WO1996007910A1

WO1996007910A1 - Diagnostic drug for chlamydia infection

Info

Publication number: WO1996007910A1
Application number: PCT/JP1995/001755
Authority: WO
Inventors: Kohsuke Kino; Toshifumi Oshima
Original assignee: Meiji Milk Products Company Limited
Priority date: 1994-09-02
Filing date: 1995-09-04
Publication date: 1996-03-14
Also published as: AU3355095A

Abstract

A diagnostic drug for chlamydia infection comprising a peptide containing a B cell epitope specific for each of Chlamydia trachomatis and C. pneumoniae or an antibody against the peptide. As this drug is species-specific, it permits the infection with C. trachomatis or C. pneumoniae, which has been difficult of definite diagnosis, to be diagnosed more accurately and more easily.

Description

Specification

Title of invention

Diagnostic agent for Chlamydia infection

Industrial applications

The present invention relates to a diagnostic agent for Chlamydia infection. More specifically, the present invention relates to a diagnostic agent that is effective for the selective diagnosis of patients infected with Chla mydia trachomatis or Chlamydia pneumoniae.

Conventional technology

In general, chlamydia infects humans and causes various diseases. Chlamydia is a wounded intracellular parasitic bacterium that has a unique 增 ring with morphological changes. When it is outside the host cell, it is resistant to the outside world and is infectious but has no metabolic activity.It has a form of elementary body (EB) with a diameter of 30 O nm and enters the host cell. It is non-infectious but metabolically active, with a morphological change to a fragile reticulate body (RB) with a diameter of about 450-: L50O nm. RB repeats two-way growth in host cells and assembles into cells to form inclusion bodies. Then, the RB changes its form from RB to EB via an intermediate form (intermediate form), and when the host cell is broken down, EB is released to the outside world.

Currently, four species of chlamydia are known, and among them, those that infect humans and cause disease are «C. trachomatis, C. pneumoniae and C« psit taci i ?.

C. trachomatis is known as a pathogen that causes granulomatous conjunctivitis (trachoma), sexually transmitted diseases (STDs), and infertility. STDs cause urethritis, Sugao-maruitis, and prostatitis in men, but only in women It has been reported that childhood appendicitis (tubulitis), bone, and peritonitis may develop, causing infertility. In addition, it is also known that vertical transmission to newborns via the birth canal during delivery may cause the newborns to develop conjunctivitis or pneumonia.

The incidence of trachoma is more common in developing countries, and according to a WHO study in 1981, 500 million people in developing countries suffer from trachoma per year, of which 10 million are due to this. I'm blind. On the other hand, STDs caused by trachomatis are common in developed countries.For example, in the United States, 400,000 women a year are at risk of infertility due to peritonitis due to C. trachomatis infection and the risk of infertility. C. trachomatis has been called as one of the STDs to prevent its infection from a public health standpoint. In Japan, trachoma was widespread for some time after World War II as an infectious disease of C. trach omatis, but recently STD has been added as in the United States, and early detection of infection has become an important issue. ing.

C. pneumoniae has recently been identified as a species of chlamydia22, and subsequent studies have shown that antibody prevalence is high (several tens of percent) even in healthy people, indicating that it is a common infectious microorganism in humans. found. The pathogenicity of acute respiratory harbor infections has been reported, and it is reported that more than 10% of pneumonia cases are pathogenic microorganisms. Under such circumstances, as in C. trachomatis, an early diagnosis method for infection is required.

For the diagnosis of Chlamydia infection, an antigen assay for detecting Chlamydia cells or their constituents using a specimen (scratch specimen) or urine collected from the affected area by abrasion, and a serum sample using Chlamydia antibodies. There is an antibody measurement method to detect.

Antigen testing methods include the isolation culture method, fluorescent antibody method, enzyme antibody method, DNA Diagnostic methods have been developed. In the separate culture method, the scraped sample is inoculated into He229 or McCoy cells, cultured, fixed and stained, and the presence or absence of Chlamydia inclusions is determined under a microscope. This is the most reliable diagnostic method,

(More than 2 days), it is difficult to perform in a general laboratory because of the skill and special equipment. In the fluorescent antibody method, a scraped sample is smeared on a slide glass, stained with an anti-chlamydia monoclonal antibody labeled with fluorescein isothiocynate (FITC), and the microbial subsp. Detect the body. This method can detect the presence of even a single EB, but it is not common because it requires a mature itt to make a decision. In the enzymatic antibody method, a sample is reacted with an anti-chlamydia antibody labeled with an enzyme (/ 3-D-galactosidase or peroxidase), and 4 MU G (4-methylujn belliferyl-β-D-galactopyranoside) is labeled with the labeled enzyme. Or ΤΜ Β (3, 3 ', 5, o-tetramethylbenzidine). This method is fast and can process large numbers of samples. However, the antibodies used in the enzymatic antibody method are polyclonal antibodies against Chlamydia cells themselves or monoclonal antibodies against lipopolysaccharide (LPS), one of the components of the EB outer membrane. Since it is a specific antibody, it has the disadvantage of causing cross-reaction between Chlamydia species. In DNA diagnostics, chlamydia ribosomal RNA in a sample or plasmid DNA universal to chlamydia and a DNA probe complementary thereto are hybridized, and the probe that contributes to hybrid formation is detected by chemiluminescence or color development. Things.

