In-house ELISA protocols for capsid p24 detection of diverse HIV isolates

Abstract

Background The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates. Methods Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes. Results Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype. Conclusions These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits.

QuickFit: A high-throughput RT-qPCR-based assay to quantify viral growth and fitnessin vitro

“…Individual viral growth assays rely on measuring viral proteins, genetic material, physical particles, or reporter gene expression (38). These methods are usually time-consuming, expensive, and have different degrees of sensitivity between different viral isolates resulting in inconsistent growth rates (25)(26)(27)39). Dual infections or competitive growth assays employ a reference and a test strain to infect cells in the same culture, and the two strains are monitored via env gene heteroduplex tracking assays, allowing quantification of their growth and fitness (37,40).…”

“…Particularly, HIV NL4-3 exhibited the strongest signal, while HIV BF520 was only detectable at the highest inoculums, leading to wider measurement error. Protein alignment of the p24 capsid proteins of HIV NL4-3 and HIV BF520 results in pairwise identity of 91.3%, emphasizing that even relatively minor differences in antigens can have large impacts on the sensitivity of p24-based ELISAs (25).…”

“…Longitudinal samples are subjected to either ELISA-or RT-qPCR-based methods to measure viral antigens or genetic material, respectively (22). However, these approaches are time-consuming, expensive, and have varying degrees of sensitivity (23,24), which leads to inconsistent reports of growth rates in the literature (25)(26)(27).…”