Immuno-chromatographic Analysis for HPV-16 and 18 E7 Proteins as a Biomarker of Cervical Cancer Caused by Human Papillomavirus

Abstract

Among the more than 120 different types of human papillomavirus (HPV), types 16 and 18 have been known to be high risk agents that cause cervical cancer. We examined, in an immuno-chromatographic analysis, the potential of using the early gene product, E7 protein, as a diagnostic marker of cervical cancer caused by HPV. We developed monoclonal antibodies specific to HPV-16 and 18 E7 proteins that were produced from bacterial cells using gene recombinant technology. For each E7 protein, the optimal antibody pair was selected using the immuno-chromatographic sandwichtype binding system based on the lateral flow through membrane pores. Under these conditions, this rapid testing assay had a detection capability as low as 2 ng/mL of E7 protein. Furthermore, since viral analysis required the host cell to be lysed using chemicals such as detergents, it was possible that the E7 protein was structurally damaged during this process, which would result in a decrease in detection sensitivity. Therefore, we examined the detrimental effects caused by different detergents on the E7 protein using HeLa cells as the host. In these experiments, we found that the damage caused by the detergent, nonylphenylpolyethylene glycol (NP-40), was minimal relative to Triton X-100 commonly used for the cell lysis. Temperature also affected the stability of the E7 protein, and we found that the E7 protein was stabilized at 4 o C for about 2 h, which was 4 times longer than at room temperature. Finally, a HPV-infected cervical cancer cell line, which was used as a real sample model, was treated using the optimized conditions and the presence of E7 proteins were analyzed by immuno-chromatography. The results of this experiment demonstrated that this rapid test could specifically detect HPV-infected samples.

Significance of serum antibodies against HPV E7, Hsp27, Hsp20 and Hp91 in Iranian HPV-exposed women

“…As known, E6 and E7 transforming proteins are consistently expressed in HPV life cycle [39–41]. Several studies have shown the induction of antibody responses against E6 and E7 oncoproteins in patients with cervical cancer higher than in healthy subjects [39, 42, 43].…”

Literatur Review : Rapid Immunochromatography Sebagai Metode Skrining Kanker Serviks Berbasis Deteksi Onkoprotein HPV pada Urin

“…Protein E6 dihitung menggunakan metode Bicinchoninic Acid (BCA). Langkah tersebut diulang untuk menghasilkan antigen protein E6 HPV-18 hingga didapatkan keseluruhan protein rekombinan yang dibutuhkan (Yetisen et al, 2013;Kim et al, 2009). Untuk mendapatkan antibodi monoklonal digunakan mencit sebagai hewan coba.…”

“…Pada test line di NC membrane diletakkan antibodi yang telah disiapkan sebanyak 1 μL. Antibodi protein E6 HPV-16 diletakkan pada T1 dan antibodi protein E6 HPV-18 pada T2 (Kim et al, 2009). Control line mengandung antibodi spesifik terhadap konjugat partikel emas.…”