On the other hand, antibody measurement has been developed using the Micro-IF "method (micro-immuno 丄 uorescence technology), the MFA method microplate immunofluorescence antiboay technique), the enzyme immunoassay, and the Western blotting method. Have been. In the micro-IF method, purified EB of 15 serotypes of C. trachomatis is used as an antigen, and measurement is performed by an indirect fluorescent antibody method. This method allows the determination of the serotype of C. trachomatis to be purified, but is not common due to the need for special equipment and techniques. In the MFA method, an inclusion body is used as an antigen, and measurement is performed by an indirect fluorescent antibody method. Although this method does not require purified EB, the inclusion bodies do not have species specificity, so cross-reaction between species occurs, and special equipment and techniques are required as in the case of the Micro-IF method. The enzyme immunoassay is performed using chlamydia-infected cells, purified EB, and EB outer membrane protein as antigens. This method is quick and simple, and can process a large number of samples. However, since none of the antigens used is species-specific, there is a problem that cross-reaction may occur between Chlamydia species.ゥ In the X stamp lotting method, EBs are subjected to electrophoresis, and then the electrophoretic proteins are transferred to a membrane. A sample serum is reacted with the membrane, reacted with an enzyme-labeled anti-human immunoglobulin antibody, and then colored with TMB or the like, and the presence or absence of a band having high species-specific chlamydia is determined. Although this method is highly confusing and allows for fairly species-specific diagnosis, it requires a messy operation and is difficult to perform in a general laboratory. Nishin super that the invention tries to solve

As mentioned above, the Chlamydia infection diagnosis method other than the isolation culture method and the DNA diagnosis method in the antigen test method uses a Chlamydia specific antibody or antigen and binds it to the Chlamydia specific antigen or antibody in the sample, respectively. It is a fundamental principle. However, any of the conventionally used antigens or antibodies have cross-reactivity between species due to insufficient species specificity. In particular, when diagnosing C. trachomatis infection, false positives are likely to occur because pneumoniae is universally transmitted to humans. Problem. Therefore, the development of a new diagnostic drug that solves this cross-reactivity has been required.

Therefore, an object of the present invention is to provide a species-specific diagnostic agent for Chlamydia ^: to increase the antigen specificity of each of C. trachomatis and C, pneumoniae, or to increase the antigen specificity of an antibody. To provide a diagnostic for Chlamydia infection.

Means for solving the problem

In order to solve the above problem, a B cell epitope specific for Chlamydia is searched for, and a diagnostic drug for Chlamydia infectious disease containing a peptide containing the same or an antibody against the peptide is contained. The present inventors thought that it would suffice to produce a diagnostic agent for Chlamydia infection.

It is said that the immunogenicity of C trachomatis is mainly in the major outer membrane protein (MOMP, molecular weight of about 40 KD), which is the most abundant component in the EB outer membrane component. From the comparison of the amino acid sequence of MOMP of trachomatis, it was found that MOMP contains four regions called VDs (Variable domains), which have many mutations, especially between serotypes (the four VDs are VDI and VD V, VD ffl and VD IV), of which VDI, VDΠ and VD IV may be immunogenic specific to the serotype or species of trachomatis. Known for the mouse system. Yuan et al. Determined the amino acid sequence of VD for 15 serotypes of C. trachomatis (Yuan Y, et al., Infect. I. un., 57, 1040-1049, 1989). In an experiment using human sera, Jones and colleagues synthesized partial peptides of VDI and VDΠ for six serotypes of C. trachomatis and examined their reactivity with sera of patients with C. trachomatis infection

(Jones H. Μ · et al., J. Infect. Dis., 166, 915-919, 1992). Jon According to es et al., there was no reactivity in the VD I region, but in many patients the VDI region was responsive. On the other hand, the gene of pneumoniae has been cloned by Melgosa et al. And the amino acid sequence has been determined.We reported that pneumoniae, like C. trac homatis, has four regions corresponding to VD. (Melgosa MP, et al., Infect. Irninun., 59, 2195-2199, 1991). Melgosa et al. Compared the amino acid sequences with C, pneumoniae C. trachomatis and found that the homology of both sequences was about 73% (compared to serotype L2 of trachomatis). Reveals little homology.

Based on the above findings, the present inventors focused on VD as a site having low amino acid sequence homology between trachomatis and C. pneumoniae, and furthermore, VD I to VD IV and VD I, focusing on VD as a site involved in antigenicity. Synthesizes peptides with a common structure to 15 serotypes of C. trachomatis present in the vicinity including the N-terminus and with low homology to C. pneumoniae (hereinafter referred to as VDI near peptides) Then, the reactivity with Chlamydia-infected patients' sera was compared.

