The most prevalent STI in the US is HPV (McLendon et al., 2021). Which is extremely harmful to an individual's social life. All sexually active men and women will eventually obtain HPV, even if they do not also catch any other Papillomaviridae-related illnesses. HPV is a tiny, double-stranded DNA virus that causes anogenital infections, cutaneous warts, oropharyngeal (tongue, tonsil, and throat) cancers, and anogenital (cervical, anal, vulvar, vaginal, and penile) cancers. In women, CC is the third most common type of cancer (Kombe Kombe et al., 2021). The pathogenesis of HPV infections is dependent on the type of virus, the host immune system, and the local environmental conditions. HPV infections are linked to a number of benign and malignant disorders. Condylomata acuminata, also known as anogenital warts, is a STI that is mostly caused by LR HPV genotypes11 and 6. However, co-infections with HR HPV genotypes have also been reported (Stuqui et al., 2023). The treatment for anogenital warts, which are benign proliferative diseases that cause visible lesions like single or multiple papules on the vulva, vagina, cervix, penis, scrotum, perineum, and perianal area, involves freezing or vaporizing the tissue with a CO2 laser, as well as applying topical immune-modulating, antimitotic, antiviral, and anti-carcinogenic medications (Bornstein, 2019). ELISA is widely recognized as the most widely used technique in hospitals and clinics worldwide. In the biotechnology sector, it has also been extensively utilized for the precise identification and measurement of biological agents (mostly proteins and polypeptides), and its significance in therapeutic, food safety, and environmental applications is growing. It is thought that ELISA has been used in some capacity in every laboratory; it offers highly repeatable and quantitative data, making it a useful biotechnological tool for clinical diagnosis and scientific study (Al-Taie et al., 2018).

Sample collection 

A total of one hundred fifty specimens ( PBS embedded tissue, cervical swab, cervical brush, and blood  ) were taken from women with genital warts who were referred to Al-Zahrawy Hospital in Mosul/Iraq and Mosul General Hospital from August /2023 to April /2024. And their ages range between (19- 58) years old

HPV Antigen Detection Using ELISA Technique

In this investigation, we identified HPV antigen from the HPV-positive samples that were previously analyzed by PCR. The Advanced Immunology Laboratory of the University of Mosul's Department of Biology, College of Science, conducted an ELISA test. The test was conducted in accordance with the guidelines provided by the manufacturer for the Human Papillomavirus (HPV) ELISA Kit (Catalogue Number: SL1332Hu SunLong Biotech Co., LTD China).

Principle of The Test:

TThe ELISA was reliant on the qualitative method of enzyme immunoassay. The kit's microplate was pre-coated with an HPV-specific antibody, which transformed it into a solid-phase antibody.. The microplate wells were filled with samples, and the corresponding antibody was then added. Subsequently, each microplate well was filled with an HPV-specific Horseradish Peroxidase (HRP)-conjugated antibody, which was then incubated to generate an antibody-antigen-enzyme-labeled antibody complex. The TMB substrate solution was then added to each well after a wash to get rid of any unbound reagents. Only the wells containing HRP-conjugated HPV antibodies and HPV will initially show blue before becoming yellow upon the addition of the stop solution. Using spectrophotometry, the Optical Density (OD) was determined at 450 nm in wavelength. The qualitative determination of HPV was determined by comparing it with the CUTOFF value

According to our information, this study was the first therefore we can’t make a comparison with another study.  (Table 1) show two types of samples were used to investigate the HPV antigen, for all HPV-positive samples. It has been previously reported that women with late stages of cervical cancer have circulating HPV DNA, which may serve as a prognostic indicator for metastases and recurrences of the illness. The bloodstreams of patients with low-grade or precancerous cervical lesions have only rarely been shown to have HPV. Only a small number of oncogenic HPV types primarily HPV-16 and 18—have been identified in blood samples from women with CC (Cocuzza et al., 2017). The current study found that HPV antigen was negative in sera; this meant there were no oncogenic types in blood samples, and only 3% were positive in tissue samples.

