The SeroCT IgA ELISA Assay kit is intended for the detection of IgA antibodies specific to C. trachomatis in human serum. This kit is a new generation qualitative ELISA test which is based on Chlamydia trachomatis specific synthetic peptides. SeroCT is used as an aid in the diagnosis of C. trachomatis specific infection. SeroCT IgA is intended to be run and interpreted in conjunction with the Savyon SeroCT IgG kit. The Eagle Biosciences SeroCT IgG ELISA Assay Kit is for Research Use Only and is not intended for diagnostic or therapeutic purposes.
The Eagle Biosciences SeroCT plates are coated with C. trachomatis specific peptides. Serum to be tested is diluted and incubated with the precoated SeroCT™ plate 1h at 37°C. In this step C. trachomatis specific antibodies are bound to the immobilized C. trachomatis specific peptides. Non specific antibodies are removed by washing. Anti-human IgA conjugated to horseradish peroxidase (HRP) is added and incubated 1h at 37°C. In this step the HRP-conjugate is bound to the prebound antigen-antibody complex. Unbound conjugate is removed by washing. Upon the addition of TMB-substrate, the substrate is hydrolyzed by the peroxidase, yielding a blue solution of the reduced substrate. Upon the addition of the stop solution, the blue color turns yellow and should be read by an ELISA reader at a wavelength of 450nm. The absorbance is proportional to the amount of the specific antibodies which are bound to the immobilized peptides.
Chlamydia is a gram negative obligate intracellular bacteria that causes acute and chronic disease in mammalian and avian species. The genus Chlamydia is comprised of four species: C. trachomatis, C. pneumoniae,C. psittaci and C. pecorum.
C. trachomatis is divided into 15 serovars. Serovars A, B, Ba and C are agents of trachoma, the leading cause of preventable blindness, endemic in third world countries. Serovars L1-L3 are the agents of lymphogranuloma venereum. Serovars D-K are the common cause of sexually transmitted genital infection worldwide: cervicitis, endometritis/salpingitis in females and urethritis in both males and females. Endometritis/salpingitis can lead to tubal occlusion with a higher risk of extrauterine pregnancy and infertility. Genital infection may cause an acute and persistent infection occasionally without any clinical signs. Generally, these infections are treatable once they are diagnosed. However, without any treatment the infection may progress to a severe chronic inflammation leading to infertility, ectopic pregnancy, induced abortion or child delivery. Moreover, infants to infected mothers can be infected during birth leading to conjunctivitis or pneumonia. The serology of C. trachomatis is more interesting in cases of chronic infections than in acute infections.
C. pneumoniae is an important respiratory pathogen in humans and causes up to 10% of community-acquired pneumonia cases. It has been associated with acute respiratory diseases, pneumonia, asthma, bronchitis, pharyngitis, acute chest syndrome of sickle cell disease, coronary heart disease and Gullain-Barré syndrome. C. psittaci infects a diverse range of host species from molluscs to birds to mammals and also causes severe pneumonia. In animals, C. psittaci and C. pecorum are capable of inducing diverse disease syndromes, like pneumonia, enteritis, polyserositis, encephalitis and conjunctivitis.
Serological testing, now an established approach in many countries, has been shown to provide a comprehensive answer for the detection of C. trachomatis infection. In suspected deep-seated infections, serum sampling reduces the necessity for invasive procedures which are required for direct antigen detection. In cases of lower urogenital infections, collection limitations such as effectiveness of scrape sampling procedure, specimen handling and transportation difficulties have to be weighted. Above all, there remains the issue that most Chlamydial infections are asymptomatic. Therefore, an infection may persist for a long time, ascend the upper genital tract causing deep and chronic infection, and increase the probability of false negative results in direct antigen detection.
Serological testing for C. trachomatis, through the detection of various specific antibodies, is today an effective and highly accepted method option. New and accurate technologies apply the immuno markers IgM, IgA and IgG to characterize the presence and stage of the infection.
Specific IgM is indicative of acute Chlamydial infection. Absence does not, however, preclude the presence of on-going infection, especially in recurrent and chronic cases. The use of specific IgA as a marker for active Chlamydial infection has been shown to have an important role because of its short half life time, while persisting as long as antigenic stimulation exists. IgA, however, is more suitable for post therapy follow-up. IgG is a marker for Chlamydial positive immune-response in either current, chronic or past infections.
Serological cross reactions occur between the three different species of Chlamydia. Most of the serological diagnostic assays for Chlamydia use either purified elementary bodies: microimmunofluorecence (MIF) and ELISA tests, lipopolysaccharide (LPS), or purified major outer membrane protein, (MOMP) as antigens. Genus specific epitopes are present in all the above antigens, therefore, low species specificity is observed. Moreover, a large proportion of the population has been exposed to C. pneumoniae (with no clinical signs), the prevalence of anti-Chlamydia antibodies is very high. Therefore, the differentiation between C. pneumoniae and C. trachomatis specific antibodies using conventional serological screening tests (MIF, ELISA, EIA etc.) is insufficient.
The SeroCT trachomatis ELISA Assay has been developed in which C. trachomatis species specific epitopes, derived from different serotypes, are used in an Enzyme – Linked Immunosorbent Assay (ELISA). The test excludes cross-species reactive epitopes and enables more accurate and more specific determination of C. trachomatis IgG and IgA antibodies.
Sensitivity and Specificity of SeroCT™ – IgA compared to microimmunofluorescence (MIF)
The study was carried out on patients suspected of having a C. trachomatis infection. SeroCT™ – IgA was compared to a commercial MIF test kit.
MIF
SeroCT™ – IgA
Positive
Negative
Positive
21
20
1
Negative
49
5
44
Total
70
25
45
Sensitivity: 20/21 x 100 = 95%
Specificity: 44/49 x 100 = 90%
Overall Agreement: 64/70 x 100 = 91%.
References
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Tsunekawa, T. and Kumamoto, Y. (1989). A study of IgA and IgG titers of C. trachomatis in serum and prostatitic secretion in chronic prostatitis. J. J A. Y. Inf. Dis. 63 (2) : 130-137.
Kaneti, J. et al., (1988). IgG and IgA antibodies specific for Chlamydia trachomatis in acute epididymitis. Europ. Urol. 14 : 323-327.
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