As a result, the peptides containing trachomatis species-specific B-cell epitopes include peptides near the VD I of C. trachomatis and VD IV of C. tracho matis, and species-specific C. pneumoniae. B-cell episode! ^ As a peptide containing one peptide, VD IV of C. pneumoniae was found, respectively. That is, as the peptide containing the species-specific B cell epitope of C. trachomatis !, a peptide having an amino acid sequence of the following (1) to (15):

(1) Ser-Ala-Glu- Thr- Ile-Phe- Asp-Val- Thr- Thr-Leu- Asn-Pro- Thr-Ile-Ala-Gly-Ala-Gly-Asp-Val-Lys-Thr-Ser -Ala-Glu-Gly-Gln-Leu-Gly, GluAla Iler ThrT¾ILe-l—i

yyyG AllLGvasl ThralvalserserAlGluG AaaAsnluLel --- l-, l--l --- t

"

yyyyGGGGlGvLAaAsnAlalullnLeuGGllualsl AllAla: lea ------ l .l ------ () Aplvl As Tr Tllr Tllr LeusnΓΟ T Al Ile! e Ale Tllr4 L-eurl-illΙ * l

yyypyGGlGGllVLAlaSerAlaulnLeuGAalsGAlalse Alal: l --------------- PhThrLeuAsnproThrheASTrThrAlaIlepAlaThr ser ------------- yyyyypyG GaGluGlGlnLeulvLslAlGlAsalGAlaleAlal I --------- l --- P:) hLpASThrThrTreuAsnroThrAlIpheThrale sAla2er ------------- -Ile-Ala-Gly-Lys-Gly-Thr-Val-Val-Ala-Ser-Gly-Ser-Glu-Asn-Asp-Leu -Ala,

(13) Leu-Ala-Glu-Ala-Ile-Leu-Asp-Val-Thr-Thr- and eu-Asn-Pro-Thr-Ile-Thr-Gly-Lys-Gly-Ala-Val-Val-Ser- Ser-Gly-Ser-Asp-Asn-Glu-Leu -Ala,

(14) shi eu-Ala-Glu-Ala-Va] ~ Leu-Asp-Val-Thr-Thr-Leu-Asn-Pro-Thr

-lie-Ala-Gly-Lys-Gly-Ser-Val-Val-Ala-Ser-Gly-Ser-Glu-Asn-Glu-Leu -Ala,

(15) Val-Leu-Gln-Thr-Asp-Val-Asn-Lys-Glu-ie-Gln,

The peptide containing the species-specific B cell epitope of C pneumoniae is a peptide having the following amino acid sequence of (16):

(ID Leu—Pro-Thr—Ala-Val—Leu—Asn—Leu—Thr ^ ia— rp-Asn-Pro-ber eu— then en— ϋ 丄 y— Asn— Ala— Thr— Ala— and eu — Ser— Thr— Thr— Asp— Ser— Phe— Ser.

Here, the peptides (1) to (14) correspond to the C. trachomatis strains B, D, L, L1 wL2, r rest, Crtt, A¾ :, H, I, J, respectively. The VD IV region of the strain, K strain and L3 strain, (15) the peptide near the VDI of trachomatis, and (16) the VD IV region of the pneumoniae. The sequence of the peptide in the VDIV region of the C. trachomatis Ba strain is the same as that of the B strain.

The present invention relates to C. trachomatis, in which the peptide represented by any of the amino acid sequences (1) to (15) and the peptide represented by the amino acid sequence or a peptide immunologically equivalent to the peptide represented by the amino acid sequence are used. And any of the peptides represented by the amino acid sequences of (1) to (15) and peptides that are immunologically equivalent to the peptides represented by the amino acid sequences. The deviated ones are linked together, and for C. pneumoniae,

A peptide represented by the amino acid sequence of (16) and / or a peptide immunologically equivalent to the peptide represented by the amino acid sequence; and a peptide represented by the amino acid sequence of (16) and / or Peptides that are immunologically equivalent to the indicated peptide are linked together (hereinafter sometimes collectively referred to as a specific antigen), or an antibody against the specific antigen (hereinafter sometimes referred to as a specific antibody). It is used as a diagnostic for Chlamydia infection.

In addition, the “Γimmunologically equivalent peptide” refers to the peptide represented by the amino acid sequence of (1) to (16) above, wherein one or more amino acid residues are deleted and / or substituted and Z Or a peptide having an inserted and / or added amino acid sequence, which reacts immunologically equivalent to the original unmodified peptide to the serum of a patient with Chlamydia trachomatis infection positive or serum of a patient with Chlamydia pneumoniae infection positive Refers to those indicated, especially those with a cut-off index of 1.50 or more as measured by enzyme-linked immunosorbent assay for infection-positive patient sera.

The 15 serotypes of C. trachomatis were classified into groups B (B, Ba, D, E, L1, L2) and C (C A, H, I, J, K, and L3 strains) and an intermediate group (F and G strains). Therefore, as shown in Example 3, by selecting peptides in one or more strains of the VD IV region from each of these groups and using a mixture of these as an antigen, the detection sensitivity of the antibody test can be increased. Can be increased.

For this reason, a combination of peptides in the VD IV region of one or more strains of each of the group B, group C and intermediate group is particularly preferably used in the present invention. The peptide in the VDI region, which Jones et al. Considered to be reactive with the serum of a patient infected with C. trachomatis, was not reactive in the experiments of the present inventors. This is because non-specific reactions were judged to be positive because the patients were white and Japanese, or because they did not have a negative control (reactivity with healthy human serum) experimental system. Possible causes are possible, but unknown.