Table 1 shows the type of samples and results of ELISA

This suggests that the HPV proteins involved in genital wart infections never enter the bloodstream and stay in cervical keratinocytes. It also confirms that the expression of viral genes is limited to keratinocytes; no evidence suggests that viral genes are expressed in any other type of cell (Stanley, 2008). Other earlier research revealed that only oncoproteins, such as E6 and E7, derived from oncogenic genotypes, could be identified in whole cells by ELISA (Yang et al., 2012). This means that 3% of HPV antigen-positive genotypes in tissue are carcinogenic HPV genotypes. The virus titer may have been too low for the ELISA to detect, which would explain the false negative. A study demonstrates that the reactions of immunoglobulin-M and-G (IgG and IgM) to HPV16 in various sample types of women with cervical intraepithelial neoplasia (CIN) operate independently of each other. Therefore, choosing the right sample type for the diagnosis of malignancies positive for HPV is challenging (Dong et al.,2021).

The HPV never reaches the blood, so ELISA was not efficient in detecting the HPV antigen in sera, while in tissue samples, the HPV antigen was very low to detect by ELISA in most tissue samples for genital warts infections.

1. McLendon, L., Puckett, J., Green, C., James, J., Head, K. J., Yun Lee, H., Young Pierce, J., Beasley, M. and Daniel, C. L. (2021). Factors associated with HPV vaccination initiation among United States college students. Human Vaccines and Immunotherapeutics, 17(4): 1033–1043.

2. Kombe Kombe, A. J., Li, B., Zahid, A., Mengist, H. M., Bounda, G. A., Zhou, Y. and Jin, T. (2021). Epidemiology and Burden of Human Papillomavirus and Related Diseases, Molecular Pathogenesis, and Vaccine Evaluation. Frontiers in Public Health, 8: 552028

3. Stuqui, B., Provazzi, P. J. S., Lima, M. L. D., Cabral, Á. S., Leonel, E. C. R., Candido, N. M., Taboga, S. R., da Silva, M. G., Lima, F. O., Melli, P. P. D. S., Quintana, S. M., Calmon, M. F. and Rahal, P. (2023). Condyloma acuminata: An evaluation of the immune response at cellular and molecular levels. PloSone, 18(4),e0284296. https://doi.org/10.1371/journal.pone.0284296

4. Bornstein, J. (2019). Vulvar disease. Cham: Springer International Publishing, 343-367

5. Yang, Y. S., Smith-McCune, K., Darragh, T. M., Lai, Y., Lin, J. H., Chang, T. C. and Cheng, S. (2012). Direct human papillomavirus E6 whole-cell enzyme-linked immunosorbent assay for objective measurement of E6 oncoproteins in cytology samples. Clinical and Vaccine Immunology, 19(9), 1474-1479

6. Cocuzza, C. E., Martinelli, M., Sina, F., Piana, A., Sotgiu, G., Dell’Anna, T. and Musumeci, R. (2017). Human papillomavirus DNA detection in plasma and cervical samples of women with a recent history of low grade or precancerous cervical dysplasia. PloS one, 12(11), e0188592

7. Al-Taie, A. A., Abdullah, B. A. and  Al-Attar, M. Y. (2018). Serological and molecular comparison study for diagnosis of cytomegalovirus infection in aborted pregnant women in iraq. Raf. J .27(5): 49-57

8. Dong, Z., Hu, R., Du, Y., Tan, L., Li, L., Du, J. and Cui, H. (2021). Immunodiagnosis and immunotherapeutics based on human papillomavirus for HPV-induced cancers. Frontiers in Immunology, 11, 586796

9. Stanley, M. (2008). Immunobiology of HPV and HPV vaccines. Gynecologic oncology, 109(2), S15-S21