JPH0434363A - Detection of antibody to microorganism - Google Patents

Abstract

PURPOSE:To enhance the specificity in the detection of the antibody to an objective microorganism by preliminarily reacting an antibody with the antigen of other microorganism of a relative and removing or reducing the antibody activity of non-specific reactivity. CONSTITUTION:In a method for detecting the antibody titer to the microorganism of a specimen by the antigen-antibody reaction of the surface antigen of a microorganism with the antibody in the specimen being the partener of the reaction thereof, the antibody is reacted with the antigen of the microorganism generating cross reaction of a relative. Subsequently, when the antibody is reacted with the crude surface antigen reagent of an objective microorganism, the specific antibody to the objective microorganism in the specimen receives no effect due to the antibody to the microorganism capable of generating cross reaction of the relative and the specificity in the detection of antibody can be simply enhanced using a crude microorganism antigen as a reagent without purifying a specific antigen as compared with a non-treatment method.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for detecting specific antibodies against microorganisms.
More specifically, blood from patients infected with microorganisms.

This invention relates to a method for specific detection of antibodies appearing in urine and other body fluids.

[Conventional technology]

BACKGROUND ART Conventionally, methods have been known in which antibodies appearing in blood, urine, and other body fluids of patients infected with microorganisms are detected by immunological means based on antigen-antibody reactions using microorganisms and their membrane components as antigen reagents. Radioimmunoassay (RIA) is the main immunological detection method.

Enzyme-linked immunosorbent assay (ELISA), Fluorescence immunoassay (FI)
A) Latex immunoturbidimetry 1 indirect fluorescent antibody method, complement fixation reaction, hemagglutination inhibition reaction, etc. are used. These immunological detection methods are widely used in the field of clinical urine testing.

However, in conventional immunological detection methods, it is often necessary to avoid non-specific reactions other than the reaction with the target antibody to be detected, such as cross-reactions with taxonomically related and similar microorganisms. I couldn't. For such non-specific reactions,
For example, in the case of chlamydia, it is taxonomically classified as a member of order 1 [Chlamydiales].
mydia-1es)), family L [Chlamydiaceae (C
hlamydiaeeae):l.

1 genus [Chlamydia (Chlaweydiagenus)]
)), 3 types [Chlamydia trachomatis species (Chl
amydia tracho-*atis) t Chlamydia psittaci
i) Chlamydia pneumoniae spp.
apneυ■oniae)), as described in Caldwell, H. D., et al.
nfect, Immun, 1981, launch, 1161
-1176 and U.S. Pat. No. 240,223 address the issue of specificity in detecting Chlamydia trachomatis species by cross-reacting with conspecific and heterogeneous Chlamydia infection species. (Ma
protein)
) has been shown to be purified. In addition, the basics and clinical practice of chlamydial infections (Metal Publishing Co., Ltd., Etsuaki Kumamoto, So Hashisuke)
Edited February 20, 1986, 1st edition published, p. 82)
reported that MOMP should be used as an antigen to measure serum antibodies against Thalamydia trachomatis species.

[Problem to be solved by the invention]

However, this method of completely amending specific antigens and turning them into reagents requires a large number of antigens and a lot of labor and time for purification. It was impossible to detect microorganisms for which specific antigens had not been discovered. As a result of various studies aimed at improving these points, the present invention was developed by first reacting an antibody with a closely related cross-reacting microorganism antigen, and then reacting it with a roughly purified surface antigen reagent of the target microorganism. The present inventors have discovered that the specificity of a specific antibody against a target microorganism can be easily improved without being affected by antibodies against closely related microorganisms that can cross-react with each other, and have completed the present invention.

[Means to solve the problem]

That is, the present invention provides a method for detecting the antibody titer against a microorganism in a specimen by an antigen-antibody reaction between the surface antigen of a microorganism and an antibody in the specimen that is a partner of the reaction, in which the antibody is reacted with a closely related microorganism antigen in advance. The present invention relates to a method for detecting antibodies against microorganisms, which is characterized by removing or reducing non-specifically reactive antibody activity and then reacting with the surface antigen of the microorganism of interest.

