JPH08304397A - Detecting method for pathogen infection - Google Patents

Abstract

PURPOSE: To make it possible to conduct sure diagnosis always from an earlier time than the formation of an antibody and also even after the formation of a solid body by using a principle of an immunity complex transition method. CONSTITUTION: Components, which are respectively peculiarly coupled with at least one antigen of a pathogen and at least one antibody against a different antigen of the same pathogen, are added to the same so as to form an immunity complex containing the antigen to be measured and/or the immunity complex containing the antibody to be measured. The formed immunity complex is captured on first solid phase which is formed by insolubilizing a peculiarly reactive component against a functional group. The immunity complex is separated from the first solid phase, and the immunity complex is captured on second solid phase which is formed by insolubilizing a peculiarly reactive component against a position in the immunity complex different from the functional group so as to measure the captured immunity complex captured on the second solid phase. The formation, the capture, and the measurement of the immunity complex are conducted simultaneously.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an immunoassay for diagnosing an infectious disease by simultaneously measuring an antigenic substance of a specific pathogen and an antibody substance sensitized to the pathogen by using an immune complex transfer method. Concerning the law. Also, human immunodeficiency virus-1 (HIV-1) p24 antigen, anti-p1
The present invention relates to an immunoassay method for diagnosing HIV-1 infection by simultaneously measuring 7 antibody and anti-reverse transcriptase (RT) antibody.

[0002]

2. Description of the Related Art As a method for measuring and analyzing an antigen substance or an antibody substance, the immunoassay method has the advantage of being simple and highly specific. In particular, the immunocomplex transfer immunoassay (hereinafter, referred to as “immunocomplex transfer method”) is suitable for adsorbing a contaminant protein on a solid phase, which is a drawback of the sandwich method, and generating a background signal due to the adsorption. It is known as a method that can be suppressed and can measure an antigenic substance or an antibody substance in a sample solution with high sensitivity. This immunocomplex transfer method is an immunoassay including performing trapping (capture) twice using a pair of solid phases. Next, one pattern of each operation procedure for measuring an antigen substance or an antibody substance by the immunocomplex transfer method will be schematically described as an example. Also,
Hereinafter, in the present specification, for the sake of explanation, among the pair of solid phases,
The solid phase used for the first trap is called the first solid phase, and the solid phase used for the next trap is called the second solid phase.

(1) In the case of antigenic substance measurement (FIG. 1) In the case of an antigen to be measured (reference numeral 11 in FIG. 1, the same applies hereinafter), a specific antibody (12) modified with two different functional groups and a label are labeled. It reacts with the specific antibody (13) thus formed to form a three-way immune complex. The complex is trapped in the first solid phase (14) in which the receptor for one functional group of the antibody 12 is insolubilized. Even if the immune complex is trapped after being completed in the analyte solution, the antibody 12 is first trapped on the solid phase 14,
It may be completed for one end. After washing the solid phase 14, the immune complex is released from the first solid phase in a transfer reaction solution for transferring the immune complex. The immune complex is trapped in the second solid phase (15) in which the receptors for the different functional groups of the antibody 12 are insolubilized. After washing the solid phase 15, the antigen to be measured by the immune complex is detected and quantified by using the label introduced in.

(2) In the case of antibody substance measurement (FIG. 2) The antibody to be measured (reference numeral 21 in FIG. 2, hereinafter the same) is labeled with a specific antigen (22) modified with a functional group. It reacts with the antigen (23) to form an immune complex consisting of the three. The complex is trapped in the first solid phase (24) in which the receptor for the functional group of the antigen 22 is insolubilized. The immune complex may be trapped after being completed in the analyte solution, or may be one in which the antigen 22 is first trapped on the solid phase 24 and completed at one end thereof. After washing the solid phase 24, the immune complex is released from the first solid phase in a transfer reaction solution for transferring the immune complex. The immune complex is trapped in the second solid phase (25) in which a component that specifically reacts with a site in the antibody 21 different from the functional group is insolubilized. After washing the solid phase 25, the antibody to be measured by the immune complex is detected and quantified by using the label introduced in.

[0005] Further detailed explanations and examples of the immune complex transfer method are described in the following documents. (1) ISHIKAWA, E., HASHIDA, S., KOHNO, T., (1991): D
evelopment of ultrasensitive enzyme immunoassay re
viewed with emphasis on factors which limit the sen
sitivity. MOLECULAR AND CELLULAR PROBES, 5, 81-95 (2) ISHIKAWA, E., HASHIDA, S., KOHNO, T., et al. (19
93): Principle and applications of ultrasensitive
enzyme immunoassay (Immune complex transferenzyme
immunoassay) for antibodies in body fluids. J. CLI
N. LABORATORY ANALYSIS, 7, 376-393. (3) JP-A-1-254868 "Measuring method for ultra-sensitive antigenic substance" (4) JP-A-2-28558 "Measuring method for ultra-sensitive specific antibody" (5) Eiji Ishikawa (1993) "Ultrasensitive enzyme immunoassay" (Society Press Center)

[0006] As described above, the immune complex transfer method can detect either antigen or antibody with higher sensitivity than conventional techniques, and therefore can detect infectious diseases earlier and with higher accuracy. Infection can be determined. For example, regarding the detection of the antigen, a report on the detection of p24 antigen of human immunodeficiency virus-1 (HIV-1) by the immunocomplex transfer method [Hashida et al., Journal of Clinical Microbiology (J. Clin. Mi
crobiol. ), 33, 298 (199
5)], regarding the detection of antibody, p of HIV-1 was also used.
Report on Detection of Antibodies to 17, p24 and Reverse Transcriptase [Hashida et al., Journal of Clinical Laboratory Analysis (J. Clin. Lab. An
al), Vol. 8, p. 237 (1994)] and the like.

