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 ...
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$^{\circ}C$ 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.
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$^{\circ}C$ 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.
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제안 방법
The aim of this study was to develop a potential alternative HPV detection method, the immuno-chromatographic assay, which may not only be highly accurate, but also rapid, easy-to-use, and inexpensive. The assay involves a one-step procedure where the complex antigen-antibody reactions are sequentially carried out by the lateral flow along the membrane strip upon the addition of a specimen.
The conjugate pad was prepared by spraying the two detection antibodies on the glass fiber membrane and drying it at 35 ℃ overnight. The signal generation pad was prepared by immobilizing the following three different antibodies on spatially separate sites of the NC membrane (from the bottom): the capture antibody to HPV-16 E7 (clone #119), the capture antibody to HPV-18 E7 antibody (#36), and an anti-mouse IgGpolyclonal antibody as the control. They were dispensed on eachpre-determined site ina linearpattern onthe NC membrane and then incubated at 37 ℃ for 20 min.
성능/효과
17 Stand ard samples of the target proteins were prepared and used to analyze the immunochromatographic assay according to the pre-determined protocol. The results of this experimentrevealed that the produced signals were proportional to the analyte concentration and detectable by the naked eyes at an analyte concentration as low as 2 ng/mL for the both of HPV-16 and 18 E7 proteins (Table 1).
The infection-free samples, MCF-7 cell and A549 cell, did not exhibit any signals at the analyte lines. These results demonstrated that the rapid testing was not only highly specific, but also capable of detecting E7 proteins produced from cancer cells infected with HPV.
참고문헌 (25)
Walboomers, J. M.; Jacobs, M. V.; Manos, M. M.; Bosch, F. X.; Kummer, J. A.; Shah, K. V. J. Pathol. 1999, 189, 12
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