JP2005061910A - Immunoassay instrument and method for measuring HIV antigen antibody - Google Patents

Abstract

【Task】
Reagents and instruments that detect HIV infection as early as possible without the need for specific diagnostic equipment or measuring equipment and can obtain measurement results in a short period of time. An apparatus for measuring is provided.
[Solution]
An HIV antigen detection part in which at least one kind of anti-HIV antibody is immobilized on a band-like matrix that can be infused by capillary action, and at least one kind of synthetic HIV peptide antigen and / or gene recombinant antigen carried on a polyamino acid carrier The measurement is performed by an immunoassay device for HIV antigen antibody measurement having a detection zone comprising an HIV antibody detection section in which is immobilized.
[Selection] Figure 1

Description

  The present invention comprises an HIV antigen detection part in which at least one anti-HIV antibody is immobilized on a band-like matrix that can be infused by capillary action, at least one synthetic HIV peptide antigen carried on a polyamino acid carrier, and / or The present invention relates to an immunoassay device for HIV antigen antibody measurement having a detection zone having an HIV antibody detection unit immobilized with a recombinant antigen, and an HIV antigen antibody immunoassay method using the immunoassay device.

  Conventionally, as a method for confirming the presence or absence of HIV (human immunodeficiency virus) infection, the timing of HIV infection, and the confirmation of therapeutic effects, immunoassay of anti-HIV antibodies and HIV antigens in specimens has been performed. With the recent increase in patients with acquired immune deficiency syndrome (AIDS) caused by HIV infection in recent years, HIV antigen and antibody are simultaneously measured as a reagent that can detect an infected person without omission even in an early infected person. Four generations of HIV diagnostic reagents have been developed. This reagent uses a reagent in which an HIV antigen or an anti-HIV antibody is bound to a solid phase, or a reagent in which a solid phase to which an HIV antigen is bound and a solid phase to which an anti-HIV antibody is bound, and an antibody in a specimen And the antigen can be measured simultaneously (see Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, etc.). In addition, these reagents have been widely used in recent years because they can measure antibodies and antigens at a time, and can shorten the blank period that cannot be detected by a test immediately after infection. Although these antibody and antigen simultaneous measurement reagents can detect at least one of antigen or antibody by EIA, ELISA, etc., in order to measure antigen and antibody separately, the conventional antigen measurement reagent and It was necessary to prepare a reagent for antibody measurement and perform at least two measurements.

    Furthermore, for the measurement of anti-HIV antibodies, anti-HIV antibodies in a specimen are detected using HIV-1 or 2 culture antigens, recombinant antigens, synthetic antigens, and the like. With the improvement of protein synthesis technology, anti-HIV antibody measurement reagents using an HIV synthetic antigen synthesized from an amino acid sequence of an antigenic determinant site of an HIV constituent protein have been developed. Since this reagent does not contain a structure other than an antigenic determinant that causes a non-specific reaction, it has characteristics such that a specific measurement can be performed. However, when the synthetic antigen is bound to the solid phase, there is a problem that the reactivity with the antibody in the specimen is not sufficient, a trace amount of antibody cannot be detected, and the required sensitivity cannot be obtained.

  On the other hand, an immunoassay instrument (hereinafter referred to as “immunochromatography instrument”) using a transmissible belt-like matrix has been developed, and only a sample is spotted at a predetermined location without requiring a special detection device or skilled measurement technique. An antigen or antibody in a sample can be easily measured in a short time. The immunochromatography instrument includes an immunochromatography instrument that binds a plurality of syphilis treponema antigens (TP antigens) to a detection zone, for example, and can detect a plurality of anti-TP antibodies in a specimen in separate detection zones depending on the substance to be measured. It has been developed (see Patent Document 5). The detection zone of this instrument has a plurality of zones to which different TP antigens are bound, and different anti-TP antibodies are separately detected to identify the time of infection and are used for selection of therapeutic agents. This reagent is not intended to simultaneously measure the antigen and antibody in the specimen.

JP-A-2-503950 JP-A-6-265548 JP 2001-504572 A JP 2001-514749 A JP-A-9-229938

  Thus, in order to prevent the spread of HIV infection, there has been a demand for measuring reagents and measuring instruments that can detect infected persons as early as possible. Moreover, it is a reagent or instrument that can obtain measurement results in a short time without the need for specific diagnostic equipment or measuring equipment. It checks the infection period, selects a therapeutic agent, and monitors the progress of treatment. There has been a demand for a method for easily measuring an antigen and an antibody by a single operation. Furthermore, in the reagent used for the diagnosis of AIDS, it has been widely demanded to improve the measurement sensitivity and reduce the ratio of false positives due to non-specific reactions. An object of the present invention is to provide an instrument for measuring HIV infection that satisfies all these requirements.

  Accordingly, as a result of intensive studies, the present inventors have provided a band-like matrix that can be infused by capillary action, and an HIV antigen detection unit in which at least one type of anti-HIV antibody is immobilized, and a polyamino acid carrier. A detection zone having an HIV antibody detection unit immobilized with at least one synthetic HIV peptide antigen and / or genetically modified antigen, an enzyme labeling reagent zone, a specimen spotting zone, and one longitudinal length of the matrix An immunoassay for HIV antigen-antibody measurement having a developing solution supply zone provided with a developing solution pad having a substrate reagent zone in the vicinity of the end in the direction and a developing solution absorption zone provided at the other end in the longitudinal direction of the matrix The instrument was discovered and the present invention was completed.

    Further, the present invention is to detect a signal in the detection zone after spotting a specimen in the specimen spotting zone of the immunoassay instrument, supplying the developing liquid to the developing liquid supply zone, and developing the developing liquid on the matrix. A method for simultaneous immunoassay of an HIV antigen and an antibody.

    Hereinafter, the immunoassay device of the present invention will be described in more detail. In the immunoassay device of the present invention, the belt-like matrix is made of an absorbent material that can infuse a liquid by capillary action. Examples of the absorptive material include filter papers, membranes, and porous materials manufactured by mixing cellulose or a derivative thereof such as cellulose and nitrocellulose, glass fibers, or the like alone or in combination. Although there is no restriction | limiting in the magnitude | size of this matrix, A strip-shaped thing about 3 mm-10 mm in width and about 30 mm-100 mm in length is easy to handle and preferable. Furthermore, it is preferable to use a matrix having a thickness of 100 μm to 1 mm. The matrix is partially or wholly blocked with animal serum such as bovine serum albumin (BSA), casein, sucrose, etc. to prevent adsorption due to non-specific reaction of the protein derived from the sample to the matrix during measurement. Can be used.