[0007]

Generally, the method for measuring the pathogen antigen itself and the method for measuring the antibody formed against the pathogen in the immunological diagnosis of infectious diseases have their respective characteristics. One of them is selected depending on the type of pathogen to be detected and the purpose of the test. However, from the viewpoint of detection of infection, in the case of antibody testing, antibodies are not formed until a certain period of time has passed after infection, so it is difficult to detect using any high-sensitivity assay in the early stage of infection. Has a drawback. On the other hand, in the case of an antigen test, it has a drawback that it is difficult to detect it until the pathogen is actively proliferating in the living body. Especially, as represented by human immunodeficiency virus, the pathogen itself may be detected after a while. In the case where the phenotype becomes latent and becomes asymptomatic, it is difficult to detect the antigen in the biological fluid during that period by using any conventional immunoassay method.

Therefore, in order to detect the infection more accurately at any time after the infection, it is important to measure both the antigen and the antibody using a highly sensitive measuring method.
In the existing technology, two different types of assays have to be performed, and in practice, only one of them is measured due to problems such as labor of operation, time, cost, and instrument. Therefore, it has been desired to develop a measurement capable of simultaneously detecting an antigen and an antibody with high sensitivity in a single operation. The object of the present invention is a system for simultaneous measurement of highly sensitive antigen and antibody based on the principle of the immune complex transfer method, which enables reliable determination of infection in a subject at any stage in the diagnosis of infectious disease. Is to provide.

[0009]

The present invention provides a method for detecting a pathogen infection by simultaneously measuring [1] "at least one antigen of a pathogen" and "at least one antibody against different antigens of the same pathogen". Therefore, the above method comprising the following steps (A), (B), (C) and (D). Step (A): The test liquid contains the following components (1), (2) and (3)
And at least one of (4) above to form an immune complex containing the antigen to be measured and / or an immune complex containing the antibody to be measured. (1) A component modified with a functional group that specifically binds to an antigen to be measured of a pathogen (2) A labeled component that specifically binds to an antigen to be measured (3) Functional group Same pathogen modified by (1)
The component that specifically binds to the antibody to be measured against an antigen different from the antigen to be measured (4) The labeled component that specifically binds to the antibody to be measured Step (B): Step ( The immune complex formed in A)
The step of capturing the component that specifically reacts with the functional group in (1) and (3) on the insolubilized first solid phase. Step (C): The immune complex is released from the solid phase, and the immune complex is captured on the second solid phase in which a component that specifically reacts with a site in the immune complex different from the functional group is insolubilized. The process of doing. Step (D): measuring the immune complex captured on the second solid phase. [2] The method described in [1] above, wherein the step (A) and the step (B) are performed simultaneously. [3] The pathogen is human immunodeficiency virus-1 (HIV-
The method according to [1] or [2] above, which is 1). [4] The method according to [3] above, wherein the antigen to be measured is p24, and the antibodies to be measured are anti-reverse transcriptase (RT) antibody and anti-p17 antibody. Regarding The outline of the method of the present invention is shown in FIG. 3, and is a combination of the measuring method of FIG. 1 and the measuring method of FIG.

The present invention will be described below in the order of steps. Regarding the step (A) , examples of the test liquid include biological fluids such as serum, plasma, saliva, urine, and spinal fluid, and buffer solutions. Pathogens to be measured include bacteria, viruses, fungi, rickettsia, chlamydia, protozoa and the like. Examples include Mycobacterium tuberculosis, Neisseria gonorrhoeae, Salmonella, Streptococcus pneumoniae, Hepatitis virus, Immunodeficiency virus, T cell leukemia virus, Influenza virus, Mycoplasma, Syphilis spirochete, Candida.

As the component specifically binding to the antigen to be measured of the pathogen, an antibody produced against the antigen is usually used, but one of the two is, for example, It may be a lectin or a receptor that specifically binds to an antigen. Antibodies are Fab, F instead of whole antibody.
Fragments such as ab 'or F (ab') ₂ may be used and may be polyclonal or monoclonal. The antigen component is usually used as the component that specifically binds to the antibody to be measured, but one of the two may be, for example, an anti-antibody. In this case, when selecting each component, the component used as a reagent that specifically binds to the antigen to be measured and the component that specifically binds to the antibody to be measured do not react with each other. Most important. The selection of the antigen or antibody to be measured is not particularly defined, but for the purpose of more reliably determining infection, it is preferable to select a component that is expressed as early as possible after infection or a component that is maintained at a high concentration. Examples of the functional group here include haptens such as dinitrophenyl group and trinitrophenyl group, biotin and the like. The functional group introduced into the component for measuring the antigen and the functional group introduced into the component for measuring the antibody may be the same or different, but unless particularly disturbing, the same is preferable. It is preferable for producing the first solid phase in the step (B).

