight of fetal calf serum (hereinafter, FCS), and spread on three 96-well plates (200 μl). / Well). At this time, spleen cells of the same mouse individual were used as feeder cells (cells supplying a growth factor at the start of culture). CO ₂ Incubator (CO ₂ The culture was started within a concentration of 5% by volume, a temperature of 37 ° C. and a humidity of 95%. In the following cultures, cultures were performed under the same conditions unless otherwise indicated.
[0165]
(Cell sorting and cloning)
On the third and sixth days after the cell fusion, 75 μl of the culture supernatant of the fused cells was collected from each well, and whether or not the antibody was produced was confirmed by the ELISA method as described above. When the supernatant was collected, 100 μl of each HAT medium prepared with Ishikov medium containing 15% by weight of FCS was added, and the culture was continued.
[0166]
On day 8 after the cell fusion, 75 μl of the culture supernatant of each fused cell was collected from each well, and the remaining culture solution containing the fused cells was subcultured and seeded on four 24-well plates. As a medium, 900 μl of hypoxanthine / thymidine (HT) medium prepared with Ishikov medium containing 15% by weight of FCS was added.
[0167]
On day 12 after the cell fusion, 200 μl of the culture supernatant of each fused cell was collected from each well. The culture supernatant was evaluated for IgG and IgM production by the binding ELISA method, and the inhibition by CRP by the inhibition ELISA method. Based on the results, 53 wells were selected, and the fused cells were passaged into nine 6-well plates. To each well, 4 ml of hypoxanthine / thymidine (HT) medium containing 15% by weight of FCS was added. The binding ELISA method measures binding ability to an antigen (in this example, CRP), and was performed without adding an inhibitor according to the description in the section <Enzyme-linked immunosorbent assay (ELISA method) described above>. . Inhibition ELISA measures the relative affinity of a test substance between a solid phase-bound antigen and an inhibitor in a solution phase, and is described in the above section <Enzyme immunoassay (ELISA)>. Was performed with the addition of an inhibitor as described below. 100 μl / well of CRP adjusted to a concentration of 5 μg / mL with 0.1 mg / mL · BSA-PBS-Az was used as a solid phase, and CRP was used as an inhibitor. The cell culture supernatant was used as the antibody solution. As a secondary antibody, a peroxidase-labeled anti-mouse IgG antibody was used. If an antibody that binds to CRP is present in the cell culture supernatant, it binds to soluble CRP added as an inhibitor and inhibits the binding (inhibition of binding to solid-phase CRP). Is not confirmed.
[0168]
On day 14 after cell fusion, 1 ml of the culture supernatant of the fused cells was collected from each well. The culture supernatant was evaluated for IgG binding by binding ELISA, and the next day, inhibition by CRP by inhibition ELISA. As a result, 23 wells were selected from wells showing high binding ability to CRP and having been inhibited in the inhibition ELISA, and the fused cells were subcultured to a medium flask (volume 250 ml). To each flask was added 45 ml of HT medium with 15 wt% FCS.
[0169]
All evaluation data so far were combined, and 16 days after cell fusion, 4 wells were selected.
[0170]
The cells in the 4 wells are diluted (limited dilution) to a concentration containing 2 cells per well using an HT medium containing 15% by weight of FCS, and divided into two 96-well microplates. Noted. Thymocytes of 4-week-old female mice (BALB / C) were used as feeders to promote initial proliferation. The culture was advanced while increasing the size of the plate, and screening of the cell culture supernatant by the inhibition ELISA method as described below was repeated as appropriate.
[0171]
As a solid phase, CRP prepared at a concentration of 5 μg / mL with 0.1 mg / mL · BSA-PBS-Az was used at 100 μl / well, and CRP and complement C1q (HUMAN C1q ANTIGEN, Cat #: AG704) were used as inhibitors. And Amyloid P (AMYLOID P, Cat #: 0490-2752, manufactured by Biogenesis). The cell culture supernatant was used as the antibody solution. As a secondary antibody, a peroxidase-labeled anti-mouse IgG antibody was used. If an antibody that binds to the inhibitor is present in the cell culture supernatant, it binds to the inhibitor that has been solubilized in the supernatant and the inhibition is applied, so that color development in the well is not confirmed.