(Detection zone)
In the detection zone, an HIV antigen detection part in which at least one anti-HIV antibody is immobilized on the matrix, and at least one synthetic HIV peptide antigen and / or gene recombinant antigen carried on a polyamino acid carrier are immobilized. It is possible to provide at least two detection sections with the HIV antibody detection section. It is easy to determine that this detection unit is on a matrix and is provided in a line shape in a direction perpendicular to the infusion direction (longitudinal direction of the matrix) of the liquid for developing the matrix, and at least two detection units are provided in the vicinity. Is preferable. The two detection units may be either upstream or downstream in the liquid infusion direction. By immobilizing an anti-HIV antibody in the HIV antigen detection part and at least one synthetic HIV peptide antigen and / or genetically modified HIV antigen carried on a polyamino acid carrier in the HIV antibody detection part Can be created.

The anti-HIV antibody immobilized on the HIV antigen detection part can be used alone or in combination with antibodies against HIV-1 or 2, and is selected from antibodies produced by known methods and various commercially available antibodies. Can be used. As the anti-HIV antibody, a monoclonal antibody or a polyclonal antibody can be used. For example, IgG antibodies against gag, env, pol, etc. of the genetic structure constituting HIV, IgM antibodies, and Fab, F which are fragments of these antibodies (Ab ′) ₂ or the like may be used. Examples of the antibody against gag of HIV-1 include p-24 antibody, p-17 and the like, examples of the antibody against env include gp120gp41, and examples of the antibody against pol include p66, p51, p32, p10 and the like. Can be selected and used. Examples of HIV-2 antibodies against gag include p26 and p16, env antibodies include gp105 and gp36, and pol antibodies include p61, p34 and p10. be able to.

  The anti-HIV antibody detection unit is constituted by immobilizing at least one synthetic HIV peptide antigen and / or gene recombinant antigen carried on a polyamino acid carrier in a matrix. As the synthetic HIV peptide antigen, various antigens obtained by chemically synthesizing peptides constituting HIV-1 or HIV-2 can be used. For example, 11-17mer amino acids having an amino acid sequence derived from HIV-2 env peptide gp36 An HIV-2 gp36 synthetic peptide antigen consisting of residues can be used. A synthetic HIV peptide antigen can be easily produced by a synthesizer using a known solid phase method, liquid phase method, or the like. HIV recombinant antigens can also be produced according to well-known methods. Moreover, the commercially available antigen can also be used for a synthetic HIV peptide antigen and a gene recombinant antigen.

    The synthetic HIV peptide antigen and / or genetically modified HIV antigen is preferably bound to a polyamino acid carrier to constitute an anti-HIV antibody detection unit. This polyamino acid carrier includes a repeating structure of a single or plural amino acid residues having a group selected from an amino group, a carboxyl group, a sulfhydryl group or a hydroxyl group in the side chain, and all or part of the reactive group Have a structure that binds to a diol structure or an aminoalcohol structure through or without a crosslinked structure. The polyamino acid carrier allows an aldehyde group to be generated from the diol structure or aminoalcohol structure by oxidation treatment, and is reacted with the amino group of the synthetic HIV peptide antigen or the recombinant antigen, thereby allowing the polyamino acid carrier to carry the antigen. Can do. Polyamino acids include, for example, neutral amino acids such as glycine, alanine, valine, norvaline, leucine, isoleucine, norleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, feralanine, tyrosine, tryptophan, aspartic acid, glutamic acid, etc. And basic amino acids such as lysine, arginine and histidine. The amino acid constituting the polyamino acid carrier preferably has a reactive group such as an amino group, a carboxyl group, a sulfhydryl group or a hydroxyl group in its side chain. More preferably, the amino acid used in the polyamino acid carrier of the present invention is lysine or glutamic acid. In addition, the amino acid used in the polyamino acid carrier may be an amino acid derivative in which the reactive group of the side chain is protected with an appropriate protecting group. The polyamino acid used for the polyamino acid carrier typically consists of a repeating structure of a single amino acid residue or a repeating structure of a plurality of amino acid residues selected from the amino acid residues or amino acid derivatives. Examples of the polyamino acid production method include, but are not limited to, general synthesis methods, for example, emulsion polymerization or heterogeneous polymerization of the amino acid N-carbonate anhydride (Japanese Patent Laid-Open No. 9-151250). In the present invention, for example, an amino acid homopolymer or a random copolymer commercially available from Sigma can be used as the polyamino acid. Examples of polyamino acids include commercially available polyamino acids having a repeating structure of a plurality of amino acid residues such as homopolymers such as polylysine and polyglutamic acid, and lysine-glutamic acid and lysine-lysine-glutamic acid (for example, poly-L-lysine). It is also possible to use a molecular weight of 30,000-70,000 or a molecular weight of 150,000-300,000 (Sigma)).

    In the polyamino acid carrier of the present invention, it is preferable that the reactive group in the side chain of the constituent amino acid and the crosslinked structure, and / or the crosslinked structure and the diol structure or amino alcohol structure are bonded by an amide bond. Alternatively, the reactive group and the diol structure or aminoalcohol structure may be directly bonded via an amide bond without using a crosslinked structure.

  In the present specification, the “diol structure or amino alcohol structure” includes a compound having a chemical structure capable of generating an aldehyde group by oxidation. Here, the “diol structure” refers to a structure in which two hydroxyl groups are bonded to two different carbon atoms. The “aminoalcohol structure” is a structure having an amino group and an alcoholic hydroxyl group in the same molecule, having an amino group on one of adjacent two carbon atoms and an alcoholic hydroxyl group on the other. The structure which has is preferable. The polyamino acid carrier for peptide bonding of the present invention can generate an aldehyde group by oxidation treatment of “diol structure or aminoalcohol structure”, and bond the peptide to the aldehyde group moiety.

