Using recombinant Chlamydia trachomatis OMP2 as antigen in diagnostic ELISA test.

BACKGROUND AND OBJECTIVES: The obligate intracellular bacterium Chlamydia trachomatis causes sexually transmissible diseases in human. Timely and sensitive detection of this pathogen is very important. There are many cross-reactions in bacteriological and serological methods in detection of this type of pathogens. The aim of this study was to achieve a more specific antigen for serological tests.
MATERIALS AND METHODS: Blood samples were taken from 192 women with suspected chlamydial infection and sera were isolated. ELISA plate wells were coated with recombinant C. trachomatis OMP2 as antigen. Cut-off system was determined with 40 negative sera. The final results of this research were compared with Euroimmun commercial kit.
RESULTS: The ELISA system cut-off was calculated at 0.27 using negative sera samples. ODs of positive samples were higher than 0.27 and negative samples were lower than it. We obtained 30 samples (15.62%) as positive and 162 cases (84.37%) as negative. Sensitivity and specificity of the recombinant antigen were 90% and 86%, respectively. This antigen showed no cross-reactivity with sera of patients infected with Hydatid cyst, HCV, Epstein barr virus, HBV, Helicobacter pylori, Toxoplasma gondii, Cytomegalovirus, Mycoplasma, Measles and Varicella zoster virus.
CONCLUSION: The sensitivity and specificity of rOMP2 in ELISA for detection of C. trachomatis were 90% and 86%, respectively. Though the sensitivity was higher than results of Euroimmun commercial kit, its specificity was calculated lower than reference kit.

INTRODUCTION

Chlamydia trachomatis is responsible for trachoma and sexually transmissible diseases in human. This bacterium is obligate intracellular parasite and use host cell energy system. Genital tract infection with chlamydia is common in some countries (1).

Serotypes L3, L2, L1, D, and K of C. trachomatis cause genital tract infections. Some infections of genital tract of women are often asymptomatic. With the progresses of the disease, symptoms may appear (2). C. trachomatis is causative of cervicitis, endometritis, salpengitis, prehepatitis bartonellosis in women (3). Complications of untreated chlamydial infections may occur and these include pelvic inflammatory disease, ectopic pregnancy, fallopian tube obstruction, infertility, cervical dysplasia, and prehepatitis (4). Prevalence of chlamydial infections of genital tracts among pregnant women in Western countries is about 5-13 percent (5). 33-50% of infants of infected mothers get chlamydial infections such as conjunctivitis, nasopharyngitis, otitis media and neonatal Chlamydia pneumonia during passage through the birth canal (6-9). Studies showed 35-50% of non-gonococcal urethritis (NGU) and a maximum of 70% of subsequent gonorrhea urethritis (post gonococcal urethritis = PGU) are caused by chlamydia (10).

Because of the importance of early and accurate diagnosis of chlamydial infection and its role in the health system, this study was aimed to use recombinant chlamydia OMP2 protein as an antigen in diagnostic ELISA Kit.

MATERIALS AND METHODS

Sampling

Blood samples from 192 women with suspected C. trachomatis infection refereed by physician and 40 healthy women (controls) were collected. Sera samples were separated and divided into small tubes and maintained at -20° C.

Preparation of antigen

BL21 E. coli bacterial colony containing C. trachomatis OMP2 gene construct (11) was cultured. When the turbidity of the culture reached to OD600 = 0.6 plasmid promoter was induced by 0.5mM IPTG (USA, Sigma) and bacteria were precipitated after 5 h and lysed by sonication. OMP2 C. trachomatis recombinant protein was purified by Ni-NTA affinity chromatography (12). The wells of ELISA plates were coated with recombinant protein as previously described (11).

Cut off determination

Forty negative C. trachomatis sera were examined by ELISA to determine system Cut-off. The graph and table of samples optical density at 1/200 dilution and standard deviation were calculated.