The present invention will be explained in detail below. In the present invention, "microbial surface antigens" refer to antigens extracted and purified from particles or membranes of bacteria, viruses, etc. that cause all infections that produce antibodies, and generally belong to the following taxonomic families: and groups.

1. Bacteria 1. Micrococticaceae 2. Lactobacillus family 3. Neisseriaceae 4. Enterobacteriaceae 5. Achromobacteraceae 6. Mycobacteriaceae 7. Bacteroideae 8. Corynebacteriaceae 9. Lactobacillus majorae 10. Green! a Mycidae 11. Spirillum family ]2. Other pathogenic bacteria 13. Mycoplasmadaceae 14. Mycobacteriaceae 15. Actinobacteriaceae 16. Spirochetae 17. Rickettsiaceae 18. Chlamydiaceae ■、Virus 1. Box, virus group 2. Herpes virus group 3. Adeno, virus group 4. Baboba, virus group 5. mixo, virus group 6. Fulvic, virus group 7. Leo, virus group 8. Picorna, virus group 9. Rhabdo, virus group 10. Hepatitis virus group 11. Rubella, virus group 12, retro, virus group 1.HTLV-I 2.HTLV-II 3.HTLV-m Each of these families and groups also has a taxonomic genus.

There are species and types. For example, in the case of Chlamydia species, there are three major types (Chlamydia trachomatis species, Chlamydia trachomatis species,
Chlamydia pneumoniae and Chlamydia pneumoniae are known, and each species has different antigenicity and infectivity.

These closely related microorganisms share common antigenicity with each other, which causes cross-reactivity and non-specific reactions.

In the present invention, the "reaction partner" is a specific antibody present in the specimen to be detected, that is, in a body fluid such as blood, serum, or tissue fluid of a host or patient who is thought to be infected with the target microorganism.

In the present invention, "another closely related microbial antigen" refers to a microorganism that is taxonomically related to the microorganism to be used as a reagent for antibody detection, and immunologically has a cross-reacting antigen epitope that exhibits common antigenicity. It is a microbial antigen.

The closely related microbial antigen may be the microorganism itself that has been inactivated to prevent infection, or it may be a membrane component extracted and purified from the microorganism that contains antigenicity. In addition, the closely related microbial antigen is
It can be used with one type of microorganism or multiple microorganisms. The amount of closely related microbial antigen to be used varies depending on the type of antigen and the conditions of the measurement system, such as the antibody content in the sample used for assay. It is necessary to set a sufficient amount.

After reacting a closely related microbial antigen with an antibody against it in the sample and removing or reducing non-specifically reactive antibody activity,
Used as a sample for the detection of specific antibodies 8. As the sample (a sample obtained as a result of the reaction between a closely related microbial antigen and the antibody in the sample against it), the resulting antigen-antibody combination is used as it coexists. However, it is preferable to use a product from which the bound substances have been removed by centrifugation (for example, 3,000×g, 15 minutes). Furthermore, in order to simplify the operation, if necessary, closely related microbial antigens can be physically or chemically immobilized on carrier particles such as latex particles or test tubes (blood collection tubes) using known methods. Antibodies in the specimen against closely related microbial antigens that cause cross-reactivity can also be removed by using a microbial antibody.

Next, the specimen is subjected to an antigen-antibody reaction with a surface antigen reagent of the microorganism of interest.

This antigen-antibody reaction is generally used as an immunological detection method to detect the antibody titer against a microorganism of interest. Highly sensitive radioimmunoassay (RIA), enzyme immunoassay (EL Engineering SA), fluorescence immunoassay (FIA)
), latex immunoturbidimetry, indirect fluorescent antibody method, complement fixation reaction, hemagglutination inhibition reaction, etc. As the immunological detection method and its detailed measurement technique, methods well known to those skilled in the art can be used.

Furthermore, the present invention is of course applicable not only to the above-mentioned detection method.

