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Establishment of a programmatic detection method for Trichomonas vaginalis based on double antibody sandwich ELISA
Yuhua Li1, Fakun Li1, Wenjie Tian1
1Department of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan 453003, PR China; inxiang Key Laboratory of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan 453003, PR China.
Background:
Trichomonas vaginalis (T. vaginalis) is a common sexually transmitted parasite that colonizes the human urogenital tract. Programmed and precise detection of T. vaginalis is a key step in preventing and treating trichomoniasis. However, the current detection methods of T. vaginalis, including wet mount microscopy, culture, nested PCR, loop-mediated isothermal amplification, and recombinant enzyme polymerase amplification, have some shortcomings. Therefore, it is urgent to establish a programmed, sensitive, and specific method for detecting T. vaginalis.
Methods:
T. vaginalis cysteine protease 39 (TvCP39) was expressed in segments as TvCP39-1 and TvCP39-2, and the polyclonal antibodies were prepared by immunizing rats and rabbits. The concentration of the polyclonal antibodies of anti-rTvCP39-2 and anti-rTvCP39-1 was determined by square matrix titration. The sensitivity and specificity of double antibody sandwich ELISA were analyzed and evaluated by detecting rTvCP39 and T. vaginalis excretory-secretory proteins (TvESPs) diluted in multiple ratios and detecting excretory-secretory proteins of T. vaginalis and other pathogens, respectively. The detection efficiency of wet mount microscopy, nested PCR, and double antibody sandwich ELISA was compared by testing sixty-two clinical samples from vaginal secretions.
Results:
The natural TvCP39 protein could be specifically recognized by anti-rTvCP39-1 and anti-rTvCP39-2 antibodies. The concentrations of anti-rTvCP39-2 and anti-rTvCP39-1 polyclonal antibodies were determined to be 0.58 μg/mL and 0.45 μg/mL, respectively. The results of the sensitivity test showed that the detection limits of rTvCP39 and TvESPs by double antibody sandwich ELISA were 1.76 ng/mL and 107.125 μg/mL, respectively. The specificity test results showed that the double antibody sandwich ELISA had a high specificity for the detection of T. vaginalis and did not cross-react with Escherichia coli, Staphylococcus aureus, Candida albicans, and Lactobacillus. The positive detection rate of clinical samples by double antibody sandwich ELISA was higher than that by wet mount microscopy, and was the same as nested PCR. The sensitivity of double antibody sandwich ELISA was consistent with that of nested PCR. The coincidence rate between double antibody sandwich ELISA and nested PCR was 100% (Kappa=1, P < 0.001).
Conclusion:
The double antibody sandwich ELISA detection method for T. vaginalis established in this study had the advantages of high sensitivity and specificity, and did not require the extraction of genomic DNA. This programmatic and simple detection method was suitable for batch testing of clinical samples and exhibited the potential value in the treatment and prevention of trichomoniasis.