[0172]
Finally, one strain was selected that showed binding ability to CRP and did not cause a cross-reaction with complement C1q or serum amyloid P. In addition, one strain showing a binding ability to CRP and not causing a cross-reaction with complement C1q but showing a weak cross-reactivity with serum amyloid P was selected.
[0173]
A strain that shows binding ability to CRP and does not cause a cross-reaction with complement C1q or serum amyloid P is designated as cell line name: MH-C5, and is a Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. Was deposited on September 18, 2002 (Accession No. FERM BP-8185).
[0174]
A strain that shows binding ability to CRP and does not cause a cross-reaction with complement C1q, but shows a weak cross-reactivity with serum amyloid P was named cell line name: MH-C6. Deposited at the Research Institute Patent Organism Depositary on September 18, 2002 (Accession No. FERM BP-8186).
[0175]
The antibodies produced by the MH-C5 strain and the MH-C6 strain are referred to as MH-C5 antibody and MH-C6 antibody, respectively.
[0176]
(Preservation of cells)
The finally selected cell line was centrifuged to remove the supernatant, and 1 × 10 ⁷ The cells were suspended in 1 mL of a solution of FCS: dimethylsulfoxide = 9: 1 (volume ratio) at a concentration of cells / mL, preliminarily frozen at -80 ° C, and then transferred to liquid nitrogen for long-term storage.
[0177]
(Purification of antibody)
The selected strain was mass-cultured in Ishikov medium containing 15% by weight of FCS and centrifuged to obtain a culture supernatant. The two selected strains were injected intraperitoneally into female BALB / C mice to proliferate, accumulate ascites, and were collected. The culture supernatant or ascites of each strain is subjected to affinity chromatography using a protein A binding gel (Protein A Sepharose 4FF, manufactured by Pharmacia) to purify each monoclonal antibody (MH-C5 antibody and MH-C6 antibody) under the following conditions. did.
[0178]
The column packed with protein A binding gel was equilibrated with binding buffer (1.5 M glycine / 3 M NaCl, pH 8.9). The culture supernatant or ascites was diluted about 3 times with a binding buffer, and then applied to an equilibrated column. The column was washed with binding buffer while monitoring the eluate from the column at 280 nm until the elution of the impurities was complete. After washing, an elution buffer (100 mM citric acid, pH 4) was applied to the column (linear flow rate: about 20 cm / hour), and an IgG-containing eluate was collected. The concentration of the antibody was determined by measuring the absorbance of the collected IgG-containing eluate at 280 nm with an absorptiometer and converting the measured absorbance by an extinction coefficient.
[0179]
From comparison with the standard protein by SDS polyacrylamide gel electrophoresis, the purified fractions of these monoclonal antibodies (MH-C5 antibody and MH-C6 antibody) were all found to have a heavy chain of about 50,000 and a molecular weight of about 25,000. It was confirmed to be an IgG consisting of an L chain. In addition, contamination on the electrophoresis was below the detection limit.
[0180]
(Evaluation of antibody)
The monoclonal antibody purified by the affinity chromatography was subjected to antibody evaluation using the CRP dilution series under the same conditions as the inhibition ELISA method in the selection in the second step.
[0181]
FIGS. 6A-C show CRP (FIGS. 6A and 7A), complement C1q (FIGS. 6B and 7B), and serum amyloid for MH-C5 antibody and FIGS. 8 is a graph showing the results of measuring the binding ability to P (FIGS. 6C and 7C) by a competitive reaction (inhibition reaction). In each figure, the vertical axis represents absorbance (wavelength: 492 nm), and the horizontal axis represents CRP concentration (mol / L, hereinafter referred to as M), complement C1q concentration (mg / ml), serum amyloid P concentration (mol / L). L, hereinafter referred to as M).