〔Example〕

Examples of specific antibody titer detection for Chlamydia species that are unaffected by infection antibodies from other species (■ Method for detection of Chlamydia trachomatis species infection antibodies by removing cross-reactivity of Chlamydia shitashi species infection antibodies, and ■ Chlamydia trachomatis species infection antibody titer detection) The present invention is not limited to the following examples.

a) Chlamydia trachomatis elementary body (EB)
The strain of Chlamydia trachomatis sp. is L2/' 43
4/Bu strain (hereinafter abbreviated as L2 strain) was used.

HeLa229 cells were used as infected host cells, and HeLa229 cells were used as infected host cells.
La229 cells were incubated with 10% tallow serum (FCS).
agleMEM with cycloheximide (Cyeloh
Eximide) was added to the culture medium at a concentration of 1 μg/mn, diluted 6 times every 3 days, and subcultured. 1. For normal passage, a 200 mff1 plastic medium bottle (
The culture was carried out by static culture using a culture bottle (culture bottle).

H e La 2291 grown in monolayers in culture bottles
Approximately 2 days after inoculating one cell with the L2 strain, most of the infected HeLa229 cells formed inclusion bodies.

The supernatant of the culture solution was discarded, and the remaining cells were scraped off with a rubber policeman, suspended in 10 mQ of Ranks' BSS, pooled, and sonicated (20 KHz, 60 see;
Nippon Seiki Seisakusho Co., Ltd. US-300).

Here, Chlamydia traphmatis was inactivated by ultraviolet irradiation to prevent infection. This cell suspension was centrifuged (50QXg, 15 minutes, 4°C), and the supernatant was collected as described in Caldwell, H.D., et al., Infect, Imw+un, 1981, 3rd Eng., 1.
Chlamydia traphmatis EB was purified by discontinuous density gradient centrifugation using Reggrain (diadrizoate meglumine and diadrizoate sodium, 76% for injection; Nippon Schering Co., Ltd.) according to the method described in 161-1176.
was purified. That is, the centrifuged supernatant was mixed with lOmM HEPES (N-2-hydr) containing 0.15M NaCQ.
roxysthylpiperazine-N'
-2-ethanesulfonic acid) diluted with 35% (V/V) leggrain, and heated at 45,000Xg at 4°C in an ultracentrifugation sweater (SPR28SA-536 type 9 manufactured by Hitachi Koki Co., Ltd.). Centrifuged for 1 hour. Precipitate with 0.25M sucrose and 5mM
Resuspended in 10 mM sodium phosphate buffer (SPG, pH 7,2) containing L-glutamic acid, collected and resuspended on a Renografin discontinuous density gradient (Renografin 40
% 15mQ, 44% 10mQ, 52%5mQ
) and an ultracentrifuge rotor (SPR28SA-536
Type 9 (manufactured by Hitachi Koki Co., Ltd.) at 4℃ and 45,000Xg
Centrifuged for hours. Rhenographine Discontinuous Density Gradient Concentration 4
The white turbidity located at the interface between 4% and 52% was collected and diluted with 3 volumes of SPG.

The remaining leggrain was removed by repeated centrifugation at 30 v OOOX g for 30 minutes to obtain purified EB of Chlamydia traphmatis.

b) Preparation of Chlamydia Shitashi elementary body (EB) Budgerigar-1 strain (hereinafter abbreviated as Bud strain) was used as the Chlamydia Shitashi bacterial strain. Furthermore, L cells were used as host cells for culture. L cells were treated with 10% tallow serum (
Fe2) 6 every 3 days using EagleMEM
It was diluted twice and subcultured. Normal subculture was carried out by static culture using a 200 mQ plastic medium bottle.