  The compound having a “diol structure or aminoalcohol structure” used in the present invention further has a group that can be directly bonded to a crosslinked structure or a reactive group in the side chain of the amino acid constituting the polyamino acid carrier. Is desirable. For example, when a crosslinked structure or a reactive group (hereinafter sometimes referred to as “crosslinked structure or the like”) has an amino group, a compound having a “diol structure or amino alcohol structure” has a carboxyl group, It is preferable to form an amide bond. When the crosslinked structure or the like has a carboxyl group, the compound having a “diol structure or amino alcohol structure” preferably has an amino group and can form an amide bond with the crosslinked structure or the like. The compound having a “diol structure or amino alcohol structure” is preferably a hydroxy amino acid, most preferably a serine residue or a threonine residue.

  In the present specification, the “crosslinked structure” means that a reactive group in the side chain of the amino acid or amino acid derivative constituting the polyamino acid of the present invention is linked to a diol structure or aminoalcohol structure for generating an aldehyde group. This is the structure of the crosslinking agent.

  The crosslinking agent used in the present invention is a compound capable of binding the reactive group to a diol structure or amino alcohol structure. Preferably, it is a compound in which one end of the crosslinked structure is a carboxyl group and the other end is an amino group, or a compound (diamino compound) in which both ends of the crosslinked structure are amino groups. For example, when one end of the crosslinked structure is a compound composed of a carboxyl group and the other end is an amino group, and the reactive group is an amino group, the carboxyl group in the crosslinked structure forms an amide bond with the reactive group. The amino group in the crosslinked structure preferably forms an amide bond with the diol structure or amino alcohol structure having a carboxyl group. Alternatively, when the both ends of the crosslinked structure are compounds (diamino compounds) and the reactive group is a carboxyl group, the other amino group in the crosslinked structure is preferably a diol structure or amino group having a carboxyl group. Forms an amide bond with the alcohol structure. A preferred cross-linked structure is γ-aminobutyric acid (GABA) or ethylenediamine.

  The cross-linked structure may be composed of a cross-linking agent in which one or more and the same or different compounds are bonded in a linear or branched manner. On the other hand, as the object of the present invention, as long as the activity of the low molecular weight antigen when bound to the carrier is not impaired, the above-mentioned cross-linked structure may be omitted, and the reactive group of the polyamino acid carrier may have a diol structure or amino alcohol. Structures may be combined.

    In the polyamino acid carrier of the present invention, the reactive group bonded to the diol structure or aminoalcohol structure is preferably 3% to 30% of the reactive groups in the side chain, with or without a cross-linked structure. More preferably, it is 5% to 20%. According to the method for producing a peptide-bonded carrier of the present invention, the oxidation treatment for generating an aldehyde group from a diol structure or an amino alcohol structure is not particularly limited. For example, a periodate oxidation method can be used. The aldehyde group produced on the polyamino acid carrier is bound to the amino group in the HIV synthetic antigen. For this binding, considering the efficiency of binding of the peptide to the polyamino acid carrier, the activity of the HIV synthetic peptide antigen, and the reactivity of the antibody to the HIV synthetic peptide antigen, etc. It is preferred to use an N-terminal amino group rather than a chain. Alternatively, it is preferable to use an amino acid having an amino group bound to the N-terminus or C-terminus of the peptide. More preferably, in order to enhance the selective binding between the aldehyde group of the polyamino acid carrier and the amino group of the HIV synthetic peptide antigen, an HIV synthetic peptide antigen in which a hydrazino group is introduced at the end of the HIV synthetic peptide antigen is prepared and used. . As a reagent used for introducing a hydrazino group, hydrazinobenzoyllysine having a hydrazino group at the ε-amino group is used. A recombinant HIV antigen can also be produced according to the case of a synthetic HIV peptide antigen.

  As described above, in the detection zone on the matrix, at least one kind of HIV antigen detection part immobilized with anti-HIV antibody, at least one kind of synthetic HIV peptide antigen and / or genetically modified HIV carried on a polyamino acid carrier. There can be provided at least two detection parts with the HIV antibody detection part in which the antigen is immobilized. These detection units are downstream of the enzyme labeling reagent zone, the specimen spotting zone, and the developing solution supply zone, and upstream of the additive solution absorption zone in the infusion direction of the matrix. Any order may be sufficient. The detection unit can be provided with a plurality of lines adjacent to each other in a line shape having a width of about 0.5 mm to 2 mm, preferably about 1 mm, in the matrix. In the case of a matrix having a width of about 5 mm, the antibody or antigen is usually spotted from about 0.1 μg to 10 μg, respectively, and dried to prepare a detection unit.

(Enzyme labeling reagent zone)
The enzyme labeling reagent zone can be provided by spotting an enzyme labeling reagent such as a labeled HIV antigen, a labeled anti-HIV antibody or the like in a zone provided on the matrix so as to be movable by a developing solution. This zone is provided upstream of the detection zone in the infusion direction of the developing liquid from the developing liquid supply zone. This zone consists of a method of spotting an enzyme labeling reagent on the matrix, a method of laminating a water-absorbing pad containing the enzyme labeling reagent on the matrix, or an enzyme labeling reagent together with the pad on part or all of the matrix part that is in close contact with the pad It is comprised by containing. When enzyme-labeling reagents are added to both the matrix and the pad, the labeled HIV antigen and the labeled anti-HIV antibody can be spotted separately or mixed.

    As the antigen constituting the enzyme-labeled HIV antigen, one or more kinds of antigens can be selected from the HIV-1 and HIV-2 antigens described above and can be produced according to a known method. Examples of the HIV-1 and HIV-2 antigens include cultured antigens obtained from HIV-infected strains, recombinant antigens, chemically synthesized antigens, and the like, and from the same antigen as the HIV antigen exemplified in the detection zone. You can select and use.

    The labeled anti-HIV antibodies are monoclonal antibodies and polyclonal antibodies against HIV-1 and HIV-2. These antibodies can be selected from the same antibodies as the anti-HIV antibodies exemplified in the detection zone.