[0182]
As shown in FIG. 6A, for the MH-C5 antibody, the half-value of the inhibition to CRP was about 1 × 10 ^-10 M and 2-3 × 10 ^-11 The possibility of detecting M CRP was shown. As shown in FIGS. 6B and C, binding of the MH-C5 antibody to solid phase CRP was not inhibited by the presence of C1q and amyloid P as inhibitors. This indicates that the MH-C5 antibody has no binding ability to serum amyloid P and complement C1q. No reaction was observed with complement C1q and serum amyloid P, confirming that no cross-reaction occurred.
[0183]
As shown in FIG. 7A, for the MH-C6 antibody, the half-value of the inhibition to CRP was about 3 to 5 × 10 ^-10 M and 2 × 10 ^-10 The possibility of detecting M CRP was shown. As shown in FIG. 7C, the binding of the MH-C6 antibody to CRP on the solid phase is inhibited by amyloid P as an inhibitor, and inhibition is applied. At this time, the half value of the inhibitor is about 1 × 10 ^-7 M and the detection limit is about 1 × 10 ^-8 It is about M. Normal plasma levels of human serum amyloid P are about 30 mg / L, or about 2.4 x 10 ^-7 M. When an antibody is incorporated into a test kit, various factors usually reduce the sensitivity of the antibody. However, the above sensitivity of the MH-C6 antibody to serum amyloid P is such that even when this antibody is incorporated into a test kit, almost no reaction occurs with serum amyloid P in normal plasma, When used together with an antibody such as MH-C5, there is no practical problem.
[0184]
<Example 4>
In this example, taking into account the fact that the present inventors have a track record in the laboratory, and that BALB / C strain mice are most often used in mass culturing of antibodies by ascites after establishing a monoclonal antibody-producing cell line. BALB / C strain mice were used for immunization.
[0185]
(Immunity)
C-reactive protein (CRP) (manufactured by Chemicon International Inc.), which is an immunogen, was adjusted to 2 mg / mL using a phosphate buffer solution (PBS) at a physiological saline concentration. To this PBS solution of CRP, the same volume of a complete adjuvant (ADJUVANT COMPLETE FREUND, manufactured by DIFCO LABORATORIES) was added, and the mixture was sufficiently emulsified with a homogenizer at 1,000 rpm to obtain an adjuvant emulsion containing an immunogen.
[0186]
Four 7-week-old female mice (BALB / C) were intraperitoneally injected with 400 μl of an adjuvant emulsion containing an immunogen. Two weeks later, a CRP solution adjusted to 2 mg / mL using PBS and an incomplete Freund's adjuvant (ADJUVANT INCOMPLETE FREEUND, manufactured by DIFCO LABORATORIES) of the same volume were emulsified with a homogenizer, and this emulsion was used for BALB / C mice. 400 μl was injected into the peritoneal cavity.
[0187]
Thereafter, 4 and 6 weeks after the start of the immunization, mice were injected subcutaneously with 400 μl of the emulsion similarly prepared using incomplete Freund's adjuvant containing the same composition and concentration of CRP as the immunization 2 weeks later. Blood was collected one week after the second injection and one week after the fourth injection, and the following antibody production was confirmed.
[0188]
(Confirmation of antibody production)
Serum was separated from the collected blood, and antibody production was confirmed by enzyme immunoassay (ELISA) using the obtained serum. As a solid phase, 0.1 mg / mL · BSA ·· mL · CRP was dispensed at 100 μl / well and coated overnight at room temperature. A peroxidase-labeled anti-mouse IgG antibody or a peroxidase-labeled anti-mouse IgM antibody was used as the second antibody. Color development in the wells confirms the presence of antibody binding to CRP in the antibody sample.