Regarding step (B), the component that specifically binds to the functional group of step (A) is
For example, if the functional group is a hapten, the anti-hapten antibody is
In the case of biotin, (strept) avidin can be mentioned. However, the binding of the functional group and the specific component is performed in the step (C).
A combination that easily dissociates is preferable. When the functional group introduced into the component for measuring the antigen in the step (A) and the functional group introduced into the component for measuring the antibody are different from each other, two types of groups that specifically bind to the respective functional groups are used. The ingredients must be insolubilized. As the solid phase, any solid phase used in conventional immunoassays can be used. For example, polystyrene, polyacryl, glass, agarose, etc. may be mentioned. Moreover, any shape may be used. The method for insolubilizing the above components on the solid phase may be any method used in conventional immunological assay methods. After capturing the immune complex on the solid phase, it is preferable to wash the solid phase before proceeding to step (C). The washing method is performed by a method used in a usual immunological assay method. In capturing the immune complex on the solid phase in steps (A) and (B), even if it is captured on the solid phase after the immune complex is completed in the analyte solution, the component having a functional group is first immobilized. It may be captured on a phase and completed for one end thereof. Moreover, you may perform process (A) and (B) simultaneously.

Regarding step (C) The dissociation of the immune complex is preferably carried out without decomposing the complex, for example, at the same site as the functional group involved in the binding of the immune complex with the component on the solid phase or This is achieved by adding a substance having a similar structure in a large excess. The binding of the immune complex to the second solid phase is performed via a component that is insolubilized on the solid phase and specifically reacts with a site different from the functional group in the complex. If necessary, a functional group for capturing in the second solid phase may be introduced into one of the components used in the step (A) in advance. When different sites are used for capturing in the complex containing the antigen to be measured and the complex containing the antibody to be measured, it is necessary to insolubilize two kinds of components specific to each site. . After capturing the immune complex on the solid phase, it is preferable to wash the solid phase before proceeding to step (D).

Regarding the step (D), the measurement of the immune complex captured on the second solid phase is carried out using the label introduced in the step (A) in advance. Labels include those used in conventional immunological measurements, such as enzymes, fluorescent substances, radioactive substances and the like. The label used in the system for measuring an antigen and the label used in the system for measuring an antibody may be the same or different, but if the purpose is to determine the presence or absence of infection, it is better to operate the same. Above all preferred. In addition, the above step (A)
The reactions (D) to (D) are carried out under the conditions generally used for immunological measurement.

Further, the present invention is a method for detecting HIV-1 infection by simultaneously measuring p24 antigen, anti-reverse transcriptase (RT) antibody and anti-p17 antibody, which comprises the steps (A) and (B) below. ) And (C). Step (A): The test liquid contains the following components (1), (2) and (3)
And (4) together to form an immune complex containing the p24 antigen and / or an anti-reverse transcriptase (RT) antibody and / or an anti-p17 antibody. (1) Functional group-modified component that specifically binds to p24 antigen (2) Labeled component that specifically binds to p24 antigen (3) Functional group-modified anti-antibody Component that specifically binds to reverse transcriptase (RT) antibody and component that specifically binds to anti-p17 antibody modified with functional group (4) Labeled anti-reverse transcriptase (RT) Component that specifically binds to antibody and labeled component that specifically binds to anti-p17 antibody Step (B): the immunocomplex formed in Step (A)
The step of capturing the component that specifically reacts with the functional group in (1) and (3) on the insolubilized solid phase. Step (C): measuring the immune complex captured on the solid phase. In capturing the immune complex on the solid phase in steps (A) and (B), even if it is captured on the solid phase after the immune complex is completed in the analyte solution, the component having a functional group is first immobilized. It may be captured on a phase and completed for one end thereof. Moreover, you may perform process (A) and (B) simultaneously.
When p24 antigen, anti-p17 antibody and anti-RT antibody are selected as the antigen and the antibody to be measured, they are captured in the first solid phase which is the stage before the transfer without the immune complex transfer method. Even if the measurement is performed, a sufficient effect can be obtained to diagnose the infection.

[Examples] [0016] In order to explain the usefulness of the present invention, HIV in a positive conversion serum panel of a human immunodeficiency virus-1 (HIV-1) -infected individual is demonstrated by an immune complex transfer method as an example. -1 antigen and anti-HIV-1
An example of simultaneous antibody measurement is shown, but the present invention is not limited to this.