    The enzyme-labeled HIV antigen or enzyme-labeled anti-HIV antibody can be produced by binding the antigen or antibody and an enzyme. Examples of the enzyme include various enzymes used in well-known enzyme immunoassay (EIA). Examples of the enzyme include alkaline phosphatase, peroxidase, β-D-galactosidase, and the like.

    In addition, the method for binding an enzyme to an HIV antigen or an HIV antibody can be produced using a known method for making a covalent bond or a non-covalent bond. Examples of the binding method include the gutalaldehyde method, the periodic acid method, the maleimide method, the pyridyl disulfide method, and a method using various crosslinking agents (for example, “Protein Nucleic Acid Enzyme”, Vol. 45 (1985)). In the bonding method using a crosslinking agent, examples of the crosslinking agent include N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidohexanoic acid, and N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1. -Carboxylic acid etc. can be used. In the covalent bond method, functional groups present in antigens or antibodies can be used. For example, functional groups such as thiol groups, amino groups, carboxyl groups, and hydroxyl groups are introduced by a conventional method, followed by enzyme labeling by the above-described binding method. HIV antigens or enzyme-labeled anti-HIV antibodies can be produced. Examples of the noncovalent bonding method include a physical adsorption method.

    The enzyme-labeled HIV antigen and the enzyme-labeled anti-HIV antibody contained in the enzyme-labeled reagent zone are contained in either the matrix or the water-absorbing pad, or the enzyme-labeled HIV antigen or enzyme is contained in both the matrix and the water-absorbing pad. A labeled anti-HIV antibody can be included. When measuring HIV antigen antibody simultaneously, since many labeling reagents are used, it is advantageous for highly sensitive measurement to contain the enzyme labeling reagent alone or in a mixture in both the matrix and the pad. The amount of enzyme-labeled HIV antigen and enzyme-labeled anti-HIV antibody can be appropriately changed according to the expected amount of the test object, but when used for the matrix, it is usually about 1 ng to 100 ng in dry weight. . The enzyme-labeled HIV antigen and the enzyme-labeled anti-HIV antibody can be applied together with a stabilizer such as animal serum and sugar, a dissolution regulator, and the like when contained in the enzyme labeling zone.

(Specimen spotting zone)
The specimen spotting zone can be provided on the matrix downstream of the development liquid supply zone in the infusion direction of the development liquid and upstream of the detection zone. This specimen spotting zone is: 1) a predetermined location upstream of the enzyme labeling reagent zone on the downstream side of the developing solution infusion direction of the developing solution zone; 2) a predetermined location upstream of the detection zone on the downstream side of the labeling reagent zone; 3) It is preferably provided at a predetermined location on the labeling reagent zone. In addition, in an apparatus in which a sample spotting zone is provided in the enzyme labeling reagent zone, it is preferable to attach a water absorbing pad containing an enzyme label as described above for efficient analysis. In the device to which this pad is added, a large amount of sample liquid can be spotted, so that a trace component in the sample can be measured with high detection sensitivity. As this water-absorbing pad, for example, a material composed of a porous synthetic or natural polymer compound such as polyvinyl alcohol (PVA), non-woven fabric, and cellulose can be used alone or in combination. The size, thickness, density, etc. of the pad are not limited, but it is usually preferable to use a pad having a length and width of about 3 mm to 12 mm and a thickness of about 0.5 mm to 4 mm for efficient measurement. .

(Development liquid supply zone)
The developing liquid supply zone is a zone provided at one end in the longitudinal direction of the matrix and supplied with the developing liquid. To start the measurement, it is possible to immerse the developing solution supply zone in a container containing at least an amount of the developing solution that reaches the developing solution absorption zone. Further, for supplying the developing liquid, a liquid tank containing the developing liquid is added to the developing liquid zone, and the measurement can be started by breaking the cover of the liquid tank and bringing the developing liquid into contact with the matrix. The developing solution can appropriately contain a surfactant, a buffer, a stabilizer, an antibacterial agent and the like. Examples of the buffer solution containing a buffer include an acetate buffer solution, borate buffer solution, Tris-HCl buffer solution, diethanolamine buffer solution and the like. Further, it is preferable to attach a developing solution pad to the developing solution supply zone in order to stably and continuously supply the developing solution to the matrix.

(Developing solution absorption zone)
The developing solution absorption zone is installed at the other end with respect to the developing solution supply zone provided at one end of the matrix. This zone is provided in order to absorb the developing solution supplied to the matrix and perform analysis smoothly. The developing solution absorption zone can be secured by forming a long matrix. Further, a water absorbing material can be attached to the matrix to promote the development. As this water-absorbing material, a filter paper, sponge, etc. having high water retention composed of a natural polymer compound, a synthetic polymer compound or the like can be used. The developing fluid absorption zone is a pad-shaped absorbent material with a volume that absorbs all of the developing fluid. However, a compact immunoassay device can be manufactured by laminating the absorbent material on or under the matrix. preferable.

(Substrate reagent zone)
The substrate reagent zone can be provided near one end of the matrix. The substrate reagent zone may be provided on the matrix, but it is preferable that the substrate reagent zone is contained in the developing solution pad attached to the developing solution supply zone so as to contain a large base mass. The substrate zone can be provided at one place or two or more places depending on the substrate to be used.

    In the substrate reagent zone, various chromogenic substrates, fluorescent substrates, luminescent substrates and the like shown below corresponding to the enzyme of the enzyme labeling reagent can be used.

(A) For chromogenic substrate peroxidase: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) combined with hydrogen peroxide, 3,3 ′, 5,5′-tetra Methylbenzidine (TMB), Diaminobenzidine (DAB)
For alkaline phosphatase: 5-bromo-4-chloro-3-indolyl phosphate (BCIP)

(B) For fluorescent substrate alkaline phosphatase: 4-methylumbelliferyl-phosphate (4MUP)
For β-D-galactosidase: 4-methylumberylphenyl-β-D-galactoside (4MUG)

(C) Luminescent substrate for alkaline phosphatase: 3- (2′-spiroadamantane) -4-methoxy-4- (3 ″ -phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD)
For β-D-galactosidase: 3- (2′-spiroadamantane) -4-methoxy-4- (3 ″ -β-D-galactopyranosyl) phenyl-1,2-dioxetane (AMGPD)
For peroxidase: Luminol, isoluminol combined with hydrogen peroxide

    The substrate can be formed by dissolving the substrate in an aqueous solution and applying it to a developing solution pad in a line, followed by drying. If desired, a substrate signal enhancer, stabilizer, dissolution regulator, etc. can be added. It can also be added. The substrate zone is not particularly limited as long as it is in the developing solution pad attached to the end of the matrix. Although the base mass added to a developing solution pad can be determined by measurement conditions, about 100 mg-400 mg can normally be used per instrument. The substrate may be contained in the developing solution and supplied to the matrix. In addition to providing the substrate zone, the developing solution may contain a part or all of the substrate.