[0189]
As a result, production of anti-CRP antibody was observed in all four mice. Furthermore, in all the mice, it was confirmed that the antibody production shifted from IgG to IgM after the second injection, and the IgG / IgM ratio was 100 or more after the fourth injection, and the class switching occurred sufficiently. It was confirmed.
[0190]
(Cell fusion)
In order to enlarge the two spleens of the immunized mice, the immunogen CRP was adjusted to a concentration of 5 mg / mL using PBS, and the mice were injected with 100 μl each without adjuvant to perform final immunization. Was.
[0191]
The spleen cells of each of two mice (mouse numbers # 3-2 and # 3-3) three days after the final immunization were extracted. Using polyethylene glycol having an average molecular weight of 1,500, spleen cells and a mouse myeloma-derived cell line (P3X63
Each of the fused cells was suspended in hypoxanthine / aminopterin / thymidine (HAT) medium prepared in Ishikov medium containing 15% by weight of fetal calf serum (hereinafter referred to as FCS), and spread on three 96-well plates. (200 μl / well). At this time, spleen cells of the same mouse individual were used as feeder cells (cells supplying a growth factor at the start of culture). CO ₂ Incubator (CO ₂ The culture was started within a concentration of 5% by volume, a temperature of 37 ° C. and a humidity of 95%. In the following cultures, cultures were performed under the same conditions unless otherwise indicated.
[0192]
(Cell sorting and cloning)
On days 5 and 7 after the cell fusion, 100 μl of the culture supernatant of the fused cells was collected from each well, and the production of the antibody was confirmed by ELISA. When the supernatant was collected, 100 μl of HAT medium prepared with Ishikov medium containing 15% by weight of FCS was added, and the culture was continued.
[0193]
On day 9 after the cell fusion, 100 μl of the culture supernatant of the fusion cells was collected in each well, and the remaining culture solution containing the fusion cells was subcultured into 12 24-well plates, and 1 ml of 15% by weight was added to each well. HAT medium prepared with Ishikov medium containing FCS was added. The collected culture supernatant was measured for its binding ability to CRP by ELISA.
[0194]
Thereafter, the supernatant was evaluated every few days to select wells, scale up, and continue culturing in HT medium.
Three days after the fused cells were subcultured into a 6-well plate, cell culture supernatant was collected at 200 μl / well for each well, and the binding ability to CRP was measured by the following ELISA method.
[0195]
A cell culture supernatant was used as a solid phase at 0.1 mg / mL · BSA ··· Antibody solution. A peroxidase-labeled anti-mouse IgG antibody was used as the second antibody. If an antibody that binds to the inhibitor is present in the cell culture supernatant, the inhibition occurs, and no color development in the wells is confirmed.
[0196]
Wells that were inhibited only by CRP and were not affected by complement C1q and serum amyloid P were selected from the wells derived from mouse # 3-2 for 2 wells, and the wells derived from mouse # 3-3 were selected. One well was selected.
[0197]
The cells in the above three wells are diluted (limit dilution) to a concentration containing two cells per well using HT medium containing 15% by weight of FCS, and dispensed into three 96-well microplates. did. Thymocytes of 4-week-old female mice (BALB / C) were used as feeders to promote initial proliferation. The culture was advanced while increasing the size of the plate, and the above-described ELISA screening was repeated for the cell culture supernatant at appropriate times.
[0198]
Cell lines that show high titers against CRP, are not inhibited by complement C1q, serum amyloid P, and show good growth are finally selected and 5% in 200 mL of medium. × 10 ⁵ Culture was advanced to a concentration of cells / mL.
Finally, two strains that show binding ability to CRP and do not cause a cross-reaction with complement C1q and serum amyloid P were derived from mouse strain # 3-2 and strains derived from mouse strain # 3-3 Three shares were selected, one for each share.
