JPH0534344A - Immunological assay method for C-reactive protein and reagent for assay - Google Patents

Abstract

(57) [Summary] [Objective] To provide an immunological assay method and assay reagent for human C-reactive protein. [Structure] As an anti-human C-reactive protein antibody, an antibody derived from hen's egg is used instead of mammalian antiserum. [Effect] It is possible to suppress the occurrence of non-specific reaction, and it is possible to perform more accurate and accurate CRP measurement than ever before. Also, it is not necessary to kill the immunized animal to obtain the antibody.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological assay method for human C-reactive protein and a reagent for assaying the antibody derived from chicken egg as an anti-human C-reactive protein antibody.

[0002]

2. Description of the Related Art C-reactive protein (C-reacti)
ve protein: hereinafter abbreviated as CRP may be used), which is one of the plasma protein components, and its blood concentration increases in the case of inflammatory disease or tissue disintegrating disease. Therefore, the CRP test is useful for determining the presence or absence of an inflammatory disease or tissue-disintegrating disease, the severity of the disease, the observation of the progress, and the prognosis.

As a CRP measuring method, a capillary method as a qualitative method and a single immunodiffusion method (S) as a quantitative method have been conventionally used.
The RID method) was mainly used, but all of these require a long time before the determination. Therefore, recently, a CRP measurement method using an automatic analyzer using a latex method or an immunoturbidimetric method, which is highly rapid and quantitative, has been widely used. Human CR in these immunological CRP measurements
It is necessary to use an anti-CRP antibody that specifically reacts with P, and as the anti-CRP antibody, generally, antiserum of animals such as rabbits and goats has been widely used.

[0004]

However, since the sera of animals such as rabbits and goats have an immunological commonality with human sera, human CRP in the sample to be tested and rabbit antiserum or goat antiserum as a reagent. A reaction other than the antigen-antibody reaction with the antibody contained therein, that is, a nonspecific reaction may occur.
In addition, it is necessary to kill the immunized animal to obtain antiserum. Therefore, it has been desired to find an anti-CRP antibody other than rabbit and goat antisera that can be used for immunological CRP measurement.

The present inventor has conducted diligent research to obtain a more preferable anti-CRP antibody.
It was found that the above-mentioned drawbacks can be overcome by using P antibody. The present invention is based on these findings.

[0006]

Therefore, the present invention relates to an immunological assay method for human C-reactive protein, which comprises using an antibody derived from chicken egg which specifically reacts with human C-reactive protein.

Furthermore, the present invention also relates to a reagent for immunologically measuring human C-reactive protein, which comprises an antibody derived from chicken egg which specifically reacts with human C-reactive protein.

In the present invention, in the immunological measurement of CRP, animal antiserum was used as an anti-CRP antibody in the conventional method, but instead of using an antibody derived from chicken eggs as the anti-CRP antibody, Unlike the method, the conventional CRP measurement technique can be applied as it is in other points.

The anti-CRP antibody used in the present invention is human CR.
It can be obtained by separating from the yolk of the egg laid by the chicken immunized with P and purifying it. Chicken immunization uses purified human CRP,
It can be carried out subcutaneously, intradermally or intravenously, optionally together with a suitable adjuvant. The specific antibody titer of the yolk of the egg laid by the immunized chicken is measured by, for example, the ELISA method, and the yolk having a high antibody titer is selected.

The anti-CRP antibody is contained in the water-soluble protein component of egg yolk. Therefore, the anti-CRP antibody can be obtained by using any known separation method for obtaining a water-soluble protein component from egg yolk. For example, a method of extracting egg yolk with chloroform to obtain the remaining solution (TK
ramer et al., Immunology, 19, 157,
1970, etc.); A method of adding polyethylene glycol to egg yolk liquid to precipitate egg yolk lipoprotein and obtaining a water-soluble protein from the supernatant; A method for obtaining water-soluble protein from the above (Journal of the Japanese Society of Agricultural Chemistry, Vol. 62, No. 3); Kaihei 2-188
No. 533). Particularly preferred is a method in which a λ-carrageenan aqueous solution is added to precipitate lipoproteins, and water-soluble proteins are obtained from the supernatant.

Next, the supernatant is subjected to column treatment (for example, D
EAE-Sephacel column) to remove impurities,
An anti-human CRP antibody can be obtained by performing a salting-out operation and dialyzing the salted-out product. If necessary, it may be further treated with a normal human serum-Sepharose column. This anti-human CRP antibody specifically reacts with human CRP, and almost no non-specific reaction occurs. The thus obtained IgG type anti-human CRP antibody (hereinafter referred to as anti-CRP-IgY
May be referred to as), and may be stored in powder form by freeze-drying.

The present invention can be used to measure, ie, detect and / or quantify CRP in serum. The test sample used in the present invention is one that may contain human CRP, such as blood, serum, spinal fluid, or synovial fluid.