(How to use the measuring instrument)
The measurement instrument of the present invention can measure HIV antigens and / or anti-HIV antibodies in various specimen samples. The measurement is performed by first supplying a sample of about 5 μl to 50 μl to the sample spotting zone of the measuring instrument of the present invention, and then developing the developing solution on a matrix. The developing solution moves through the matrix by capillary action, reaches the developing solution absorption zone, and the components in the specimen, enzyme labeling reagent, etc. that are not bound to the detection zone are absorbed, and the development is completed. After elapse of a predetermined time (usually 10 to 20 minutes), the detection zone having an HIV antigen detection part and an HIV antibody detection part is observed, and the HIV antigen detection part in an amount depending on the HIV antigen or anti-HIV antibody in the sample liquid; HIV antigen and anti-HIV antibody can be measured by detecting a signal generated by the reaction between the enzyme label immobilized on the HIV antibody detection unit and the substrate. This detection can be carried out by using a measuring device such as a colorimeter, a fluorimeter, a photon counter, a photosensitive film, etc., in addition to the visual measurement by the substrate used in the measuring instrument of the present invention. For the measurement, for example, a method of qualitatively visually measuring the presence or absence of a dye produced by a chromogenic substrate is simple. Further, in this method, a semi-quantitative analysis can be performed by using a color chart (color chart) corresponding to the amount of HIV antigen or anti-HIV antibody contained in the specimen. Further, the coloration in the detection zone can be converted into a numerical value by a colorimeter or the like for quantitative determination.

  Furthermore, it is preferable to provide a developing solution confirming unit for confirming whether the developing solution is developed and the measurement is normally performed on the matrix between the detection zone and the developing solution absorption zone. The developing solution confirmation unit includes an indicator that reacts with the developing solution or a reagent contained in the developing solution, an indicator that reacts with the developing solution and disappears in color, and an antibody or antigen that reacts with a part of the reagent of the labeling reagent. Etc. can be immobilized. When an antibody or antigen that reacts with the labeling reagent is bound, the developing solution confirmation unit can confirm the color by color reaction with the substrate contained in the developing solution.

    Further, the matrix can be used by being laminated and fixed on a support member such as plastic, metal or paper. The matrix is fixed to a case of plastic or the like, a liquid tank containing a developing solution is attached to the developing solution supply zone, and a cover with a case with a hole in each zone portion is configured to constitute a device that is easy to handle. be able to. Examples of the sample measured with the measuring instrument of the present invention include body fluids such as serum, plasma, whole blood, saliva, urine.

  Since the HIV antigen and / or anti-HIV antibody in a specimen can be measured simultaneously by using the immunoassay instrument for measuring HIV antigen antibodies of the present invention, HIV infection can be measured easily and easily. In addition, in the device of the present invention, infection can be detected earlier in the early stage of infection than in the conventional antibody test, and since the proportion of false positives is very small, highly sensitive and accurate diagnosis can be performed. Furthermore, since the device of the present invention can be determined in about 15 minutes after the start of the test and does not require a special measuring device for the determination, HIV infection can be easily determined and is useful for preventing the spread of HIV infection. In addition, it is useful for specifying the time of infection and selecting a therapeutic agent from the measurement results of HIV antigen and anti-HIV antibody obtained at the same time.

Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to the examples.

Reference Example 1 Preparation of HIV-1 gp41 Recombinant Antigen An E. coli strain into which a plasmid in which the genes from amino acids 540 to 683 of HIV-1 gp41 were incorporated into an expression vector was introduced.
Preparation of recombinant HIV-1 antigen (gp41) Culture was carried out by aeration and agitation at 37 ° C. using a production medium, and IPTG (isopropyl-thio-β-D-galactopyranoside) was monitored while monitoring the growth of recombinants for turbidity. Induced with. After completion of the culture, the recombinants were collected by a membrane concentration method, and the cells were washed by adding a buffer solution to the concentrated solution and further performing a concentration operation. For purification of the antigen, the cells are crushed and centrifuged, and the precipitate is recovered. The recovered precipitate was suspended in a buffer solution, a surfactant was added, and the precipitate was washed. The obtained precipitate was solubilized, further purified by chromatography, and finally desalted by gel filtration to obtain a purified recombinant.

Reference Example 2 Preparation of Alkaline Phosphatase-Labeled HIV-1 gp41 Recombinant Antigen 0.2MDTT ((±) -dithiothreitol, Wako) was added to a purified recombinant antigen solution containing a disulfide bond and incubated at 37 ° C. for 2 hours. Gel filtration purification was performed, and the free thiol group (-SH) was coupled with alkaline phosphatase into which N-succinimidyl-4-maleimidobutyric acid (GMBS) was introduced. Finally, it was purified by gel filtration to obtain alkaline phosphatase labeled HIV-1 gp41 recombinant antigen.