[0199]
One of the strains derived from mouse number # 3-2, which shows binding ability to CRP and does not cause a cross-reaction with complement C1q and serum amyloid P, was designated as a cell strain name: MH-IIC2. It was internationally deposited on March 24, 2003 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Accession No. FERM BP-8338). Another strain was designated as cell strain name: MH-IIC4, and was deposited at the Depositary Center on March 24, 2003 (Accession No. FERM BP-8339).
[0200]
One of the strains derived from mouse number # 3-3, which shows binding ability to CRP and does not cause a cross-reaction with complement C1q and serum amyloid P, was named cell line name: MH-IIC5, It was internationally deposited on March 24, 2003 at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (Accession No. FERM BP-8340).
Antibodies produced by the MH-IIC2, MH-IIC4 and MH-IIC5 strains are referred to as MH-IIC2, MH-IIC4 and MH-IIC5 antibodies, respectively.
[0201]
(Preservation of cells)
The finally selected cell line is centrifuged to remove the culture supernatant, and about 1 × 10 ⁷ The cells were suspended in 1 mL of a solution of FCS: dimethylsulfoxide = 9: 1 (volume ratio) at a concentration of cells / mL, preliminarily frozen at -80 ° C, and then transferred to liquid nitrogen for long-term storage.
[0202]
(Purification of antibody)
Each of the three selected strains was cultured in large amounts in an Ishikov medium containing 15% by weight of FCS, and the supernatant was collected by centrifugation. The culture supernatant of each strain was subjected to affinity chromatography using a recombinant protein A binding gel (recombinant protein A Sepharose 4FF, manufactured by Pharmacia) under the following conditions to obtain each monoclonal antibody (MH-IIC2 antibody, MH-IIC4 antibody, MH-IIC). -IIC5 antibody) was purified.
[0203]
The column packed with protein A binding gel was equilibrated with binding buffer (1.5 M glycine / 3 M NaCl, pH 8.9). The culture supernatant was diluted about 3 times with a binding buffer, and then applied to an equilibrated column. The column was washed with binding buffer while monitoring the eluate from the column at 280 nm until the elution of the impurities was complete. After washing, an elution buffer (100 mM citric acid, pH 4) was applied to the column (linear flow rate: about 20 cm / h), and an IgG-containing eluate was collected. The concentration of the antibody was determined by measuring the absorbance of the collected IgG-containing eluate at 280 nm with an absorptiometer and converting the measured absorbance by an extinction coefficient.
[0204]
From the comparison with the standard protein by SDS polyacrylamide gel electrophoresis, the purified fractions of these monoclonal antibodies (MH-IIC2 antibody, MH-IIC4 antibody, MH-IIC5 antibody) were all H chains having a molecular weight of about 50,000. And about 25,000 L chains. In addition, contamination on the electrophoresis was below the detection limit.
[0205]
(Evaluation of antibody)
The three types of monoclonal antibodies purified by the affinity chromatography were evaluated using the dilution series of CRP, serum amyloid P, and complement C1q under the same conditions as the inhibition ELISA method in the above selection.
[0206]
FIGS. 8A to 8C show CRPs for the MH-IIC2 antibody, FIGS. 9A to 9C show the same for the MH-IIC4 antibody, and FIGS. 10A to 10C show the CRPs for the MH-IIC5 antibody (FIG. 8A, FIG. 9A and FIG. 10A) A graph showing the results of measuring the binding ability to complement C1q (FIGS. 8B, 9B and 10B) and serum amyloid P (FIGS. 8C, 9C and 10C) by a competitive reaction (inhibition reaction). It is. In each figure, the vertical axis represents absorbance (wavelength: 492 nm), and the horizontal axis represents CRP concentration (mol / L, hereinafter referred to as M), complement C1q concentration (mg / ml), and serum amyloid P concentration (mg / ml). dl, hereinafter referred to as M).
[0207]
As shown in FIGS. 8A to 8C, in the MH-IIC2 antibody, the half value of the inhibitory activity against CRP is about 1.5 × 10 · M, indicating that CRP of 2-3 × 10 · M can be detected. Was done. No reaction was observed with complement C1q and serum amyloid P, confirming that no cross-reaction occurred.