Experimental Example 1 In this experiment, HIV-1 p2 was detected by the immunocomplex transfer method.
Simultaneous detection of 4 antigens, anti-p17 antibody, and anti-RT antibody was performed. Details of purification / preparation of each substance and details of experimental procedure are as follows. [ Each buffer ] Buffer A: 0.1 M sodium phosphate, pH 7.0 Buffer B: 0.1 M sodium phosphate, pH 6.0, 5
mM ethylenediaminetetraacetic acid buffer C: 0.01 M sodium phosphate, pH 7.0,
0.1 g / L bovine serum albumin (fraction V, Intergen, NY), 1 mM magnesium chloride, 1 g / L sodium azide

[ Recombinant HIV-1 antigen ] Recombinant p17 (rp17), Recombinant p24
(Rp24) and recombinant reverse transcriptase (rR
T) was prepared by incorporating an expression plasmid containing cDNA encoding these proteins into Escherichia coli and expressing it, followed by purification by salting out or column chromatography. Regarding the preparation method of each protein, regarding rp17,
[Saito et al., Microbiology & Immunol., Vol. 36, p. 105 (1992)] and [Hashida et al., Journal of Clinical Laboratory Analysis (J. Clin. Lab. Anal.), Vol. 7, p. 353 (1993)], regarding rp24 [Tanaka et al., Microbiology & Immunology, Microbiol. & Immunol.
6, p. 823 (1992)], regarding rRT [Saito et al., Microbiology & Immunology (Microbiol. & Immuno).
l. ), 34, 509 (1990)].

[Preparation of rabbit anti-rp24 antiserum ] rp2
0.5 ml of physiological saline containing 0.2 mg of 4 was suspended in 0.5 ml of Freund's complete adjuvant, and the suspension was intradermally injected into a female rabbit (New Zealand albino rabbit). After that, rp24 was set to 0. 3 times at intervals of 3 weeks.
A suspension of 0.5 ml of physiological saline containing 2 mg in 0.5 ml of Freund's incomplete adjuvant was similarly injected. Blood was collected 2 weeks after the last injection to give anti-rp2
4 antisera were obtained.

[ Purification of antibody ] Known methods using salting out and ion exchange chromatography from rabbit anti-rp24 antiserum and rabbit anti-2,4-dinitrophenyl-bovine serum albumin antiserum (Shiba goat, Gunma) [Ishikawa et al. Journal of Immunoassay (J. Immunoas
Say), Vol. 4, p. 209 (1983)].
The p24 antibody and anti-2,4-dinitrophenyl-bovine serum albumin antibody were purified respectively.

[Preparation of thiol group-introduced protein ] Introduction of a thiol group into a protein is carried out by introducing N- via the amino group of the protein.
A known method of reacting succinimidyl-S-acetylmercaptoacetate (Boehringer Mannheim, Germany) [Hashida et al., Journal of Clinical Laboratory Analysis (J. Clin. Lab. An.
al. ), Vol. 8, p. 86 (1994)].

[Preparation of Maleimide Group-Introduced Protein ] A known maleimide group is introduced into a protein by reacting N-succinimidyl-6-maleimidohexanoate (Dojindo Laboratories, Kumamoto) via the amino group of the protein. Method [Hashida et al., Journal of Clinical Laboratory
Analysis, (supra)].

[ Affinity Purification of Antibody ] (1) Preparation of rp24-Rabbit Nonspecific IgG It was prepared by a method of reacting thiol group-introduced rp24 with a maleimide group-introduced rabbit nonspecific IgG. The average number of rp24 molecules bound per IgG molecule was 2.5. (2) Preparation of 2,4-dinitrophenyl-bovine serum albumin To bovine serum albumin introduced with a thiol group, ε was introduced via N-succinimidyl-6-maleimidohexanoate.
The known method of reacting N-2,4-dinitrophenyl-L-lysine [Hashida et al., Journal of Clinical Laboratory Analysis (supra)] 2,4-
Dinitrophenyl-bovine serum albumin was prepared. The average number of 2,4-dinitrophenyl groups bound per molecule of bovine serum albumin was 6. (3) Preparation of Protein-Sepharose 4B Rabbit non-specific IgG-rp24, 2,4-dinitrophenyl-bovine serum albumin, and human IgG were each
Insolubilized in CNBr-activated Sepharose 4B according to the Pharmacia (Uppsala, Sweden) manual. (4) Anti-rp24 antibody (F (ab ') ₂ ), anti-2,4-
Dinitrophenyl-bovine serum albumin antibody (IgG)
And rabbit anti-human IgG γ chain antibody (IgG) (Institute of Medical Biology, Nagoya) are rabbit non-specific IgG-
A known method of adsorbing rp24, 2,4-dinitrophenyl-bovine serum albumin and human IgG insolubilized Sepharose 4B column and then eluting at pH 2.5 [Kono et al., Journal of Biochemistry (J. Bioch).
em. ), 100, 1247 (1986)],
[Hashida et al. Analytical Letters (Anal.Le
tt. ), 16:31 (1983)].

[ Affinity purified anti-2,4-dinitro
Preparation of polystyrene beads insolubilized with phenyl group antibody ] Colored polystyrene beads (diameter 3.2 mm, immunochemical, Okayama) were immersed in buffer A containing 0.1 g / L of affinity-purified rabbit anti-dinitrophenyl-bovine serum albumin antibody at 4 ° C. The mixture was left to stand overnight at 110 ° C. for insolubilization by physical adsorption.