According to the present invention, the CRP (antigen) in the test sample causes a specific antigen-antibody reaction with anti-CRP-IgY, resulting in turbidity in proportion to the amount of CRP in the test sample. By optically measuring this turbidity, C in the test sample
The amount of RP can be calculated. Therefore, the present invention can be directly applied to the known immunoturbidimetric method, latex agglutination method, or the like. As a measuring method, visual measurement on a slide plate, an optical method (laser nephelometry,
Photometry with near infrared light or UV-visible spectrophotometry).

The measuring reagent of the present invention contains the above-mentioned anti-CRP-IgY derived from avian egg, in place of the antiserum of goat, rabbit, etc. in the conventional CRP measuring reagent. There is no difference from the configuration of the conventional CRP measurement reagent. Therefore, the measuring reagent of the present invention may contain a commonly used sensitizer, buffer and / or preservative in addition to the anti-CRP-IgY. As the sensitizer, polyethylene glycol or a surfactant (for example, a nonionic surfactant such as TWE) can be used.
EN-20 etc.) can be mentioned. Examples of the buffer solution include phosphate buffer solution, Tris buffer solution, glycine buffer solution and the like. Examples of antiseptics include sodium azide.

The measuring reagent of the present invention varies depending on the measuring principle, but the anti-CRP-IgY is 0.0 as a Becker value.
5 to 5.0 mg / ml, preferably 0.1 to 1.0 mg / ml,
Depending on the case, 0.01 to 10%, preferably 0.
01-5%, and buffer 1-500 mM, preferably 5-
It is composed of 50 mM and is preferably used at a pH value of 7-10, more preferably 7-9.

In the present invention, anti-CRP-IgY can be used by adsorbing it on a commonly used carrier. The type and particle size of the carrier are not particularly limited, but for example,
Synthetic polymer beads such as polystyrene and acrylic having a particle size of 0.05 to 1.0 μm can be used.

Although the measurement according to the present invention can be carried out manually individually, it is preferable to use an automatic analyzer as in the conventional method. It can be used for various devices that are currently in wide use. Since the turbidity is produced in proportion to the amount of CRP present in the test sample when the test sample and the antiserum are mixed, the turbidity is generally measured spectrophotometrically. The measurement wavelength can be in the range of 340 nm to 900 nm.

[0018]

In the conventional CRP measurement, as an anti-CRP antibody,
An artificial antiserum of a mammal such as a mouse, a rabbit, a goat, or a horse that had been hyperimmunized was used. These mammals are developmentally closely related to humans and have various immunological commonalities. Serum proteins share common antigens with each other, and have commonality with the complement system, coagulation system, rheumatoid factor, and the like. In addition, these mammals and humans have many similarities in infectious microorganisms and normal flora. Therefore, these mammals have an antibody having the same specificity as a human antibody as a normal antibody in addition to the immune antigen (CRP), and therefore, the target antigen (CR
It is easily expected that an antigen-antibody reaction will occur due to the involvement of an antigen other than P), and a non-specific reaction will appear from the viewpoint of the target antigen (CRP).

On the other hand, birds are located phylogenetically between fishes and mammals and are more distant from humans than mammals, but their immune defense mechanism is sufficiently advanced. Therefore, by using an antibody derived from a bird that is taxonomically distant from human, non-specific reaction is reduced, and only the antigen-antibody reaction of interest appears, and more accurate diagnosis can be performed. In birds, the antibody can be isolated and purified from the egg, and the antibody can be obtained without killing.

[0020]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention.

Example 1: Preparation of anti-human CRP antibody (1) Human CRP (manufactured by Canadian Bioclinical) used as an immunogen was dissolved in physiological saline and mixed with an equal amount of Freund's complete adjuvant to emulsify. The antigen emulsion was intramuscularly injected (1 mg CRP / wing) to a total of 4 places under the left and right wings and on the left and right thigh muscles of a laying hen (Diacross B34). Two weeks and five weeks after the initial immunization, booster immunization was carried out by the same method. C in the yolk of the egg laid by the immunized chicken after the first immunization
The specific antibody titer against RP was measured by the ELISA method (CRP coated plate). Eggs with increased specific antibody titers to CRP (6-10 after initial immunization)
The egg yolk was separated from (week) and used in the following steps.