Cells on the 3rd day of culture were used for inoculation of Chlamydia Shitashi, and the cells were collected by centrifugation at 150 x g p S for 3 minutes and centrifugally washed three times with Ranks' BSS. Adsorption is M
Cell concentration was 10'/mQ for 2 hours at 37°C with shaking in warm water. After adsorption, the cells were resuspended in FC5 supplemented medium to a cell concentration of 5 to 7 x 10 s/mQ and cultured at 37°C for 2 days. After culturing, a portion of the infected cells was collected by centrifugation, a smear was prepared, and Giemsa
) It was confirmed by staining that the EBs were sufficiently proliferating.6 Next, the infected cells were disrupted by ultrasonic waves, and Chlamydia citrus EBs were purified by Reggrain density gradient centrifugation. That is, the infected cell suspension was pooled and sonicated (2
0KHz, 60sec; Nippon Seiki Seisakusho Co., Ltd. US-
300), and this suspension was centrifuged at 700×g for 10 minutes to obtain an infected culture supernatant. The supernatant was diluted with 0.15M Na
10 m M HE P E S (N
-2-hydroxyethylpiperazin
e -N'-2-ethanesu-1fonic
76% Leggrain liquid for injection was layered on top of 8 mj2 of a solution prepared to contain 35% Leggrain using ultracentrifugation rotor (SRP2
85A-536 type 2 Hitachi Koki Co., Ltd. at 4℃, 45,
Centrifugation was performed at 000 x g for 1 hour. 0.25M
10mM sodium phosphate buffer (SPG, pH 7,2 containing sucrose and 5mM L-glutamic acid)
) and pooled in a discontinuous density gradient (Regraphine 40% 15mQ, 45% 10
mQ, 52% 5mQ) and heated at 4°C in an ultracentrifuge rotor (SRP28SA-536 type 4 Hitachi Koki Co., Ltd.).

Centrifugation was performed at 45,000×g for 1 hour. The white turbidity located at the interface between 45% and 52% of the rhenografin discontinuous density gradient concentration was collected, diluted with 3 volumes of SPG,
The remaining leg graphin was removed by repeated centrifugation at 0×g for 30 minutes to obtain purified EB.

c) Method for preparing EB immobilized ELISA plate ELI
For immobilization of EB on a microplate (96 wells) using the SA method, EB purified as described above was used as an antigen and Coa.
ting buffer, pH 9.6 (Na,
Co, 1.59 g, NaHCo.

2.93 g, NaN, containing 0.2 g),
The protein concentration of EB prepared to a concentration of 5 μg/mQ was added to each formula on a microtiter plate at 250 μΩ.
The mixture was added in batches and left to stand overnight at 4°C. This EB-fixed plate was treated with PBS-Tween, pH 7.4 (NaCQ 8.Og in distilled water IQ).

KH2PO40,2g, KCQ O,2g, Tv
een200.5mff1. (containing 0.2 g of NaNa) three times, and by adding 100 μΩ of 1% BSA-PBS solution to each formula and leaving it at 4°C overnight.
The non-EB adsorbed area on the plate surface was blocked.

Furthermore, it was washed three times using PBS-Tween, pH 7.4, to prepare an EB-immobilized ELISA plate.

d) Method for removing activity of non-specifically reactive antibodies in specimen In order to detect species-specific antibodies of Chlamydia trachomatis, the activity of antibodies against Chlamydia sitasi species was specifically removed.

As a similar microbial antigen to Chlamydia trachomatis, Chlamydia sitasiBudgerigar-1
Strain EB was used. In addition, as a similar microbial antigen to Chlamydia trachomatis,
Two strains of EB were used. The specimen used and the method for removing the activity of the non-specifically reactive antibody are shown below.

■Atsuko Bessho et al., History of Medicine, 1984, 128°571
The following patient specimens tested for IgG antibodies by the indirect fluorescent antibody method (MFA) described in Japanese Patent No. 572 were used.