[ Affinity purified anti-human IgG γ chain anti
Body and streptavidin-insoluble polystyrene beads
Preparation of (1) Preparation of Biotinyl-Bovine Serum Albumin A known method of reacting maleimide group-introduced bovine serum albumin with N-biotinyl-2-mercaptoethylamine [Kono et al., Journal of Clinical Laboratory Analysis ( J. Clin. Lab. Ana
l. ), Vol. 2, p. 19 (1988)], biotinyl-bovine serum albumin was prepared. The average number of biotin introduced per molecule of bovine serum albumin is 1
It was three. (2) Preparation of affinity-purified anti-human IgG γ-chain antibody and streptavidin-insoluble polystyrene beads White polystyrene beads (diameter 3.2 mm, immunochemical, Okayama) were affinity-purified anti-human IgG γ-chain antibody 0.1 g / L and biotinyl-bovine serum. The sample was immersed in a buffer solution A containing 0.01 g / L of albumin at the same time and left at 4 ° C. overnight to be insolubilized by physical adsorption. Then, after washing, it was prepared by reacting with a buffer solution A containing 0.1 g / L of streptavidin.

[ 2,4-dinitrophenyl-biotinyl
-Bovine serum albumin-Affinity purified anti-rp24
Preparation of Fab ' ] (1) Preparation of mercaptoacetyl-2,4-dinitrophenyl-bovine serum albumin 2,4-dinitrophenyl-bovine serum albumin with N-
Succinimidyl-S-acetylmercaptoacetate was reacted to introduce a thiol group. The average number of thiol groups per molecule of albumin was 5.2. (2) Preparation of 6-maleimidohexanoyl-biocytin Biocytin (Sigma, Missouri, USA) 7.5 μmo
buffer solution A containing 0.45 ml and N-succinimidyl-
N, N containing 5 μmol of 6-maleimidohexanoate
-Mixture with 0.05 ml of dimethylformamide, 30 ° C
And reacted for 30 minutes to prepare 6-maleimidohexanoyl-biocytin. (3) Preparation of 2,4-dinitrophenyl-biotinyl-bovine serum albumin 0.1 ml of buffer solution B containing 6.6 mg (0.1 μmol) of mercaptoacetyl-2,4-dinitrophenyl-bovine serum albumin and (2). 0.2 ml (2 μmol) of 6-maleimidohexanoyl-biocytin solution was mixed and reacted at 30 ° C. for 30 minutes. After the reaction, 0.05 ml of buffer solution B containing 5 μmol of N-ethylmaleimide was added, and the mixture was reacted at 30 ° C. for 15 minutes. Then, gel filtration purification was performed with buffer A using Sephadex G-50 (Fine, Pharmacia). The average number of biocytin molecules introduced per molecule of albumin was 5.2. (4) Preparation of 6-maleimidohexanoyl-2,4-dinitrophenyl-biotinyl-bovine serum albumin Maleimide using 2,4-dinitrophenyl-biotinyl-bovine serum albumin with N-succinimidyl-6-maleimidohexanoate The group was introduced. Albumin 1
The average number of maleimide groups introduced per molecule is 3.
There were nine. (5) Preparation of affinity-purified anti-rp24Fab 'A known method of reducing affinity-purified anti-rp24F (ab') ₂ with 2-mercaptoethylamine [Ishikawa et al.
Journal of Immunoassay (supra)]
p24Fab 'was prepared. (6) 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity purified anti-rp24 Fab '
Preparation of affinity purified anti-rp24 Fab ′ 0.61 mg
Buffer B containing (13 nmol) 0.6 ml and 6-maleimidohexanoyl-2,4-dinitrophenyl-biotinyl-bovine serum albumin 0.17 mg (2.6 nm
buffer solution B (0.1 ml) and mixed at 4 ° C. for 20 minutes.
Allowed to react for hours. After the reaction, 0.01 ml of buffer solution B containing 1 μmol of 2-mercaptoethylamine was added, and 3
The mixture was reacted at 0 ° C. for 15 minutes, and 0.02 ml of buffer solution B containing 2 μmol of N-ethylmaleimide was further added,
The reaction was carried out at 15 ° C for 15 minutes. Then, the reaction solution was added to Ultrogel A.
It was applied to cA34 (IBF Biotechnics, France), eluted with 0.01M sodium phosphate buffer containing 0.1M sodium chloride, pH 7.0, and subjected to gel filtration purification. The average number of anti-rp24 Fab 'bound per molecule of albumin was 2.0 molecules.

[ Β-D-galactosidase-affinite
Preparation of purified anti-rp24 Fab ′ ] β-derived from Escherichia coli
A maleimide group was introduced into D-galactosidase (Boehringer Mannheim, Germany) using N, N-o-phenylenedimaleimide, and affinity purified rabbit anti-r
It was prepared by a known method of reacting with p24Fab ′ [Ishikawa et al., Journal of Immunoassay, (supra)]. The average number of affinity-purified anti-rp24 Fab ′ bound per molecule of β-D-galactosidase is
It was 1.5 molecules.

[ 2,4-Dinitrophenyl-bovine serum
Preparation of Lubumin-rp17] 2,4-Dinitrophenyl-bovine serum albumin introduced with a maleimide group was reacted with rp17 introduced with a thiol group to prepare 2,4-dinitrophenyl-bovine serum albumin-rp17.
The average number of rp17 bound per molecule of albumin was 2.8 molecules.

[ 2,4-dinitrophenyl-bovine serum
Preparation of Lubumin-rRT] Introduced Maleimide Group 2,
4-Dinitrophenyl-bovine serum albumin and thiol group-introduced rRT were reacted to prepare 2,4-dinitrophenyl-bovine serum albumin-rRT. The average number of rRT bound per molecule of albumin was 1.9.

