Development Of Monoclonal Antibodies Against HIV-1p24Protein And Its Application In Immunological Assays

P24antigen is the capsid protein of human immunodeficiency virus type1(HIV-1) andplays an important role in viral core assembly and maturation. P24protein is always present atrelatively high levels in blood serum of patient during early and late stages of HIV-1infection,making it a biomarker for early diagnosis, blood donor screening and monitoring of diseaseprogression.The synthetized p24gene was amplified by PCR and constructed into pET28a vector.Recombinant p24protein was successfully expressed in E. coli in the soluble form and theoverall purity was above95%after one-step affinity chromatography purification. Balb/cmice were immunized with recombinant p24protein. After four times of immunizations, themice were sacrificed and the spleen cells were harvested and fused with SP2/0myeloma cells.33stable hybridoma cells secreting anti-p24monoclonal antibodies (mAbs) were establishedafter enzyme-linked immunosorbent assay ELISA screening and subcloning by limitingdilution. MAbs were purified from the ascites by ammonium sulfate precipitation and/or byProtein G column methods.34different coating and labeling mAb pairs that capture HIV-1p24antigen in a sandwich format were screened by a novel antibody-capture indirectsandwich ELISA. MAb pairs screened out were then applied in colloidal goldimmunochromatographic assays (GICAs) to detect p24. An antibody pair consisting ofcapture (mAb3-1D5) and detector (mAb4-8A10) that showed a relatively higher sensitivityto p24was selected for later GICA tests. Our GICA method yielded a limit of detection (LOD)of25pg/mL and could detect p24protein in all10positive samples obtained from theNational Reference of HIV-1p24antigen. Only three of HIV-1&2antibodies and p24negative samples (n=153) that were confirmed as negative by commercially available fourthgeneration HIV ELISA test kits were however reported positive by the GICA. Our resultsindicate that the developed GICA method could provide a convenient and efficient approachto detect p24antigen for early diagnosis of HIV infection and find application in the fourthgeneration HIV immunoassays.