(1) Chlamydia trachomatis infected patient serum (C, P) titer; Chlamydia trachomatis x512 Chlamydia trachomatis X4 (2) Chlamydia trachomatis infected patient serum (C, T) titer; Chlamydia trachomatis
X1024 Chlamydia Shitashi ×4 (3) Combined infection patient serum (C, P + C, T) titer;
Chlamydia trachomatis X
1024 Chlamydia trachomatis x 512 (4) Healthy human serum (N, S,) titer; Chlamydia trachomatis
x4Chlamydia x4
100 samples obtained by diluting the patient samples from ) to (4) 100 times
Microbial antigen (EB) similar to μQ (protein content approximately 10 mg)
/mQ) and reacted, incubated at 37°C for 3 hours, and centrifuged (3,000 x g, 15
) The shida supernatant was used as the treated sample. In addition, as an untreated sample (control) for the activity removal treatment of non-specific reactive antibodies, ■
The patient samples (1) to (4) shown in (1) to (4) were similarly diluted 100 times, and 100 μQ of each sample was mixed with 100 μQ of SPG and reacted. , 15 minutes) was used as an untreated specimen.

e) Inspection method and evaluation of Chlamydia species-specific antibodies For the purpose of detecting specific antibodies for Chlamydia trachomatis species and Chlamydia citasi species, the specimen obtained in d) was immobilized on the EB prepared in C) (Chlamydia trachomatis species and ELISA (separately immobilized Chlamydia spp.)
The play plate was evaluated. In the detection operation by ELISA, as is widely known to those skilled in the art, the specimens obtained in step d) are added in duplicate at 100 μΩ each to the plate prepared in step C), and the samples are incubated at room temperature for 2 hours.
Let it stand for a while. After washing three times with PBS-Tween, 100 μΩ of peroxidase-labeled anti-human IgG antibody (manufactured by KPL) was added and allowed to react at room temperature for 2 hours.

After washing three times with PBS-Tween, 40 mg of orthophenylenediamine, 40 μQ of 30% H2O was added to substrate buffer (35 molar of 0.1 M citric acid in 1.0 l of distilled water).
After color development (usually after reacting for 20 to 30 minutes after adding the substrate), add INNaOH or INH2So to stop the reaction. The absorbance at 492 nm was measured within 1 hour after the reaction was stopped using MTP-22 manufactured by Corona Electric Co., Ltd. The measurement results obtained are shown in Tables 2 and 3.

In Table 2, the experiment indicated as MOMP was shown as a control in which a specific antigen highly purified with much effort was made into a reagent. That is, the bacterial cells of Chlamydia trachomatis sp.
t, Immun, 1981, 31, 1161-1
Major outer membrane protein purified according to the method described in 176 (
Major 0uter Membrane Prot
ein) in the same manner as the EB immobilization plate for ELISA.
C)), and performed ELI in the same manner as above, except that the patient sample diluted twice with SPG was used as the sample.
Measured by SA method. The same procedure was carried out for Chlamydia sitasi species.

Table 2 in the margin below Detection table 3 of specific antibodies for Chlamydia trachomatis species
Detection of specific antibodies of Chlamydia sitasi species [Effects of the invention] As is clear from Tables 2 and 3, according to the present invention, when detecting a patient specimen, in advance, antibodies against closely related microorganisms other than the target microorganisms are By reacting antibodies with excess amounts of similar microbial antigens and removing or reducing them, we can easily increase the specificity of antibody detection using crudely purified microbial antigens as reagents without purifying specific antigens compared to untreated methods. There are 8 things to improve.

Claims (1)

[Scope of Claims] 1. In a method for detecting the antibody titer of a specimen against a microorganism by an antigen-antibody reaction between a surface antigen of the microorganism and an antibody in the specimen that is a partner of the reaction, the antibody is prepared in advance by using antibodies closely related to the microorganism. A method for detecting antibodies against microorganisms, which comprises reacting with other microorganism antigens to remove or reduce non-specifically reactive antibody activity, and then reacting with surface antigens of the target microorganism. 2. The antibody detection method according to claim 1, wherein the microorganism is Chlamydia trachomatis, and the other similar microorganism is Chlamydia citasi and/or Chlamydia pneumoniae. 3. The antibody detection method according to claim 1, wherein the microorganism is Chlamydia citasi and the other similar microorganism is Chlamydia trachomatis and/or Chlamydia pneumoniae. 4. The antibody detection method according to claim 1, wherein the microorganism is Chlamydia pneumoniae and the other similar microorganism is Chlamydia trachomatis and/or Chlamydia citasi.