In any case, since Jones et al. Have not studied the VD IV region at all, the present inventors have pointed out that the fact that peptides close to VDI and the VD IV region are common B-cell epitopes for antibodies to C. trachomatis patients suggests that the present inventors. This is the first finding.

In addition, for Chlamydia species other than C. trachomatis and C. pneumoniae (eg, C. psittaci), species-specific B cell epitopes are searched for from among the EB outer membrane components of the species. It is possible to produce a diagnostic agent for Chlamydia infection comprising a peptide containing the same, or a diagnostic agent for Chlamydia infection comprising an antibody against the peptide. The production of a diagnostic agent for Chlamydia infectious disease using the peptides (1) to (16) is described below.

The antigens used in the antibody test are as follows. Diagnostic agents for trachomatis infection include peptides represented by any one of the amino acid sequences of (1) to (15) or a combination of these peptides, and species-specific B-cell epitopes of Z or C. trachomatis. A partial peptide of the peptide represented by any one of the amino acid sequences (1) to (15), including a partial peptide, or a combination of these partial peptides is used. As the diagnostic agent for C. pneumoniae infection, use is made of the peptide represented by the amino acid sequence of (16) or a partial peptide thereof containing a species-specific B cell epitope of pneumoniae. The antibodies used in the antigen test are as follows. The diagnostic agent for C. trachomatis infectious disease includes a peptide having the amino acid sequence of any one of (1) to (15) or a portion having a species-specific B cell epitope of C. trachomatis (1). ) To (15), a peptide obtained by immunizing a partial peptide of the peptide represented by any one of the amino acid sequences of (1) to (2) with rabbits, guinea pigs, rats, mice, goats, sheep, and the like; Use a combination. For the diagnosis of C. pneumoniae infection, the peptide represented by the amino acid sequence of (16) or the partial peptide containing the portion having the species-specific B-cell epitope of pneumoniae can be obtained by using a heron, guinea pig, Use a peptide antibody obtained by immunizing a mouse, goat, sheep or the like. When these antibodies are obtained, the peptides (1) to (16) or their partial peptides are all low in molecular weight and have no immunogenicity as they are, so that serum albumin, KLH ( The peptide is bound to a carrier protein such as keyhole limpet hemocyanin) as a hapten, and a sufficient amount of a peptide to induce an immune response is injected into the animal, and the antibody is recovered from the serum of the animal. In addition, mice are immunized with a hybrid and a sodopeptide that combines the binding motif of the mouse major histocompatibility complex and a peptide to obtain the target antibody.

A method of making the body work can also be used. (Ogasawara, K. et al, Proc. Natl. Acad. Sci, USA, 89, 8995-8999 (1992)). As the antibody, any of a polyclonal antibody and a monoclonal antibody can be used, but in the case of using an antibody targeting a specific epitope as in the present invention, it is more preferable to use a monoclonal antibody.

The specific antigen or specific antibody of the present invention can be used for any immunoassay such as enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, chemiluminescence immunoassay, particle agglutination, immunochromatography, and the like.

In using the specific antigen or the specific antibody of the present invention as a diagnostic agent, the specific antigen or the specific antibody is used by binding to a carrier for immobilization or a labeled substance. Examples of the carrier for immobilization include polystyrene resin, acrylamide resin, latex particles, and gelatin particles. The labeling substance, an enzyme (Peruokishida Ichize, P- D-galactosidase and the like; an enzyme immune疫測method), fluorescence substances (FITC like; fluorescence immunoassay), radioisotopes (such as ^{1 ZS} I; Rajioi厶Noatsusi) , Chemiluminescent substances (such as luminol; chemiluminescent Imnoassay), and colloidal gold. When the specific antigen or the specific antibody is bound to the immobilizing carrier or the labeling substance, a spacer structure may be interposed between them by a covalent bond or the like.

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example

Example 1 (Search for B cell epitope in VD)

We synthesize peptides of VDI to VD IV of C. trachomatis and peptides near VDI, and these peptides and C. trachomatis infection » The reactivity with healthy human serum was evaluated.

The following eight peptides of CT # 1 CT # 8 were synthesized.

CT # 1 (VDI neighborhood peptide; L 2 strain)

Val-Leu-Gln-Thr-Asp-Val-Asn-Lys-Glu-Phe-Gln,

CT # 2 (VDI; C strain)

Thr-Ser-Asp-Val-Ala-Gly-shi eu_Glri-Asn-Asp-Pro-Thr-Thr-Asn-Val-Ala,

CT # 3 (VD II; L 2 strains)

Asp— Asn— Glu-Asn— His Ala— Thr Val-Ser— Asp-Ser— and ys— and eu— Val CT # 4 (VDΠ; strain C)

Thr-Gin-Ser-Ser-Ser-Phe_Asn-Thr-Ala-Lys-eu_Ile-Pro-Asn-Thr -Ala-Leu-Asn-Glu-Ala,

CT # 5 (VD II; L 2 strains)