[Of β-D-galactosidase-rp17
Preparation] β-D-galactosidase-rp17 was prepared by reacting β-D-galactosidase introduced with a maleimide group with rp17 introduced with a thiol group, using N, N-o-phenylenedimaleimide. The average number of rp17 bound per molecule of β-D-galactosidase is 2.
There were 5 molecules.

[ Adjustment of β-D-galactosidase-rRT
Ltd.] (1) Preparation of maleimide-beta-D-galactosidase beta-D-galactosidase 2.5 mg (4.6Nmo
0.01 ml of N, N-dimethylformamide containing 400 nmol of 4,4′-dithiodipyridine (Nacalai Tesque, Kyoto) was added to 0.19 ml of buffer A containing 1) and incubated at 30 ° C. for 15 minutes. After gel filtration purification with Sephadex G-50 (Pharmacia), a maleimide group was introduced with N-succinimidyl-6-maleimidohexanoate. The average number of maleimide groups introduced per molecule of β-D-galactosidase was 1.5. (2) Preparation of β-D-galactosidase-rRT β-D-galactosidase-rRT was prepared by reacting maleimide-β-D-galactosidase with rRT having a thiol group introduced therein. The average number of bound rRTs per molecule of β-D-galactosidase was 1.5.

[ HIV-1 Positive Converted Serum Panel ] HIV
-1 Positive Converted Serum Panels include North American Biologicals (Florida, USA) and Boston.
Purchased from Biomedica (Massachusetts, USA).

[ Activity measurement of β-D-galactosidase ]
The β-D-galactosidase was measured by a known method using 4-methylumbelliferylgalactoside as a substrate [Ishikawa et al., Journal of Immunoassay, (supra)] to produce 4-methylumbelliferone. Was measured with a spectrofluorometer (RF-510, Shimadzu Corporation, Kyoto). The fluorescence value was corrected by setting the fluorescence intensity of 1 × 10 ⁻⁸ M 4-methylumbelliferone to 100.

[ P in serum sample by immunocomplex transfer method ]
Simultaneous measurement of 24 antigens, anti-p17 antibody, and anti-RT antibody ]
0.01 ml of serum sample was added to 0.4 M sodium chloride, 10
0 fmol of anti-p24 Fab 'conjugate (2,4
-Dinitrophenyl-biotinyl-bovine serum albumin-affinity purified anti-p24Fab 'and β-D-galactosidase-affinity purified anti-p24Fa
b ′), 30 fmol of rp17, rRT conjugate (2,4-dinitrophenyl-bovine serum albumin-
rp17, -rRT and β-D-galactosidase-
rp17, -rRT), 0.1 mg rabbit non-specific F
(Ab ′) ₂ and 0.05 mg of inactivated β-D-galactosidase (mutein, Boehringer-Mayheim)
Two affinity-purified anti-2,4-dinitrophenyl-bovine serum albumin antibody-insoluble colored polystyrene beads were added to a solution mixed with 0.14 ml of a buffer solution C containing C. and incubated overnight at room temperature. After the reaction, the reaction solution was removed, and the polystyrene beads were washed twice with 2 ml of buffer C containing 0.1M sodium chloride. Subsequently, the colored polystyrene beads were 0.1M sodium chloride, 1 mM.
Incubation with 0.15 ml of buffer C containing εN-2,4-dinitrophenyl-L-lysine was performed with streptavidin and affinity-purified anti-human IgG γ chain antibody-insolubilized white polystyrene beads for 3 hours at room temperature. After the reaction, the white polystyrene beads were washed by the same method as described above, and the activity of the bound β-D-galactosidase was measured at 30 ° C for 2.5 hours. The results are shown in D of FIGS. 4 and 5 by the cutoff index and in A of FIG. 6 by the relative fluorescence intensity.

Comparative Example 1 In this comparative example, HIV-1 p was determined by the immune complex transfer method.
Twenty-four antigens were detected. The details of the purification and preparation of each substance are the same as in Experimental Example 1 except for the following. [ Preparation of streptavidin-insoluble polystyrene beads
Ltd.] white polystyrene beads (diameter 3.2 mm, immuno Chemical, Okayama) and biotinyl - in soaked 4 ° C. in buffer A containing bovine serum albumin 0.1 g / L to stand overnight insolubilized by physical adsorption . Subsequently, after washing, it was prepared by reacting with a buffer solution C containing 0.1 g / L of streptavidin.

[ Measurement of p24 Antigen in Serum Sample] 0.01 ml of serum sample was added with 0.4 M sodium chloride, 1
00 fmol 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity purified anti-p24Fa
b ′ and β-D-galactosidase-affinity purified anti-p24 Fab ′, 0.1 mg of rabbit non-specific F (a
_Two affinity-purified anti-2,4-dinitrophenyl-bovine serum albumin antibody-insoluble colored polystyrene beads were added to a solution mixed with 0.14 ml of buffer solution C containing b ′) ₂ and incubated at room temperature for 4 hours. After the reaction,
The reaction solution was removed, and the polystyrene beads were washed twice with 2 ml of buffer C containing 0.1 M sodium chloride. Subsequently, the colored polystyrene beads were incubated with streptavidin-insoluble white polystyrene beads in 0.15 ml of buffer C containing 0.1 M sodium chloride, 1 mM εN-2,4-dinitrophenyl-L-lysine for 3 hours at room temperature. After the reaction, the white polystyrene beads were washed by the same method as described above, and the activity of the bound β-D-galactosidase was measured at 30 ° C for 2.5 hours. The results are shown in D of FIGS. 4 and 5 by the cutoff index.