Evaluation of cross-reactivity with other infections

Possible cross-reactivity to sera of patients with other infections, such as: hydatid cyst (5 samples), Hepatitis B virus (5 cases), Hepatitis C virus (5 samples), Helicobacter pylori (5 samples), Toxoplasma gondii (5 cases), and Mycoplasma spp. (3 cases), Cytomegalovirus (3 samples), Measles (3 samples), Herpes zoster (1 case), Epstein barr virus (2 samples) were examined. Optical density (OD) of all samples at 1/200 dilution were obtained lower than of C. trachomatis Cut-off, demonstarting that they were negative and there are no cross reactivity between these sera samples and rOMP2 of Chlamydia trachomatis.

The samples evaluation by ELISA

192 chlamydial infections suspected sera samples were tested by recombinant omp2 coated plate and Euroimmun commercial kit. The amount of antigen was 3 mg and sera were diluted at 1/400, but better results were taken at 1/200. The sensitivity and specificity of ELISA with recombinant protein and commercial kit were calculated using formula: Cut off= mean + 2SD.

The Cut off for 1/400 serum dilution was 0.244 (0.116 + 2 X 0.064) and for 1/200 serum dilution was 0.271(0.127 + 2 X 0.072). We used 0.271 as cut- off for ELISA test carry out.

Specificity and Sensitivity were determined using the formula:

The T- student statistical method was used to analyse of the results.

Results

Sera were collected from 192 suspected patients infected with Chlamydia trachomatis. C. trachomatis rOMP2 was purified by affinity chromatography and ELISA plate wells were coated with it. Forty seronegative samples were tested and 0.271 were considered as cut- off for this ELISA system.

For specificity of system, the sera with other infections were examined by this protein. There were no cross-reactivity between rOMP2 protein and sera with other infections (Fig.1).

Fig. 1

Evaluation of cross-reactivity between C. trachomatis rOMP2 and sera of other infectious disease. 1 to 5 hydatid cyst, 6 to 10 Hepatitis C virus, 11 to 15 Hepatitis B virus, 16 to 20 Helicobacter pylori, 21 to 25 Toxoplasma gondii, 26 to 28, Mycoplasma spp., 29 to 31 Cytomegalovirus, 32 to 34 Measles, 35 to 36 Epstein barr virus 37 Varicella zoster virus, 38 to 48 healthy serum samples.

Fig. 2 shows comparison of Cut Off for 40 negative sera at 1/200 and 1/400 dilutions and 3mg/ml rOMP2.

Fig. 2

Comparison of Cut off based on 1/200 and 1/400 serum dilutions. The best Cut off was obtained at 1/200 serum dilution.

We tested antigen concentration for coating on microplate wells with 3 mg/ml and 2 mg/ml and used for serum concentration (Fig.3). The OD of 192 C. trachomatis suspected serum were calculated by rOMP2 and Euroimmun commercial kit as shown in Figs. 4 and 5.

Fig. 3

Comparison of sera dilutions with 3mg/mL and 2mg/mL of rOMP2 protein.

Fig. 4

The OD of sera of C. trachomatis suspected patients in rOMP2 coated ELISA plate wells.

Fig. 5

The sera OD for C. trachomatis suspected patients in Euroimmun commercial ELISA kit Since the P-Value = 0.88 is greater than the significance level of a = 0.05, so there is no significant relationship between the rOMP2 and Euroimmun commercial ELISA kit.

According to Table 1 and figure 3, 3 mg/ml of rOMP2 and serum dilution at 1/200 is the best in this ELISA system.

Table 1

Comparison of statistical T-test against 2mg/ml of rOMP2: t = 55.1 (p <0.05) and 3mg/mL of rOMP2: t=139.2 (p <0.05). Therefore, using 2mg/ml and 3mg/ml of rOMP2 for ELISA are significant.

Test values	Test Value = 0
Concentration	t	df.	Sig. (2-tailed)	Mean Difference	95% Confidence Interval of the Difference
					Lower	Upper
Concentration 2	55.190	47	0.000	0.620652	0.59803	0.64328
Concentration 3	139.249	47	0.000	0.859917	0.84749	0.87234

The specificity and sensitivity of ELISA were calculated 86 and 90% for rOMP2 protein and 97.1 and 78.2 for Euroimmun commercial kit, respectively. Therefore rOMP2 protein for ELISA is more sensitive and less specific than Euroimmun commercial kit.