Val-Gly-Gln-Glu-Phe-Pro-Leu-Asp-Leu-Lys-Ala-Gly-Thr-Asp-Gly -Val,

CT # 6 (first half of VD IV; L2 strain)

Ala-Thr-Thr-Val-Phe-Asp-Val-Thr-Thr-Leu-Asn-Pro-Thr-Ile-Ala -Gly

CT # 7 (Late VD IV; L 2 strains)

Thr-Leu-Asn-Pro-Thr-Ile-Ala-Gly-Ala-Gly-Asp-Val-Lys-Ala-Ser -Ala-Glu-Gly-Gln-Leu-Gly,

CT # 8 (first half of VD IV; E strain)

Ala-Thr-Ala-Ile-Phe-Asp-Thr-Thr-Thr-Leu-Asn-Pro-Thr-Ile-Ala

-Gly

The above peptides were synthesized with Shimadzu Peptide Synthesizer P SSM8. Was. Synthesis was carried out by the Fmoc method, and the peptide was biotinylated by reacting NHS-biotin (Pierce) with the amino terminal of the synthesized peptide. The synthesized peptide was desorbed from the solid phase, deprotected for protecting groups, purified to a purity of 95% or more with a reversed-phase column of HP LC, and subjected to amino acid analysis to confirm the synthesized product. The enzyme immunoassay was performed as follows.

Coat 10 μg Zm1 avidin or streptavidin (both from Pierce) in a 96-well plate (Nunc) in 50 μl Zwells, and leave at 4 CC overnight to remove the same solution. As a result, a biotinylated plate or a streptavidinized plate was prepared. Next, the above-mentioned synthetic peptide was added in 50 μl Zwell each so as to have an equimolar number with avidin or streptavidin, and the mixture was left at 37 for 2 hours. Then, the solution was removed and 2% bovine serum albumin ( Phosphate-impregnated physiological saline (PH7.3) was added to 200 μl Zwell, and the mixture was allowed to stand overnight at 4 to bind the synthetic peptide to a biotinylated plate or a streptavidinized plate.

After washing the plate twice with a washing solution (sodium acid buffer + 0.05% Tween 20, pH 7.3), the plate was diluted with a serum diluent (acid g solution + 0.1% bovine serum albumin, PH 7.3). Serum samples were added at 50 i 1 / well and reacted at 37 T! For 1 hour. Serum diluent was used as a blank. Serum was used for 10 healthy patients with C. trachomatis infection (cut off index (cut off index) of 1.00 or more in antibody measurement using Cellopalizer Chlamydia IgGG manufactured by Savyon Diagnostics) and health. Serum from 10 humans (cut off index for the same diagnostic drug manufactured by the company and the cut off index was 1.00 drops) was diluted 64 times and used. After the reaction, the plate is washed 5 times with a washing solution, and then diluted 5000 times with a serum diluent. A human IgG antibody (manufactured by Dako) was added to 50 µl of Zwell, and reacted with 37 for 1 hour. After the reaction, the plate was washed 5 times with a washing solution, and 100 μl / well of TMB (manufactured by Savyon Diagnostics) diluted 10-fold with purified water was added, and the mixture was reacted at room temperature for 15 minutes. After the reaction, 100 # 1 Zwell was added with 2 N ruic acid, and the absorbance at 450 nm was measured with a microplate reader using a blank as a control.

The reactivity of each of the CT # 1 to CT # 8 peptides with serum was examined using the above measurement system.

Table 1 shows the results. Since no reaction was observed in all serum samples for CT # 3 peptide, the relative value to the absorbance of other peptides was calculated by setting the absorbance for CT # 3 peptide to 1.00, and calculated as 1.50. The above was determined to be positive. As a result, in CT # 2 to CT # 5 (VDI, VDΠ and VDΙΠ), no positive serum sample was observed, and the positive match rate with `` Cellophilizer Chlamydia IgG '' was 0%. .

^ The nature of the chlamydia specific IgG of human and に対する »¾Λώι

On the other hand, the peptide synthesized from the VD IV region (CT # 6 to CT # 8) showed a positive match rate of 40, 0% to 70.0%, and the peptide near the VD I, CT # 1, also showed a positive 20.0% The concordance rate was high (negative concordance rate was 100.0%, indicating good concordance rate).

The VD IV region shows up to 70% positive concordance, indicating that this region is a fairly common epitope among patients. The VD IV region has an amino acid sequence of about 30 residues, but CT # 6 is the first half including the N-terminal, and D # 7 is the second half including the (: terminal (CT # 6 And CT # 7 overlap each other in the central part of the VD IV region.) However, depending on the sera of positive patients, the response to one of the two Some samples did not show that at least two epitopes were present in the amino acid sequence of about 30 residues in the VD IV region. It indicates that.

Example 2 (Species-specific reactivity of VD IV and VDI neighboring peptides) The results of Example 1 revealed that two or more B cell epitopes were present in the VD IV region. In an effort to increase the efficiency, we synthesized about 30 residues of peptide C. pneumoniae. C. trachomatis strain C and C. trachoaatis strain E across the VD IV region. In addition, CT # 1 described in Example 1 was also synthesized. The synthesis, biotinylation, purification and confirmation of the synthesized product of these peptides were performed in the same manner as in Example 1. The amino acid sequence and peptide name of the peptide synthesized in this example are as follows.