Reference Example 3 Preparation of HIV-2gp3617mer peptide-binding carrier (I) Synthesis 1 of serinated polylysine 1
With ^(a) N α -t- butoxycarbonyl -O-t-butyl -L- seryl -γ- aminobutyric acid (1) .gamma.-aminobutyric acid and serine with synthetic amino alcohol structure as a crosslinking agent (GABA) Then, serine having the title crosslinking structure was synthesized (Scheme 1). A commercially available serine in which the amino group and hydroxyl group of serine were previously protected was used. N ^{alpha-t-butoxycarbonyl} -O-t-butyl -L- serine 2.61 g, N-hydroxysuccinimide 1.27g of N, N-dimethylformamide and water (hereinafter, referred to. DMF), ice Under cooling, 2.11 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and the mixture was stirred at room temperature overnight. Ethyl acetate and water were added to the reaction mixture, and the mixture was stirred well for separation. The aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with 5% citric acid aqueous solution, water, 5% sodium hydrogen carbonate aqueous solution and water in this order, and dried over anhydrous magnesium sulfate. Ethyl acetate was distilled off, the residue was dissolved in DMF, 1.03 g of GABA and 0.84 g of sodium hydrogen carbonate were dissolved in water, and the mixture was stirred overnight. Ethyl acetate and water were added to the reaction solution, and the mixture was separated with 6N hydrochloric acid to a pH of about 3. The aqueous phase was extracted with ethyl acetate, and the combined ethyl acetate phases were washed with water and dried over anhydrous magnesium sulfate. When ethyl acetate was distilled off, crystallization occurred. n-Hexane was added and the crystals were collected by filtration. Yield 2.77 g (80%).

(B) N ^α -t-butoxycarbonyl-Ot-butyl-L-seryl-γ-aminobutyric acid N-succinimide ester (2)
The carboxyl group of the compound (1) prepared above was activated with a succinimide ester (Scheme 1).

1.04 g of compound (1) and 0.38 g of N-hydroxysuccinimide are dissolved in DMF, and 0.63 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is added under ice cooling, and at room temperature. Stir overnight. Water and ethyl acetate were added to the reaction solution, and the mixture was well stirred and separated. The aqueous phase was extracted with ethyl acetate, and the combined ethyl acetate phases were washed with 5% citric acid aqueous solution, water, 5% sodium hydrogen carbonate aqueous solution and water in this order, and dried over anhydrous magnesium sulfate. When ethyl acetate was distilled off, crystallization occurred. n-Hexane was added and the crystals were collected by filtration. Yield 1.21 g (91%)
Scheme 1

(C) Polylysine {Boc-serine (t-butyl) -γ-aminobutyric acid} (3)
The compound (2) produced above and polylysine were peptide-bonded to obtain the title compound (3) (Scheme 2).

20 mg of polylysine (Sigma No. P2636, MW (viscosity) 30,300) was dissolved in 1 ml of 0.1 M aqueous sodium hydrogen carbonate solution, and 8.9 mg of compound (2) was dissolved in 200 μl of DMF and added at room temperature. Stir for hours. 100 μl of acetic acid was added to the reaction solution, transferred to a dialysis tube, dialyzed against water for 3 hours at room temperature, and then lyophilized. Yield 22.0mg
Scheme 2

(D) Polylysine {Boc-serine (t-butyl) -γ-aminobutyric acid, succinyl} (4)
The unreacted amino group of polylysine in the compound (3) produced above was reacted with succinic anhydride for modification (Scheme 3). 10 mg of the above compound (3) was dissolved in 1 ml of 1M aqueous sodium hydrogen carbonate solution, 25 mg of succinic anhydride was dissolved in 250 μl of DMF, and the mixture was stirred at room temperature for 2 hours. The reaction solution was transferred to a dialysis tube, dialyzed overnight at 4 ° C. against water, and then freeze-dried. Yield 13.7 mg

(E) Polylysine {serine-γ-aminobutyric acid, succinyl} (5)
The compound (4) produced above was deprotected to give the title compound (5) (Scheme 3). The total amount of compound (4) was dissolved in 25 μl of water and 500 μl of trifluoroacetic acid, and stirred at room temperature for 1 hour. Water was added to the reaction solution and lyophilized. It melt | dissolved in 1M sodium hydrogencarbonate aqueous solution, and dialyzed with water at room temperature for 3 hours, Then, it was freeze-dried. Yield 10.7mg
Scheme 3

  By amino acid analysis after hydrolysis, the proportion of reactive groups to which an amino alcohol structure was bonded was examined among the reactive groups of polylysine. When the reactive group of compound (2) and polylysine were mixed at a molar ratio of 1: 5, the amino alcohol structure reacted at a ratio of 1 to the reactive group of polylysine 4.6.

  (F) Furthermore, in order to prepare polylysine carriers having different proportions of reactive groups bonded to the aminoalcohol structure, the mixing ratio of the above compound (2) and polylysine was changed, and serrated polylysine was prepared in the same manner. Prepared. When the molar ratio of the above compound (2) and the reactive group of polylysine was mixed at 1:10, the amino alcohol structure reacted at a ratio of 1 to the reactive group 9.6 of polylysine. When the molar ratio of the above compound (2) to the polylysine reactive group was 1:20, the amino alcohol structure reacted at a ratio of 1 to the polylysine reactive group 19.2.

Reference Example 4 Synthesis of hydrazino peptide (1) Synthesis of 4- (t-butoxycarbonylhydrazino) benzoic acid 15.2 g of 4-hydrazinobenzoic acid was suspended in 50 ml of water under an argon stream, and 4.8 g of sodium hydroxide. To dissolve. 26.2 g of di-t-butyl dicarbonate was dissolved in 50 ml of 1,4-dioxane and added, and the mixture was stirred overnight. About half of the solvent was distilled off under reduced pressure, water was added, and the mixture was washed twice with ethyl acetate. Ethyl acetate was added to the aqueous phase, neutralized with 6N hydrochloric acid and separated. Ethyl acetate was added once more, pH was adjusted to 3 with 6N hydrochloric acid, and liquid separation was performed. The ethyl acetate phases were combined, washed twice with water, and dried over anhydrous magnesium sulfate. Crystals precipitated when the solvent was distilled off. The crystals were collected by filtration and washed with ethyl acetate. Yield 16.9 g (67%)
Crystals precipitated when the mother liquor was concentrated. It was filtered and washed with ethyl acetate. Yield 4.8g. In total 21.7 g (86%).

(2) Synthesis of 4- (t-butoxycarbonylhydrazino) benzoic acid N-succinimide ester 5.05 g of 4- (t-butoxycarbonylhydrazino) benzoic acid and 2.53 g of N-hydroxysuccinimide are dissolved in DMF. Under ice cooling, 4.22 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added and stirred overnight. Ethyl acetate was added to the reaction mixture, and the mixture was washed with water, 5% aqueous sodium hydrogen carbonate solution and saturated brine. Crystals precipitated. Filtered off and dried over diphosphorus pentoxide. The mother liquor was concentrated, and the precipitated crystals were collected by filtration and dried over diphosphorus pentoxide. Combined with the initially precipitated crystals, 6.15 g (88%) of the title compound was obtained.