(2) Preparation of anti-CRP-IgY The egg yolk obtained in (1) above was homogenized by adding an equal amount of deionized water to prepare a 2-fold diluted yolk solution. To this 2-fold diluted egg yolk, double the amount of λ-carrageenan aqueous solution (1.5 mg / m 2
l) was added, and the obtained egg yolk lipoprotein aggregate was subjected to a centrifugation treatment (10,000 × g; 10 minutes). The supernatant is suction-filtered with filter paper, and disodium phosphate 20 mM is added to the filtrate.
To dissolve, then add 3N hydrochloric acid to add p
It was adjusted to H8.0. The obtained solution was added to a DEAE-Sephacel column which had been equilibrated with 20 mM phosphate buffer (pH 8.0) in advance, and 20 mM phosphate buffer (pH
The non-adsorbed material was thoroughly washed with 8.0). Then 200
The adsorbed protein was eluted from the column with mM phosphate buffer (pH 8.0). Sodium sulfate was added to the obtained eluate so as to be 15% (w / v) and dissolved,
The obtained salted-out product was subjected to a centrifugal separation treatment (10,000 × g; 1
0 minutes). The precipitated salted-out product was dissolved in 10 mM phosphate buffer (pH 8.0), and the same salting-out operation was repeated twice. The final salted out product was 10 mM phosphate buffer (pH
After dissolution in 8.0), it was dialyzed against the same buffer.
The liquid was freeze-dried to obtain anti-CRP-IgY powder.

Example 2: Preparation of calibration curve (1) Preparation of reagent To a 10 mM phosphate buffer solution (pH 7.0), 3% polyethylene glycol 6000 and 0.5% Tween 20 were added to prepare a first reagent. Prepared. In addition, the anti-CRP-obtained in the above Example 1 (2) was added to 10 mM phosphate buffer (pH 7.0).
A second reagent was prepared using IgY powder so that the activity value (Becker titer) was 0.5 mg / ml.

(2) Dilution series CRP stock solution prepared by adding purified CRP (Canadian Bioclinical) at a concentration of 2.0 mg / dl to 10 mM phosphate buffer (pH 7.0) was diluted in 10 steps (dilution). Rate: 1/10, 2/10, 3/10, 4/10, 5/10, 6/10, 7/10, 8/1
0, 9/10, 10/10) Series A and C prepared by adding purified CRP to the same buffer as above at a concentration of 20 mg / dl.
Dilute the RP stock solution in 10 steps in the same manner (dilution ratio: 1/10, 2/1
0, 3/10, 4/10, 5/10, 6/10, 7/10, 8/10, 9/10, 10/1
The calibration curve was prepared using each of the reagents described in (1) above, using each of the 0) series B as a sample.

(3) Measurement As a measuring instrument, an automatic analyzer (Cobas Fara;
2 point end measurement was adopted as a reaction mode. Incubation was carried out at 37 ° C. for 15 minutes using 10 μl of dilution series A or dilution series B of the CRP stock solution, 175 μl of the first reagent and 25 μl of the second reagent. Turbidity was measured at 340 nm. Dilution series A
The results for (CRP stock solution concentration = 2.0 mg / dl) are shown in FIG. 1, and the results for dilution series B (CRP stock solution concentration = 20 mg / dl) are shown in FIG. Example 3: Correlation with LPIA method In order to examine whether or not the method of the present invention specifically measures CRP in a test sample, an automatic analyzer (LPIA system L) was used for 60 cases of CRP-positive human serum. -100;
A correlation experiment between the CRP measurement method (latex agglutination method and near infrared photometry) by Mitsubishi Kasei Co., Ltd. and the method of the present invention was conducted.
The results are shown in Fig. 3. In FIG. 3, the X axis (horizontal axis) is the latex method (LPIA method), and the Y axis (vertical axis) is the immunoturbidimetric method of the present invention. The correlation coefficient (r) between the two is 0.99
It was 43.

As is apparent from FIG. 3, the measuring method according to the present invention showed a high correlation with the LPIA method. Therefore, the measuring method of the present invention can specifically measure CRP in a clinical test sample.

[0027]

According to the present invention, as an anti-CRP antibody,
Since egg-derived antibodies are used instead of mammalian antisera,
It is possible to suppress the occurrence of non-specific reaction, and it is possible to perform more accurate and accurate CRP measurement than ever before. Also, it is not necessary to kill the immunized animal to obtain the antibody.

[Brief description of drawings]

FIG. 1 is a calibration curve based on a dilution series containing CRP at a low concentration.

FIG. 2 is a calibration curve based on a dilution series containing CRP at a high concentration.

FIG. 3 is a graph showing the correlation between the present invention and the conventional LPIA method.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Keigo Kabazawa             1-11-4 Higashi-Kanda, Chiyoda-ku, Tokyo Stock             In ceremony company Yatron (72) Inventor Hiroshi Kita             1-11-4 Higashi-Kanda, Chiyoda-ku, Tokyo Stock             In ceremony company Yatron (72) Inventor Hajime Hatta             1-3 Takaracho, Yokkaichi-shi, Mie Taiyo Chemical Co., Ltd.             Shikisha Research Institute (72) Inventor Makoto Ozeki             1-3 Takaracho, Yokkaichi-shi, Mie Taiyo Chemical Co., Ltd.             Shikisha Research Institute

Claims (2)

[Claims] 1. A method for immunologically measuring human C-reactive protein, which comprises using an antibody derived from chicken egg which specifically reacts with human C-reactive protein. 2. A reagent for immunological measurement of human C-reactive protein, which comprises an antibody derived from chicken egg which specifically reacts with human C-reactive protein.