CT # 1 (peptide near VDI; trachomatis L 2 strain)

Val-Leu-lain-Thr-Asp-Val-Asn-Lys-Glu-Phe-ϋΐη,

CT # 9 (VD IV whole region; trachomatis E strain)

Ser-Ala-Thr-Ala-Ila-Phe-Asp-Thr-Thr-Thr-Leu-Asn-Pro-Thr-Ile -Ala-Gly-Ala-Gly-Asp-Val-Lys-Ala-Ser-Ala- Glu-Gly-61n-Leu-Gly »CT # 10 (VD W whole region; C. trachomatis C strain)

Eu— Ala— Glu— Ala_Ile— eu— Asp— Val— Thr— Thr-Leu— Asn— Pro— Thr— lie -Ala-Gly-Lys-Gly-Ser-Val-Val-Ser-Ala-Gly- Thr-Asp-Asn-Glu-Leu-Ala, CP (VD IV region of C. pneumoniae)

Leu-Pro-Thr-Ala-Val— then eu— Asn— then eu-Thr— Ala— Trp_Asn-Pro_Ser— Leu-Leu-Gly-Asn-Ala-Thr-Ala-Leu-Ser-Thr-Thr-Asp- Ser-Phe-Ser

Enzyme immunoassay was performed in the same manner as in Example 1, except that the binding of the synthetic peptide to the biotinylated plate or the streptavidinized plate was performed for each of these peptides alone, as well as for CT #. 1, Peptide mixture of 3 kinds of peptides of CT # 9 and CT # 10 (Hereinafter referred to as CT mixture). As a negative control, a system in which the above-mentioned synthetic peptide was not added at all to a biotinylated plate or a streptavidinized plate was used.

As a serogroup,

ΓCerroparaizer Chlamydia IgG ”is a positive serum, and

Serogroup (hereinafter referred to as CT (+) serogroup), ¾celloparaizer chlamydia IgG ”|

and

ΓCeloperizer Chlamydia IgG-negative serogroup (hereinafter referred to as CT (1) serogroup)

of,

Three serum samples were measured in each group, for a total of nine serum samples.

CT # 1, CT with the absorbance of the negative control as 1.00

The relative values of the absorbance of the # 9, CT # 10, CP, and CT mixtures were determined. Table 2 shows the results.

Table 2 C. trachoBiatis¾¾ * 4¾ttl6L using peptide as ¾¾M

C

Examination of ¾tt

a | ¾¾® ¾ ^ when the peptide weight is 1.00. The underline is judged to be ¾¾¾) ¾¾1.50.

All three samples of the CT (+) CP (+) serogroup tested positive for CP only and reacted with any of CT # 1, CT # 9, CT # 10, and their mixture, cτ mixture. Did not show sex. Therefore, the result of the C, trachomatis © property by the r cellopalizer Chlamydia IgGG is that the possibility that the C «pneumoniae antibody cross-reacts with the C, trachomatis is possible. On the other hand, in the enzyme immunoassay using the peptide of the present invention, crossed antibodies could be differentiated.

In the CT (+) serogroup, all specimens were positive for CT # 10 or the CT mixture. Two out of three samples were positive for CT # 9. Whether serum samples in this group contain antibodies to C. pneumoniae Although it is unknown, all three serum samples showed a negative reaction to the CP peptide.

The CT (1) serogroup did not react with any of the CT peptides and showed good agreement with r cellophylizer chlamydia IgGj. It is unknown whether the serum samples in this group also have antibodies against C. pneumoniae, but all three serum samples showed a negative reaction to the CP peptide.

A close examination of the reactivity to trachomatis in the CT (+) serogroup revealed that two specimens, sample numbers 4 and 5, were more reactive with the CT mixture, a mixture of these, than with CT # 9 or CT # 10 alone. Relative absorbance was high. Although there are 15 serotypes of C. trachomatis, it is expected that the reactivity of a serum sample to a CT mixture will be higher than the reactivity to a single peptide. It was confirmed by a real example.

Therefore, a combination of peptides in the VD IV region of multiple serotypes of C. trachomatis, or a combination of peptides near the VD I, may be used as a diagnostic agent for Chlamydia infection with higher reactivity. It is possible.

In addition, in the above two examples, the VD IV region was a peptide of the entire VD IV region or a combination of these peptides or a species-specific C. trachomatis in the L2 strain, E strain, and C strain of C. trachomatis. High partial-reactivity of these partial peptides, including those with a typical B-cell epitope, was confirmed. However, other strains of C. trachomatis (B strain, Ba strain, D strain) Strains, L1, F, G, A, H, I, J, K and L3 strains) peptides in all regions of VD IV or species-specific B cell peptides of C. trachomatis — These partial peptides, including those with a loop, also have high homology to each other In view of the above, it is considered that it has the same type-specific reactivity as the L2 strain, E strain, or C strain. Therefore, peptides of the entire VD IV of a strain other than L2, E, or C strains, or a combination of such peptides, and those containing a portion having a Z- or C. trachomatis species-specific B-cell epitope It is also possible to develop a highly reactive diagnostic agent for Chlamydia infectious disease using a partial peptide or a combination of these partial peptides.