DISCUSSION

C. trachomatis is one of the main causes of urinary tract - genital infections (13). C. trachomatis is causative of 30-50% of non-gonococcal urethritis (NGU) and the most common causative of bacterial STD (14). The bacterial causative diseases such as trachoma, keratoconjunctivitis and lymphogranuloma syndrome (LGV) are Chlamydia trachomatis. Due to eye, neurological and genital diseases, timely and sensitive detection of C. trachomatis is most importance. C. trachomatis detection is done based on molecular and serological diagnosis, inoculation of suspected material into 8-7 day chick embryo yolk sac and culture in cells such as Hela229T, McCoy, BHK2, BGMK. Chlamydia LPS antibodies show cross reaction with Salmonella and E. coli LPS by immunoperoxidase and immunofluorescent techniques (14).

Recombinant cysteine-rich protein (OMP2) of C. trachomatis was prepared by Goodall et al. and detected activated immune response against OMP2 in peripheral blood and synovial fluid (14). Bas et al. used serological tests with OMP2 and MOMP antigens for detection of C. trachomatis and declared that OMP2 has more sensitivity and specificity than MOMP (15). Gdoura et al. used cell culture, PCR and ELISA methods for detection of C. trachomatis in men's semen and no difference was found between the methods (16).

Culture of this bacterium is difficult in the common media and it is not possible practically. Serological and molecular methods are the best tools for C. trachomatis detection in human fluid and tissues. Access the specific antigen is necessary for a successful serological test.

In this study, the protein of C. trachomatis named rOMP2 (11) was designed as specific antigen and coated on wells of ELISA microplates. Sera of patients with Hepatitis C virus, Hepatitis B virus, Epstein barr virus, helicobacter pylori, Toxoplasma gondii, Cytomegalovirus, Measles, Mycoplasma, hydatid cyst, Varicella zoster virus were examined and no cross reaction were found with ELISA by rOMP2 protein and Euroimmun commercial kit.

There were 7 positive samples (3.83%) and 173 negative samples (96.1%) of 180 suspected C. trachomatis sera by Euroimmun commercial ELISA kit. The specificity and sensitivity of Euroimmun ELISA kit were 97.1% and 78.2% respectively, whereas those of rOMP2 were 86% and 90%. That means ELISA assay with recombinant protein rOMP2 antigen is more sensitive but the commercial kit is more specific. Bas et al. reported 76% sensitivity and 85% specificity for recombinant protein and synthetic peptides in ELISA system (15).

OMP2 is an extra membrane protein of C. trachomatis and was described as cysteine-rich protein (containing 24 disulfide bonds) with 60 kDa by Allen in 1989 for the first time (17). Portig et al. declared that OMP2 is a properly antigen in ELISA system for detection of chlamydial infections (18).

In conclusion, the sensitivity and specificity of rOMP2 in ELISA for detection of C. trachomatis are 90% and 86%, respectively, which is more sensitive than Euroimmun commercial and its specificity is lower than Euroimmun commercial kit

Table 2

Results of ELISA test with rOMP2 on 192 sera samples of C. trachomatis suspected patients.

Total	Negative		Positive
	Percent	Number	Percent	Number
192	84.37	162	15.62	30

Table 3

Results of ELISA test with Euroimmun commercial ELISA kit on 180 sera samples of C. trachomatis suspected patients.

Total	Negative		Positive
	Percent	Number	Percent	Number
180	96.1	173	3.8	7

Table 4

Results of ELISA with Euroimmun commercial kit and C. trachomatis rOMP2.

rOMP2 ELISA(-)		rOMP2 ELISA (+)		rOMP2 ELISA2 (-)		rOMP2 ELISA (+)
Euroimmun (+)		Euroimmun (-)		Euroimmun (-)		Euroimmun (+)
Percent	Number	Percent	Number	Percent	Number	Percent	Number
45.4	169	54.3	102	89.7	173	9.9	37