[0208]
As shown in FIGS. 9A to 9C, in the MH-IIC4 antibody, the half value of the inhibition to CRP is about 2.5 × 10 · M, indicating that the possibility of detecting CRP of 2 to 3 × 10 · M can be detected. Was done. No reaction was observed with complement C1q and serum amyloid P, confirming that no cross-reaction occurred.
[0209]
As shown in FIGS. 10A to 10C, in the MH-IIC5 antibody, the half value of the inhibition to CRP was about 2 × 10 · M, indicating the possibility of detecting 2-3 × 10 · M CRP. . No reaction was observed with complement C1q and serum amyloid P, confirming that no cross-reaction occurred.
[0210]
【The invention's effect】
According to the present invention, it is possible to provide an anti-CRP monoclonal antibody and a CRP detection kit capable of accurately and easily detecting and measuring CRP.
[Brief description of the drawings]
FIG. 2A
1 is a graph showing the results of an inhibition ELISA showing the ability of the anti-CRP monoclonal antibody (MH-IIC1 antibody) of the present invention to bind to CRP.
FIG. 2B
1 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-IIC1 antibody) of the present invention to complement C1q.
FIG. 2C
1 is a graph showing the results of an inhibition ELISA showing the ability of the anti-CRP monoclonal antibody (MH-IIC1 antibody) of the present invention to bind to serum amyloid P.
FIG. 3A
Fig. 4 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-IIC3 antibody) of the present invention to CRP.
FIG. 3B
1 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-IIC3 antibody) of the present invention to complement C1q.
FIG. 3C
1 is a graph showing the results of an inhibition ELISA showing the ability of the anti-CRP monoclonal antibody (MH-IIC3 antibody) of the present invention to bind to serum amyloid P.
FIG. 4A
4 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-C3 antibody) of the present invention to CRP.
FIG. 4B
1 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-C3 antibody) of the present invention to complement C1q.
FIG. 4C
1 is a graph showing the results of an inhibition ELISA showing the ability of the anti-CRP monoclonal antibody (MH-C3 antibody) of the present invention to bind to serum amyloid P.
FIG. 5A
4 is a graph showing the results of an inhibition ELISA showing the ability of the anti-CRP monoclonal antibody (MH-C4 antibody) of the present invention to bind to CRP.
FIG. 5B
Fig. 4 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-C4 antibody) of the present invention to complement C1q.
FIG. 5C
It is a graph which shows the result of the inhibition ELISA which shows the binding ability with respect to the serum amyloid P of the anti-CRP monoclonal antibody (MH-C4 antibody) of this invention.
FIG. 6A
1 is a graph showing the results of an inhibition ELISA showing the binding ability of an anti-CRP monoclonal antibody (MH-C5 antibody) of the present invention to CRP.
FIG. 6B
Fig. 4 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-C5 antibody) of the present invention to complement C1q.
FIG. 6C
1 is a graph showing the results of an inhibition ELISA showing the ability of the anti-CRP monoclonal antibody (MH-C5 antibody) of the present invention to bind to serum amyloid P.
FIG. 7A
It is a graph which shows the result of the inhibition ELISA method which shows the binding ability with respect to CRP of the anti-CRP monoclonal antibody (MH-C6 antibody) of this invention.
FIG. 7B
Fig. 4 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-C6 antibody) of the present invention to complement C1q.
FIG. 7C
Fig. 4 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-C6 antibody) of the present invention to serum amyloid P.
FIG. 8A
It is a graph which shows the result of the inhibition ELISA method which shows the binding ability with respect to CRP of the anti-CRP monoclonal antibody (MH-IIC2 antibody) of this invention.
FIG. 8B
It is a graph which shows the result of the inhibition ELISA method which shows the binding ability with respect to complement C1q of the anti-CRP monoclonal antibody (MH-IIC2 antibody) of this invention.