Example 3 (Improvement of trachomatis positive concordance rate by combination of VD IV peptide)

The 15 serotypes of C. trachomatis were compared in the sequence of the VD IV region and found to be group B (strains B, Ba, D, E, L1, L2), group C (strain C, A Strains, H, I, J, K, and L3 strains) and an intermediate group (F and G strains). Therefore, it is considered that by selecting one or more peptides of the VD IV region from each of these groups and using a mixture thereof as an antigen, the detection sensitivity of the antibody test can be further improved. .

Therefore, when the peptide of the VD IV region of one strain (L2 strain) was used as an antigen, one or more of the peptides of the VD IV region of one or more strains were selected from each of these groups, and a mixture thereof ( L2 strains, C strains, E strains, and G strains) were used as antigens, and a comparison of the positive rate agreement rate with the Western blotting test result was performed.

Synthesis, biotinylation, purification, and confirmation of the synthesized product of these peptides were performed in the same manner as in Example 1. The amino acid sequence and the peptide name of the peptide synthesized in this example are as follows.

CTL2 (all regions of L2 strain VD IV)

Ser-Ala-Thr-Thr-Val-Phe-Asp-Val-Thr-Thr-Leu-Asn-Pro-Thr-Ile -Ala-Gly-Ala-Gly-Asp-Val-Lys-Ala-Ser-Ala-Glu-Gly-Gln-Leu-Gly CTC (all regions of CD strain VD IV)

Leu-Ala-Glu-Ala-lie-shi eu-Asp-Val-Thr-Thr-Leu-Asn-Pro-Thr-lie -Ala-Gly-Lys-Gly-Ser-Val-Val-Ser-Ala-Gly -Thr-Asp-Asn-Glu-Leu-Ala CTE (E-strain VD IV whole region)

Ser-Ala-Thr-Ala-Ile-Phe-Asp-Thr-Thr-Thr-Leu-Asn-Pro-Thr-Ile -Ala-Gly-Ala-Gly-Asp-Val-Lys-Ala-Ser-Ala- Glu-Gly-Gln-Leu-Gly CTG (all regions of G strain VD IV)

Eu— Ala— then ys— Pro— Val— Val— Asp— lie— Thr_Thr— Leu— Asn— Pro— Thr— lie-Ala-Gly-Cys-Gly-Ser-Val-Val-Ala-Ala-Asn- Binding of Ser-Glu-Gly-Gln-Ile-Ser synthetic peptide to streptavidinated plate is achieved by mixing CTL2 alone and four peptides of CTL2, CTC, CTE and CTG in 1 Z4 amount. The peptide mixture (hereinafter referred to as a four-type mixture) was used.

The enzyme immunoassay was performed in the same manner as in Example 1. >> Spontaneous / negative was judged as positive if the OD " ₀ value of the specimen was divided by the cut-off value (cut-off index; COI) was 1.0 or more, and negative if not 1.0.

The test by the Western blotting method was performed as follows.

The antigen (EB of L2 strain) diluted 10-fold with the blotting buffer was applied to a 4 to 12% gradient gel (TEFCO) in an amount of 180 μl and subjected to electrophoresis with one 15 AZ sheet. After the electrophoresis, the electrophoretic protein was transferred to the membrane at 180 A / sheet using a blotting unit (manufactured by TEFCO). The membrane was washed twice with PBS, dried and stored at 4. Then PBS containing 5% wZv skim milk For 2 hours, washed twice with PBS containing 0.05% w / v Tween 20, and then set on a screen plotter (manufactured by SAN PLATEC). Sample serum diluted 100-fold with PBS containing 5% wv skim milk was applied at 2501 per lane and allowed to react at room temperature for 2 hours. Remove the membrane from the screen plotter.

1 After washing 5 times with PBS containing 6020, and reacting for 1 hour in 2 Oml of peroxidase-labeled anti-human IgG V chain antibody (manufactured by TAGO) diluted 2000-fold with PBS containing 1% wZv BSA. Washing was performed 5 times. Finally, the cells were immersed in 5 OmM Tris-HC 1 _P H7.2 containing 0.05% 3,3 'diaminobenzidine and 0.01% 11 ₂ 〇 ₂ to determine the species specificity of MOM P. The band (about 40KD) was checked for color development.

Using the above measurement system, the reactivity of CTL2 alone or a mixture of four of them with 44 sera was examined, and ゥ: I was compared with the test result by the stamp lotting method. As a result, in 40 samples, the judgment was the same for CTL 2 alone and the mixture of 4 types, but for 4 samples, CTL 2 alone was negative and 4 types of mixture were sexual. When these four samples were tested by Western plotting, they were all positive. Table 3 shows the measurement results of the four samples.

Table 3

4 mixture

Sample No. L 2 (L 2 + C + E + G) Western

COI judgment COI judgment Blotting

9 0.63 1 7.18 +

15 0.64 1 3.24 + +

35 0.39 one 1.64 + +

48 0.98 1 5.39 + Based on the above, rather than using the peptide of the VD IV region of one strain (L2 strain) as an antigen, the VD IV region of one or more strains from each of the groups B, C, and the intermediate group was used. It became clear that the selection of peptides and the use of a mixture of them as antigens enabled more accurate determination of C. trachomatis antibodies.

