Preparation,Identification And Application Of Monoclonal Antibody Against Tat Protein Of HIV-1

Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). Currently HIV tests conclude screening assays and confirmed tests. ELISA is the mostly useful screening assays, and some rapid HIV tests are also available. Any HIV positive result given by an ELISA test must therefore be confirmed using a second test. The confirmed tests conclude Western Blot Assays (WB), radioimmunoprecipitation assay (RIPA), immunofluorescent assay (IFA), and so on. WB is the usual method for the confirmed test internal. These methods have nice sensitivity and specificity, but are all HIV antibody tests. They cannot test the antibody during the window period (the time between initial infection and the development of detectable antibodies against the infection), so antigen test and PCR test are as the assistant tests. The antigen on HIV that most commonly provokes an antibody response is the protein p24. Early in the infection, p24 is produced in excess and can be detected in the blood serum. However p24 antigen often varies to cause false test. PCR tests are not often used to test for HIV in adults, as they are very expensive and more complicated to administer than a standard antibody or p24 test. So looking for a new testing target to establish a new testing method can be as a useful HIV assistant test. HIV-1 Tat(trans-activator of transcription) is one of regulative proteins, and is also a small nuclear protein binding with RNA. Tat vastly increases the level of transcription from the HIV DNA, and is one of the most early expression proteins. In conclusion, Tat is more and more to be pay attention of as a target of AIDS test.In this study we generated three cloned mAbs from BALB/c mice immunized with the Tat, named A10,E2 and C10. We tested specific of the mAbs and found that the three mAbs could bind Tat specially, but gp41-5 and Bcsp31. The relative affinity sequence of the mAbs is C10>E2> A10. The results of epitope analysis with competitive ELISA showed that the three mAbs could recognize the different epitopes.Then we choose mAbs A10 as the capture mAb and mAb C10 as the anzyme-conjugate mAb in order to establish an antibody-sandwich enzyme linked immunosorbent assay (ELISA) for detection of HIV-1 Tat antigen. The method has good stability. By antibody-sandwich ELISA, the positive rates of HIV positive serums were 30% (6/20).All these results gained in this research will be effective tools in early detection and therapy of HIV-1 infection.