| Research Abstract |
At present, though PCR, bDNA assay and NASBA are used in the measurement of provirus DNA and virus RNA such as HIV and HCV, detection limit is tens of thousand copies from several hundred. This is because the signal by the nonspecific binding of conjugates of DNA probe prevents the sensitive measurement. Then, using the novel method of 2-site binding complex transfer method which applicants developed, the specific signal was maintained, and by almost perfectly removing the non-specific signal, it was made that the ultrasensitive measurement is enabled to be a purpose. The sensitivity of RT-PCR method and the ultrasensitive 2-site binding complex transfer method of HIV-1 p24 antigen was compared by the using seroconversion panel serum. As a result, the detection period of the p24 antigen and RNA were most same time. The detection limit of RT- PCR of this experiment was the 400 copies/ml. In this time, anti-p17 IgG and IgM were early detected from p24 antigen and the virus RNA. This result showed that virus RNA was existed before the detection time of IgG and IgM. Then, the supersensitive method of amplified DNA after RT-PCR using DNA prove was developed. After hybridization of amplified DNA with both FITC and DNP labeled avidin-biotinyl DNA prove I and β-D-galactosidase labeled avidin-biotinyl DNA prove II, the complex was trapped on the anti-DNP IgG - solid phase. By excess DNP- lysine addition, the complex is eluted from the solid phase, and it is transfed on the anti-FITC IgG -solid phase. Finally, the enzyme activity on solid phase is measured.
The detection limit of this transfer method was 10 amol (10^<17> mol). Because the amplification of 10^<6-8> 8copies of DNA was possible, an outlook to the detection of DNA of 6 copies was obtained.
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