Construction of a p24 epitope homogeneous immunoprobe based on FRET and its application in rapid clinical detection of anti-HIV antibodies
Objective To construct and clinically validate a fluorescent protein probe for HIV p24 antigenic epitope detection based on genetically encoded fluorescence resonance energy transfer(FRET)technology,laying the foundation for the development of a homogeneous immunoassay reagent for the rapid detection of anti-HIV P24 antibodies.Methods The fluorescent proteins ECFP and EYFP were used as FRET donors and acceptors,respectively,and nucleic acid sequences corresponding to p24 antigenic epitopes were inserted into an ECFP-Linker-EYFP probe backbone.A p24 fluorescent protein dimerization interface was constructed using targeted mutagenesis,and a recombinant plasmid was constructed using the pET28a vector,which was transformed into E.coli BL21 recipient cells to induce the expression of p24 antigenic epitope fluorescent protein probes,and purified using Ni columns and molecular sieves.The optimal concentration of the probe was screened,and the binding ability and spectroscopic changes of the probe to anti-p24 polyclonal antibodies were detected.Sixty sera from HIV-positive patients and 58 sera from normal subjects were selected for detection by the probe,and the efficacy of the probe for the detection of anti-HIV p24 antibody was evaluated by specificity,sensitivity,positive predictive value,negative predictive value,and diagnostic efficiency.Results High-throughput sequencing results demonstrated that the p24 antigen epitope fluorescent protein probe was successfully constructed,its theoretical molecular weight was approximately 67 kDa,and the optimal concentration was 0.20 mg/mL.The fluorescence intensity ratio at 530/477 nm was reduced after binding the p24 polyclonal antibody to the probe(2.13 vs.1.47).The 530/477 nm ratio(1.42±0.21)of HIV-positive patient sera was significantly lower than that of normal patients(1.76±0.11),P＜0.001.ROC curve analysis of the ratio between the two groups yielded an area under the curve of 0.97 with a standard error of 0.02,P＜0.001.The sensitivity of the p24 antigenic epitope fluorescent protein probe for the clinical detection of the HIV p24 antibody was 88.33％,and the specificity was 100.00％.The positive predictive value was 88.33％,the negative predictive value was 100.00％,and the diagnostic efficiency was 94.07％.Conclusions The successful construction of a FRET-based p24 antigen epitope homogeneous immunoassay probe for the detection of anti-HIV p24 antibody in HIV/AIDS patients exhibited good sensitivity and specificity,significantly shortened the detection time,and provided insights for further improvements in sensitivity and timeliness of anti-HIV p24 antibody laboratory testing.
HIVp24antibody detectionfluorescence resonance energy transfer(FRET)
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