Development Of A Recombinant Antigen-based Capture Enzyme Immunoassay To Detect Recent HIV-1 Infection

Objective:To construct and express an HIV-1 recombinant protein and label it for use in HIV-1 incidence capture enzyme immunoassay(CEIA).To initially establish a new assay with advantages of BED HIV-1 Capture Enzyme Immunoassay(BED CEIA)and Limiting antigen avidity enzyme immunoassay(LAg-Avidity EIA)for detecting recent HIV-1 infection.Methods"(1)One hundred and seventy HIV-1 antibody-positive plasma specimens were screened using the BED CEIA kit,and 81 specimens were selected to construct a recent infection detection reagent evaluating panel with representative normalized absorbance(ODn)values.(2)The nucleic acid sequence for HIV-1 recombinant protein(named as HIV-Ag-R03)was designed and synthesized with reference to the core structure of the antigen rIDR-M used in the LAg-Avidity EIA kit,and then ligated into the pET30a-Trx vector backbone to construct a recombinant plasmid.The recombinant plasmid was transformed into BL21(DE3)cells,and the protein was induced by isopropylthiogalactoside(IPTG).Plasma specimens from 24 HIV-1 antibody positive and 24 HIV negative individuals were used to analyze the antigenicity of the recombinant protein after renaturing and purifying.(3)HIV-Ag-R03 labeled with horseradish peroxidase(HRP)was applied to replace the biotinylated BED peptide and subsequent components from the BED CEIA kit.The results of detecting recent infections in 18 HIV-1 positive specimens were compared with those using the BED CEIA kit to explore the feasibility of the new assay.(4)The new CEIA assay was evaluated preliminarily by detecting the recent infection detection reagent evaluating panel,as well as by analysing the correlation between the results of CEIA and the results of BED CEIA or LAg-Avidity.Results:(1)A recent infection detection reagent evaluating panel(named as BEDn81)was constructed with considering the representativeness of the source of specimens and the ODn values of the BED CEIA.(2)The recombinant plasmid for HIV-Ag-R03 was constructed,and the nucleic acid sequence was confirmed.Inclusion body of the recombinant protein was obtained,and the size of Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis(SDS-PAGE)analysis was consistent with the predicted value.Using an indirect enzyme immunoassay coated with HIV-Ag-R03 to detect 48 plasma specimens,the absorbance(OD)values for HIV-1 antibody positive specimens(0.794±0.193)were significantly higher than those for HIV negative specimens(0.117±0.073),the difference was statistically significant(P<0.01),which means that HIV-Ag-R03 has good antigenicity.(3)To replace the biotinylated BED peptide and subsequent components from the BED CEIA kit,the optimal dilution of HIV-Ag-R03-HRP was 1:2500.The P50(P25-P75)of ODn values,2.401(1.341～4.160),for 18 HIV-1 positive specimens obtained with this new assay correlated well with those,0.884(0.369～1.942),by using BED CEIA kit(r=0.939,P<0.01).The new assay is feasible.(4)Microwell plates were coated with goat anti-human IgG(diluted 1:500).HIV-Ag-R03-HRP was used and other components were debuged to develop a new CEIA initially.The results of detecting 81 specimens of the panel BEDn81 by using new CEIA show that the ODn values,2.913(1.761～4.297),of the new CEIA are strongly correlated with those,1.114(0.557～1.675),of BED CEIA(r=0.869,P<0.01).And the ODn values of the new CEIA are strongly correlated with those,2.493(1.609～3.716),of LAg-Avidity(r=0.810,P<0.01).The ODn values of new CEIA are higher,which suggests the new CEIA has stronger capability to distinguish recent infection and long-term infection.If we use ODn=2.5 as the Cutoff value of the new CEIA to distinguish recent infection and long-term infection,the coincidence rate of result of the new CEIA was 85.2%compared with result of BED CEIA(Kappa=0.698,P<0.01),which suggests that the assay needs further optimization and evaluation,including setting the Cutoff value of recent infection,and researching the core technical parameters applicable to HIV-infected people in China(Window Period,False Recent Rate,etc.).Conclusions:An antigenic and labelable recombinant antigen HIV-Ag-R03 was constructed and expressed.And a new assay to detect recent HIV-1 infection with the advantages of the BED CEIA and the LAg-Avidity EIA was initially established.The new CEIA needs to be further optimized and evaluated.