The invention's effect

According to the present invention, it is possible to provide a diagnostic agent for species-specific Chlamydia infection, and thus, it is possible to more reliably and easily infect C. trachomat is or C. pneumoniae, which has been difficult to confirm until now. It is possible to make a diagnosis immediately.

Claims

The scope of the claims

1 · Diagnostic agent for Chlamydia infection, which contains a peptide containing the species-specific B cell epitope of Chlamydia.

2. The diagnostic agent for chlamydial infection according to claim 1, wherein Chlamydia is Chlamydia trachomatis.

3. The diagnostic agent for chlamydia infection according to claim 1, wherein the Chlamydia is Chlamydia pneumoniae.

4. A diagnostic agent for Chlamydia infection comprising an antibody against a peptide containing a species-specific B cell epitope of Chlamydia.

5. The diagnostic agent for chlamydial infection according to claim 4, wherein the Chlamydia is Chlamydia trachomatis.

6. Chlamydia power Chlamydia pneumoniae is a diagnostic drug for chlamydial infection according to claim 4.

7. The peptide containing the species-specific B cell epitope of Chlamydia trachomatis is immunologically linked to the peptide represented by the following amino acid sequence (1) to (15) and / or the peptide represented by the amino acid sequence. 6. The diagnostic agent for chlamydial infection according to claim 2 or 5, wherein the diagnostic agent is selected from peptides equivalent to:

(1) Ser-Ala-Glu-Thr-Ile-Phe-Asp-Val-Thr-Thr-Leu-Asn-Pro-Thr

-lie-Ala-Gly-Ma-Gly-Asp-Val-shi ys-Thr-Ser-Ala-Glu-Gly-Gin-Leu-Gly,

(2) Ser-Ala-Thr-Ala-lie-Phe-Asp '' Thr-Thr-Thr-eu-Asn-Pro-Thr -Ile-Ala-Gly-Ala-Gly-Asp-Val-Lys-Thr- Gly-Ala-Glu-Gly-Gln-Leu-Gly.

(3) Ser-Ala- Thr- Ala- Ile-Phe- Asp- Thr-Thr- Thr-Leu- Asn-Pro- Thr

-lie-Ala-Gly-Ala-Gly-Asp-Val-Lys-Ala-Ser-Ala-Glu-Gly-Gln- then evrGly,

(4) Leu-Ala-Thr-Ala-Ile-Phe-Asp-Thr-Thr-Thr-Leu-Asn-Pro-Thr

yyyyPyGGGLG IlGvalAlasrAlGllneuleAlalAlalASLsealul ---------- l-l-

() In VAl5AlnuvRieAS Thrnutusn roer selerlliral-ellilil Ilfli-- (14) Leu-Ala-Glu-Ala-Val-Leu-Asp-Val-Thr-Thr-Leu-Asn-Pro-Thr-Ile-Ala-Gly-Lys-Gly-Ser-Val-Val-Ala-Ser -Gly-Ser-Glu-Asn-Glu-Leu -Ala,

(15) Val-Leu-Gln-Thr-Asp-Val-Asn-Lys-Glu-Phe-Gln

8. The peptide containing the species-specific B cell epitope of Chlamydia trachomatis is a peptide represented by the amino acid sequence of (1) to (15) according to claim 7, and a peptide represented by the amino acid sequence of Z or the amino acid sequence thereof. 6. The diagnostic agent for chlamydial infection according to claim 2, wherein a peptide selected from peptides immunologically equivalent to the peptide is linked to each other.

9. The diagnosis of Chlamydia infection according to claim 7 or 8, wherein the immunologically equivalent peptide has a cut-off index of 1.50 or more as measured by the serum of a patient infected with Chlamydia trachomatis by enzyme immunoassay. Abstinence.

10. The peptide containing the species-specific B cell peptide of Chlamydia pneumoniae contains the peptide represented by the following amino acid sequence and Z or the peptide represented by the amino acid sequence and immunology. 7. The diagnostic agent for chlamydial infection according to claim 3 or claim 6, wherein the diagnostic agent is a substantially equivalent peptide.

Leu-Pro-Tr-Ala-Val-Leu-Asn-Leu-Thr-Ala-irp-Asn-Pro-aer-Leu eu—Gly—Asn—Ala_Thr—Ala—and eu—Ser—Thr—Thr — Asp— Ser— Phe— Ser

11. A peptide containing a species-specific B cell peptide of Chlamydia pneumoniae! Is immunized with a peptide represented by the amino acid sequence according to claim 10 and / or a peptide represented by the amino acid sequence. 7. The diagnostic agent for chlamydial infection according to claim 3, wherein the chemically equivalent peptides are linked to each other.

12. The chlamydial infection according to claim 10 or claim 11, wherein the immunologically equivalent peptide has a cut-off index of 1.50 or more as measured by serum of an infectious patient of Chlamydia pneumoniae by enzyme immunoassay. Diagnostics.