Comparative Example 2 In this comparative example, HIV-1 anti-p24 antibody, anti-p17 antibody and anti-RT antibody were detected by the immunocomplex transfer method. The method for purifying and preparing each substance is the same as in Experimental Example 1 except for the following. For details on each experimental method, see [Hashida et al., Journal of Clinical
Laboratory Analysis (J. Clin. Lab. A
nal. ), Vol. 7, p. 353 (1993) and vol. 8, p. 86 (1994)], [Hashinaka et al., Journal of Clinical Microbiology (J. Cl).
in. Microbiol. ), 32, 819 (1994)]. [ Affinity purified anti-human IgG γ chain antibody insolubilized police
Preparation of Tylene Beads] White polystyrene beads (diameter 3.2 mm, immunochemical, Okayama) were immersed in buffer A containing affinity-purified anti-human IgG γ chain antibody 0.1 g / L and allowed to stand overnight at 4 ° C. to physically It became insoluble by adsorption.

[ 2,4-dinitrophenyl-bovine serum
Preparation of Lubumin-rp24] 2,4-Dinitrophenyl-bovine serum albumin-rp24 was prepared by reacting maleimide group-introduced 2,4-dinitrophenyl-bovine serum albumin with thiol-introduced rp24.
The average number of rp24 bound per molecule of albumin was 2.5.

[Of β-D-galactosidase-rp24
Preparation] β-D-galactosidase-rp24 was prepared by reacting β-D-galactosidase in which a maleimide group was introduced with rp24 in which a thiol group was introduced, using N, N-o-phenylenedimaleimide. The average number of rp24 bound per molecule of β-D-galactosidase is 2.
It was one molecule.

[ Anti-antigens in serum samples (p24, p
17, RT) Measurement of antibody ] 0.01 ml of a serum sample was added to 0.
4M sodium chloride, 100 fmol of each antigen 2,4
-Dinitrophenyl and β-D-galactosidase conjugate and 0.05 mg inactivated β-D-galactosidase (mutein, Boehringer-Mayheim)
Two affinity-purified anti-2,4-dinitrophenyl-bovine serum albumin antibody-insoluble colored polystyrene beads were added to a solution mixed with 0.14 ml of a buffer solution C containing C. and incubated overnight at room temperature. After the reaction, the reaction solution was removed, and the colored polystyrene beads were washed twice with 2 ml of buffer C containing 0.1 M sodium chloride. continue,
Colored polystyrene beads are 0.1M sodium chloride, 1
Incubation was carried out for 3 hours at room temperature with affinity-purified anti-human IgG γ chain antibody-insoluble white polystyrene beads in 0.15 ml of buffer C containing mMεN-2,4-dinitrophenyl-L-lysine. After the reaction, the white polystyrene beads were washed in the same manner as above, and bound β
The activity of -D-galactosidase was measured at 30 ° C for 2.5 hours. The result is shown in Fig. 4 by cut-off index.
It is shown in C of 5.

Comparative Example 3 In this comparative example, antibodies were detected by various existing commercially available HIV-1 antibody detection diagnostic kits. The kit used is
"Therodia-HIV" (Fujirebio, Tokyo), HTL
V-III ・ EIA "Abbott" (Abbott Lab)
oratories, Illinois, USA, Western Blot Kit "HIV-Ortho" (Ortho D
3 kits from Diagnostic Systems (New Jersey, USA) were all measured according to the operating instructions of the kit. The measurement value of each kit is "Cerodia-HIV" is the maximum sample dilution rate showing an aggregation image,
HTLV-III-EIA "Abbott" is a cutoff index, and "HIV-Ortho" is a presence or absence (+,-) of reaction to each band. The results are shown in FIG.
Shown in B.

Experimental Example 2 In this experiment, β-D- which was bound to the affinity-purified anti-2,4-dinitrophenyl-bovine serum albumin antibody insolubilized colored polystyrene beads before being subjected to complex transfer in Experimental Example 1 was bound. Galactosidase activity was measured. The purification / preparation method for each substance is the same as in Experimental Example 1. The results are shown in B of FIG.

