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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session E

Poster Y18 - Probe Development for a Novel Ultrasensitive Test for Early Acute HIV Detection

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Human Immunodeficiency Virus (HIV) continues to be a major global health concern as there are 39 million people living with HIV (PLHIV) globally. Although it is not a curable disease, it has become a manageable condition if proper prevention, diagnosis, and treatment are accessible. The Acute HIV infection (AHI) phase is the first and earliest stage of infection, and it is classified as the most contagious phase after initial infection. Detection and treatment during this stage hinders disease progression; however, if AHI goes undetected, the infection can further multiply and lead to severe health consequences. Early testing and diagnosis of an acute HIV infection (AHI) is a key strategy for placing patients in the HIV care continuum thereby limiting disease progression and transmission by initiating access to proper and effective treatment to improve patient quality-of-life and patient survival. Existing rapid diagnostic tests (RDTs) cannot detect AHI, and the current gold-standard diagnostic methods are detecting HIV RNA are impractical and not easily accessible to all. To close the gap in AHI detection, we propose to develop an ultrasensitive test antibody-initiated loop mediated isothermal amplification (ai-LAMP), depicted in Figure 1, for the detection of the HIV capsid protein p24 antigen, a biomarker present in the AHI phase at high concentrations. This novel assay combines the sensitivity of LAMP with the selectivity of p24 antibody binding in a user-friendly platform like an RDT. Here we focus on the development of the oligo-conjugated antibody probes for the initiation of the detection assay.

Materials and

Methods:  A high antibody-oligonucleotide conjugation efficiency is desired to attain a more efficient detection assay. Here, we optimize our conjugation method to determine which will yield the most efficient ai-LAMP probes. The p24 specific target antibodies and the target oligonucleotides are both functionalized in preparation for conjugation. The functionalized antibodies are then conjugated with their respective oligonucleotide; 47P to forward primer and 49P to the reverse primer. In this study, parameters such as ratios, concentrations, and incubation time and temperature are varied in both the functionalization step and the conjugation step. Moreover, the use of zeba columns and amicon columns are also tested in both steps. The conjugation is assessed via gel electrophoresis, protein staining and DNA staining. The protein-stained gel images are taken for analysis of conjugate band intensity using Fiji Image J software providing a numerical value for the calculation of the conjugation efficiency.

Results, Conclusions, and Discussions: In these studies, we demonstrate that our antibodies and oligonucleotides can be conjugated. The expected band size for our conjugate products is approximately 185-188 kDa based on the size of our antibody and oligo. From our results depicted in Figure 1, a band is present at 185 kDa, which demonstrates that a conjugate product is present. However, it can also be seen that there are fragmented bands present, which could be caused by possible degradation of the product, antibody, or oligo; this could be affecting the efficiency therefore, more experimentation is necessary to resolve the current issues and to further improve the conjugation efficiency for our detection assay.

Acknowledgements (Optional):  NIH NIDA DP2DA05191