Development of novel ultra sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for provirus DNA and virus RNA of HIV-1 (Early diagnosis of HIV-1 infection)

目前，对HIV、丙型肝炎病毒等前病毒DNA和病毒RNA的检测虽然采用了聚合酶链式反应、BDNA测定法和NASBA法，但检测下限从几百个拷贝增加到几万个拷贝。这是因为DNA探针的偶联物的非特异性结合所产生的信号阻碍了灵敏的测量。然后，利用申请人开发的新方法--2位结合络合物转移法，保持了特异性信号，并几乎完美地消除了非特异性信号，使得超灵敏测量成为可能。用血清转换板血清检测HIV-1p24抗原，比较RT-PCR法和超灵敏的HIV-1p24抗原双位点结合复合体转移法的敏感性。因此，p24抗原和RNA的检测周期最长。本实验的RT-PCR检测下限为400拷贝/毫升，可从p24抗原和病毒RNA中早期检测到抗p17抗体和IgM抗体。这一结果表明，在检测到Ig G和Ig M之前，病毒RNA就已经存在。在此基础上，建立了利用DNA Proven进行RT-PCR扩增DNA的超灵敏方法。将扩增的DNA分别与FITC和DNP标记的亲和素生物素DNA pron I和β-D-半乳糖苷酶标记的亲和素生物素DNA pron II杂交后，复合物被固定在抗DNP免疫球蛋白的固相上。通过过量的DNP-赖氨酸加成，将络合物从固相中洗脱出来，并将其转移到抗FITC抗体的固相上。最后测定了酶在固相上的活力，该转移方法的检出限为10amol(10^~lt；17~gt；m ol)。由于扩增10~8个拷贝的DNA是可能的，因此对6个拷贝的DNA检测进行了展望。

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.