Binding characteristics of selected commercial antibodies against recombinant HIV Subtype D P24 antigens

Abstract

The standard method for monitoring HIV treatment response is quantitative RT-PCR, but it is difficult to implement in low and middle income countries due to infrastructure and personnel requirements. Point of care technologies for HIV VL determination are needed, but available ones mostly rely on nucleic acid amplification with requirements that still limit their access in these areas. Earlier p24-based technologies as alternatives had low accuracy. One of the major reasons for their failure is poor binding characteristics of anti-p24 antibodies against diverse HIV subtypes, and a lack of reagents to sufficiently disrupt host p24 antigen-antibody immune complexes. Our study aimed at characterizing commercially available anti-p24 antibodies against HIV-1 p24 antigens from which the best antibodies would be used in the development of the innovative rapid diagnostic assay. From April to November, 2022, we conducted a laboratory based innovative study from Makerere University at the Department of Immunology and Molecular Biology. HIV-1 recombinant p24 protein were obtained from MyBioSource & Two anti-P24 antibodies from; Genscript and RayBioTec. We planned to design and optimize an antigen capture ELISA an use it determine the binding avidity of the commercial antibodies by the chaotropic method using Potassium Thiocyanate as the chaotropic agent. The antibody with best binding avidity would be chosen for use in the development of semi-quantitative locally relevant HIV-1 viral load assay. Controls of the antigen capture ELISA failed with ODs ≥1. Reproducibility of the experiments was very poor. Troubleshooting by variation of blocking buffers including use of 5% skimmed milk as an alternative, use of 1:1000 dilution instead of 1:500 dilution of HRP and even change of the secondary antibody from the Goat Anti-HIV-1 p24 polyclonal Antibody, HRP to Goat anti-Mouse to Goat anti-Mouse IgG Antibody, HRP did not improve the antigen capture ELISA. With the above challenges coupled with time constraints and the lack of other relevant resources like precoated plates, we failed fully trouble shoot, redesign and optimize the antigen capture ELISA and later on characterize the primary antibodies for which the study was intended. However, we designed an indirect antigen down ELISA which can be deployed for other purposes. Precoated ELISA plates, characterization of all reagents before use can be deployed in later experiments to improve the antigen capture ELISA design and optimization.