FIG. 8C
1 is a graph showing the results of an inhibition ELISA showing the ability of the anti-CRP monoclonal antibody (MH-IIC2 antibody) of the present invention to bind to serum amyloid P.
FIG. 9A
It is a graph which shows the result of the inhibition ELISA method which shows the binding ability with respect to CRP of the anti-CRP monoclonal antibody (MH-IIC4 antibody) of this invention.
FIG. 9B
Fig. 4 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-IIC4 antibody) of the present invention to complement C1q.
FIG. 9C
It is a graph which shows the result of the inhibition ELISA method which shows the binding ability with respect to the serum amyloid P of the anti-CRP monoclonal antibody (MH-IIC4 antibody) of this invention.
FIG. 10A
It is a graph which shows the result of the inhibition ELISA which shows the binding ability with respect to CRP of the anti-CRP monoclonal antibody (MH-IIC5 antibody) of this invention.
FIG. 10B
Fig. 4 is a graph showing the results of an inhibition ELISA showing the binding ability of the anti-CRP monoclonal antibody (MH-IIC5 antibody) of the present invention to complement C1q.
FIG. 10C
1 is a graph showing the results of an inhibition ELISA showing the ability of the anti-CRP monoclonal antibody (MH-IIC5 antibody) of the present invention to bind to serum amyloid P.

Claims (15)

(A) obtaining a cell line producing a monoclonal antibody by cell fusion of mammalian spleen cells immunized with the C-reactive protein and cells derived from mammalian myeloma;
Monoclonal antibodies specifically binding to C-reactive protein were assayed by immunoassay of monoclonal antibodies produced from the above cell lines for their ability to bind to C-reactive protein, complement C1q, and serum amyloid P. (B) selecting a cell line to be produced;
A method for producing an anti-C-reactive protein monoclonal antibody, comprising: A method for producing an anti-C reactive protein monoclonal antibody according to claim 1,
In the above step (b), an anti-C-reactive protein monoclonal antibody that selects a cell line that produces a monoclonal antibody that has a binding ability to C-reactive protein and does not bind to complement C1q and serum amyloid P How to make. A cell line that produces a monoclonal antibody that has the ability to bind to C-reactive protein and does not bind to complement C1q and serum amyloid P. The cell line according to claim 3, wherein
A cell strain which is Accession No. FERM BP-7941 of the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology. The cell line according to claim 3, wherein
A cell strain which is Accession No. FERM BP-7942, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. The cell line according to claim 3, wherein
A cell line under the accession number FERM BP-7939 of the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary. The cell line according to claim 3, wherein
A cell line under the accession number FERM BP-7940 of the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. The cell line according to claim 3, wherein
A cell strain which is Accession No. FERM BP-8185 of the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. The cell line according to claim 3, wherein
A cell strain which is Accession Number FERM BP-8186 of the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. The cell line according to claim 3, wherein
National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM BP The cell line according to claim 3, wherein
National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM BP The cell line according to claim 3, wherein
National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Accession No. FERM BP An anti-C-reactive protein monoclonal antibody that has a binding ability to C-reactive protein and does not bind to complement C1q and serum amyloid P. A C-reactive protein detection kit for detecting a C-reactive protein in a sample solution,
A first antibody and a second antibody,
The first antibody is an anti-C-reactive protein antibody;
The second antibody is a labeled anti-C-reactive protein antibody,
A C-reactive protein detection kit, wherein at least one of the first antibody and the second antibody is an anti-C-reactive protein monoclonal antibody that does not bind to complement C1q and serum amyloid P. A C-reactive protein detection kit according to claim 14,
The first antibody is immobilized on a solid phase,
A kit for detecting a C-reactive protein, wherein the second antibody is an anti-C-reactive protein monoclonal antibody that does not bind to complement C1q and serum amyloid P, and is contained in a mobile phase.

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