(3) N α ^- (9- fluorenyl methoxycarbonyl) -Nε- (4- (t- butoxycarbonyl-hydrazino) benzoyl) -L- synthesis of lysine N ^alpha - (9-fluorenyl methoxy carbonyl) - L-lysine (3.0 g) and sodium bicarbonate (0.65 g) were suspended in water and DMF, and under ice cooling, 4- (t-butoxycarbonylhydrazino) benzoic acid N-succinimide ester (2.7 g) was dissolved in DMF. added. Stir at room temperature overnight. Ethyl acetate and water were added to the reaction solution, acidified with 6N hydrochloric acid and separated. The aqueous phase was extracted twice with ethyl acetate, and the combined ethyl acetate phases were washed with water and dried over anhydrous magnesium sulfate. The solvent was distilled off and the resulting oily residue solidified upon drying under reduced pressure. Yield 3.50 g (75%).

(4) Synthesis of HIV-2 gp36 synthetic peptide having hydrazino group As an antigen peptide for detecting an antibody derived from HIV-2 coat protein gp36, 17 residues were selected from the amino acid sequence of HIV-2 gp36, Synthesis was performed. According to a known Fmoc method, a peptide synthesizer PSSM-8 (Shimadzu Corporation) was used to bind 17-residue amino acids, and finally N ^α- (9-fluorenylmethoxycarbonyl) -N ^ε- (4- By adding (t-butoxycarbonylhydrazino) benzoyl) -L-lysine, an HIV-2gp36 synthetic peptide having a hydrazino group at the N-terminal lysine was prepared.

Structure of hydrazino peptide 18-residue sequence
Lys (HBz) -Gln-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys (SEQ ID NO: 1)
HBz = hydrazinobenzoyl

Reference Example 5 Preparation of complex of serinated polylysine and hydrazino peptide Compound (5) produced in Example 2 above, which was reacted at a ratio of aminoalcohol structure 1 to polylysine reactive group 20.2, was added. A peptide-bonded polylysine carrier bound with the hydrazino peptide produced in Example 3 was prepared by oxidation with iodate (Scheme 4).

  2 mg of compound (5) was dissolved in 1 ml of 10 mM sodium hydrogen carbonate aqueous solution, 40 μl of 100 mM sodium periodate aqueous solution was added, and the mixture was stirred at room temperature for 20 minutes. The reaction solution was applied to a PD-10 column (manufactured by Amersham Bioscience) equilibrated with 10 mM aqueous sodium hydrogen carbonate solution and eluted with the same solution. The first 2.5 ml of eluate was not collected and the subsequent 2.0 ml was collected. To this solution, hydrazino peptide was dissolved in 10 mM aqueous sodium hydrogen carbonate solution (1 mg / ml), and stirred at room temperature for 3 hours. 10 μl of 400 mM hydroxyamine hydrochloride / 1M aqueous sodium hydrogen carbonate solution was added and stirred for 30 minutes. Acetic acid 20 μl was added and stirred for 30 minutes. Acetic acid (20 μl) was added to adjust the pH of the solution to about 5 and transferred to a dialysis tube. The solution was dialyzed against water at 4 ° C. overnight and then lyophilized.

  When the introduction rate of the peptide was examined by amino acid analysis after hydrolysis, the ratio of the peptide was 1 with respect to the reactive group 20 of polylysine (the ratio of the peptide was about 1 with respect to the amino alcohol structure 1 on the polylysine carrier). When mixed, the peptide reacted at a ratio of 1 to the reactive group 45.5 of polylysine. Further, when the peptide is mixed at a ratio of 1 to the reactive group 40 of polylysine (the ratio of peptide is about 0.5 to aminoalcohol structure 1 on the polylysine carrier), the reactive group 62. The peptide reacted at a ratio of 1 to 5.

Scheme 4

Reference Example 6 Preparation of alkaline phosphatase-labeled anti-HIV-1p24 polyclonal antibody Anti-HIV-1 gagP24 polyclonal antibody was cleaved with Feps region using pepsin to obtain F (ab ′) ₂ , and then 2ME (2-mercaptoethanol, Nacalai Tesque) F (ab ′) ₂ in which a disulfide bond was cleaved to expose a free thiol group was obtained. Next, it was coupled with alkaline phosphatase into which a maleimide group was introduced, and finally purified alkaline phosphatase-labeled anti-HIV-1 gagP24 polyclonal antibody was obtained by gel filtration.

Example 1 HIV-1, 2 antigen antibody simultaneous measurement instrument As shown in FIG. 1, in a matrix 2 of a nitrocellulose membrane (made by Millipore) having a width of 5 mm and a length of 50 mm, 16 mm from the end on the developing solution absorption zone 5 side. The HIV antibody detector 6a was prepared by spotting 0.7 μl of an aqueous solution containing the HIV-2 gp36 18mer peptide-binding carrier prepared in Reference Example 3 and the HIV-1 gp41 synthetic antigen at the position of the development solution absorption zone 5 side. 0.7 μl of an aqueous solution containing a monoclonal antibody against HIV-1 p-24 produced according to a well-known method at a position 13.5 mm from the end was spotted on a nitrocellulose membrane and dried to prepare an HIV antigen detection unit 6b. Further, an anti-alkaline phosphatase antibody (manufactured by Dako) was spotted and immobilized at a position 11 mm from the end of the matrix 2 on the side of the developing solution absorption zone 5 to prepare a developing solution confirmation unit 10. Next, 2.5 μl of the alkaline phosphatase-labeled anti-HIV-1 p24 polyclonal antibody aqueous solution (100 μg / ml) produced in Reference Example 6 was spotted and dried at a position 23 mm from the end of the developing solution absorption zone 5 side of the matrix 2, An enzyme labeling reagent part 4b was prepared. Furthermore, the alkaline phosphatase-labeled HIV-1 gp41 recombinant antigen produced in Reference Example 2 (1 μg / ml) and the alkaline phosphatase-labeled anti-HIV-2 gp36 peptide produced in Reference Example 3 (5 μg) were applied to a pad made of 10 mm × 6 mm PVA. / Ml), 5 μl spotted, and dried to prepare enzyme-labeled reagent pad 4a. As shown in FIG. 1, the enzyme labeling reagent pad 4 a was overlaid on the enzyme labeling reagent part 4 b of the matrix 2 to create an enzyme labeling reagent zone 4.