[0043]

The following three points can be confirmed by the above-mentioned Experimental Examples 1 and 2 and Comparative Examples 1 to 3. (1) Effect of Simultaneous Measurement of Antigen and Antibody by Immune Complex Transfer Method Antibody measurement by the immune complex transfer method can be diagnosed about 1 week earlier than existing antibody measurement kits. On the other hand, the antigen measurement by the immunocomplex transfer method can be diagnosed about 2 weeks earlier than existing antibody measurement kits, but the measured value becomes lower as time passes after infection. On the other hand, in the simultaneous assay method for antigen and antibody, the respective detection powers are added together, and it is about 2 times more than the antibody detection by the existing antibody measurement kit.
It was confirmed that a high signal was always obtained weekly and even after antibody detection. (2) Usefulness of anti-p17 antibody measurement in early detection of HIV-1 infection Conventionally, anti-p17 antibody was not considered as a good marker, but the p17 antigen was not directly adsorbed on a solid phase, but rather a liquid. HIV-1 by reacting in phase
It was found that among the antibodies against various antigens, the antibody against p17 rapidly rose to a high level after the infection of E. coli, confirming the usefulness of the p17 antibody measurement in early detection in a liquid phase capture system. (3) Effect of conventional sandwich assay In the combination system of p24 antigen, anti-p17 antibody, and anti-RT antibody, the state of being trapped on the first solid phase before the transfer is not necessarily performed in the immune complex transfer method. It was confirmed that a sufficient effect can be obtained even if the measurement is performed with.

[0044]

[Brief description of drawings]

FIG. 1 is a schematic view of one pattern of antigen measurement by the immune complex transfer method.

FIG. 2 is a schematic view of one pattern of antibody measurement by the immunocomplex transfer method.

FIG. 3 is a schematic view of one pattern of an antigen / antibody simultaneous measurement method by an immune complex transfer method.

FIG. 4 shows Western blot kit (A), existing agglutination kit (B), and ELISA kit (for the samples collected after a lapse of a certain number of days after the start of blood sampling, using four types of HIV-1 post-infection positive conversion serum panels. B), or the result of p24 antigen (D), anti-p24 antibody (C), anti-p17 antibody (C), anti-RT antibody (C) alone measurement by the immune complex transfer method and the p24 antigen by the immune complex transfer method, Anti p1
It is the figure which compared the result of simultaneous measurement (D) of 7 antibody and anti-RT antibody. The horizontal axis represents the number of days elapsed since the start of blood sampling, and the vertical axis represents the corrected relative value of each measurement signal value when the cutoff value obtained by the negative sample measurement in each measurement method is 1.
However, the results of Western blotting were represented by + and −, and the results of the agglutination method were represented by the maximum dilution ratio showing aggregation.

FIG. 5 is a view similar to FIG.

FIG. 6 shows a result of simultaneous measurement of p24 antigen, anti-p17 antibody and anti-RT antibody by an immune complex transfer method (A) and a result of measurement at the stage of being captured by the first solid phase in a certain serum panel ( It is a comparison figure of B). The horizontal axis is the same as in FIG. 4, and the vertical axis is the fluorescence intensity showing the β-D-galactosidase activity bound to the solid phase. The dotted line shows the cutoff value.

Claims (6)

[Claims] 1. A method for detecting a pathogen infection by simultaneously measuring "at least one antigen of a pathogen" and "at least one antibody against different antigens of the same pathogen", which comprises the following steps (A), ( B), (C)
And the method (D). Step (A): The test liquid contains the following components (1), (2) and (3)
And at least one of (4) above to form an immune complex containing the antigen to be measured and / or an immune complex containing the antibody to be measured. (1) A component modified with a functional group that specifically binds to an antigen to be measured of a pathogen (2) A labeled component that specifically binds to an antigen to be measured (3) Functional group Same pathogen modified by (1)
The component that specifically binds to the antibody to be measured against an antigen different from the antigen to be measured (4) The labeled component that specifically binds to the antibody to be measured Step (B): Step ( The immune complex formed in A)
The step of capturing the component that specifically reacts with the functional group in (1) and (3) on the insolubilized first solid phase. Step (C): The immune complex is released from the solid phase, and the immune complex is captured on the second solid phase in which a component that specifically reacts with a site in the immune complex different from the functional group is insolubilized. The process of doing. Step (D): measuring the immune complex captured on the second solid phase. 2. The method according to claim 1, wherein step (A) and step (B) are performed simultaneously. 3. The pathogen is human immunodeficiency virus-1 (H
IV-1), The method according to claim 1 or 2. 4. The method according to claim 3, wherein the antigen to be measured is p24, and the antibodies to be measured are anti-reverse transcriptase (RT) antibody and anti-p17 antibody. 5. HI by simultaneously measuring p24 antigen, anti-reverse transcriptase (RT) antibody and anti-p17 antibody.
A method for detecting V-1 infection, comprising the following steps (A), (B) and (C). Step (A): The test liquid contains the following components (1), (2) and (3)
And (4) together to form an immune complex containing the p24 antigen and / or an anti-reverse transcriptase (RT) antibody and / or an anti-p17 antibody. (1) Functional group-modified component that specifically binds to p24 antigen (2) Labeled component that specifically binds to p24 antigen (3) Functional group-modified anti-antibody Component that specifically binds to reverse transcriptase (RT) antibody and component that specifically binds to anti-p17 antibody modified with functional group (4) Labeled anti-reverse transcriptase (RT) Component that specifically binds to antibody and labeled component that specifically binds to anti-p17 antibody Step (B): The immune complex formed in Step (A) is bound to the functional group. And the step of capturing on the insolubilized solid phase the components that specifically react with each other. Step (C): measuring the immune complex captured on the solid phase. 6. The method according to claim 5, wherein step (A) and step (B) are performed simultaneously.