  The matrix 2, the developing solution pad 3, the enzyme labeling reagent pad 4a and the developing solution absorbing pad 5 are fixed to a plastic case having a developing solution tank 11, and the immunoassay instrument 1 for measuring HIV antigen antibody shown in FIGS. did.

Example 2 Measurement of HIV antigen and anti-HIV antibody An anti-HIV-1 seroconversion panel specimen (Panel) was placed in the specimen spotting zone 8 of the immunoassay instrument 1 for HIV antigen-antibody measurement (FIG. 2 or 3) produced in Example 1 above. After dropping 25 μl of BI (PRB959); manufactured by BBI), the pressing portion 12 provided on the deformable member is pressed downward to be deformed, and the developing liquid pad 3 is developed by the protrusion 13 attached to the deformable member. Measurement was started by inserting the developing solution into the tank 11 and supplying the developing solution to the developing solution pad 3. 15 minutes after the start of measurement, development of the developing solution was confirmed by color development of the target reagent zone 10, and then color development of the detection zones 6a (antibody detection part) and 6b (antigen detection part) was visually measured. The results are shown in Table 1.

As controls, HIV-1 / 2 Ab (Abbott), HIV-1 Agmonoclonal-1 detection kit (Abbott), Genscreen HIVAg / Ab (Bio), obtained from Boston Biomedica, Inc. data sheet. Rad), Integral HIVAg / Ab (Dade Behring) and Uni-Form HIVAg / Ab (Organon Technica) are also shown in Table 1.
Compared with the conventional HIV-1 / 2 antibody detection kit, the measurement instrument of the present invention was able to detect two days earlier. In addition, detection was possible at the same time as the conventional HIV-1Ag detection kit and HIV-Ag / Ab simultaneous measurement kit.

Example 3
Table 2 shows anti-HIV-2 antibody positive specimens (PRF202-09; manufactured by BBI) in the specimen spotting zone 8 of the immunoassay instrument 1 for HIV antigen antibody measurement (FIG. 2 or 3) produced in Example 1 above. The sample was diluted 800-fold to 12,800-fold, 25 μl of the sample was dropped into the sample spotting zone, and measurement was performed in the same manner as in Example 2. The results are shown in Table 2. The immunoassay instrument for measuring HIV antigen antibodies of the present application showed high reactivity to HIV-2 antibody positive diluted specimens as compared with existing HIV-1 / 2 antibody detection reagents.

It is sectional drawing which shows typically an example of the measuring instrument aspect of this invention. It is a top view which shows an example when the measuring instrument of this invention is set to the cassette. It is sectional drawing of AA 'of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Immunoassay instrument 2 for HIV antigen antibody measurement Matrix 3 Developing liquid pad 4 Enzyme labeling reagent zone 4a Enzyme labeling reagent pad 4b Enzyme labeling reagent part 5 Developing liquid absorption zone 6 Detection zone 6a HIV antibody detecting part 6b HIV antigen detecting part 7 Substrate Reagent zone 8 Specimen spotting zone 9 Specimen 10 Development liquid confirmation part 11 Development liquid tank 12 Pushing part 13 Projection

Claims (10)

Provided with a band-like matrix that can be infused by capillary action,
An HIV antigen detection unit immobilizing at least one anti-HIV antibody and an HIV antibody detection unit immobilizing at least one synthetic HIV peptide antigen and / or gene recombinant antigen carried on a polyamino acid carrier A detection zone;
An enzyme labeling reagent zone;
Specimen spotting zone,
A developing solution supply zone provided with a developing solution pad having a substrate reagent zone in the vicinity of one longitudinal end of the matrix;
An immunoassay instrument for measuring HIV antigen antibodies, comprising a developing solution absorption zone provided at the other longitudinal end of the matrix.     The matrix is a matrix that infuses a developing solution from the developing solution supply zone toward the developing solution absorption zone, and includes an enzyme labeling reagent zone upstream of the detection zone and a substrate reagent zone upstream of the enzyme labeling reagent zone. The immunoassay device according to claim 1, further comprising a sample spotting zone on the enzyme labeling reagent zone.   The immunoassay device according to claim 1 or 2, wherein the enzyme labeling reagent zone is a pad containing an enzyme labeling reagent laminated on a matrix.     An enzyme labeling reagent zone in which a pad containing an enzyme labeling reagent is stacked on a matrix, and an enzyme labeling that includes an enzyme labeling reagent that is the same as or different from the enzyme labeling reagent in part or all of the matrix portion that is in close contact with the layered portion of the pad The immunoassay device according to any one of claims 1 to 3, wherein a zone is provided.     The immunoassay device according to any one of claims 1 to 4, wherein the anti-HIV antibody immobilized on the HIV antigen detection unit is one or two monoclonal antibodies against p24 of the HIV-1 gag antigen.     6. The immunoassay device according to claim 5, wherein the enzyme labeling reagent is a polyclonal antibody against p24 of the enzyme-labeled HIV-1 gag antigen.     The HIV antigen of the HIV antibody detector comprises a recombinant antigen of gp41 of HIV-1 env antigen and a synthetic HIV peptide antigen in gp36 of at least one HIV-2 env antigen carried on a polyamino acid carrier. 5. The immunoassay device according to any one of items 4 to 4.     The immunoassay device according to claim 7, wherein the enzyme labeling reagent comprises gp41 of enzyme-labeled recombinant HIV-1 env antigen and synthetic HIV-2gp36 labeled with enzyme.     The immunoassay device according to claim 7, wherein the polyamino acid carrier is polylysine.   A sample is spotted on the sample spotting zone of the immunoassay device according to claim 1, a developing solution is supplied to the developing solution supply zone, and the developing solution is developed on the matrix, and then a signal in the detection zone is detected. An HIV antigen antibody immunoassay method comprising: