Sensitive immunoassays have detection limits on the order of 1 pg/mL, corresponding to approximately one million protein molecules per sample. In contrast, PCR can sometimes detect down to a single nucleic acid molecule. PCR and related technologies are complex and expensive, and therefore not suitable for all settings (Westreich 2008). However, the detection of HIV infection is one application where an ultrasensitive immunoassay could serve as a valuable diagnostic tool.
Patients who have recently been infected with HIV contribute disproportionately to the spread of the disease. Viral loads are high in the first few weeks after infection. Newly infected patients are unlikely to be aware that they are infected and can spread the disease to others. Therefore, early detection of an acute HIV infection is of great importance for public health. Each virus particle contains approximately 2,000 copies of the p24 capsid protein, but only one copy of RNA; thus an ultrasensitive immunoassay to detect the p24 protein might be an alternative to nucleic acid detection technologies.
We are developing a novel ultrasensitive electrochemiluminescence assay format based on MSD’s MULTI-ARRAY® technology. MULTI-ARRAY technology has been widely adopted by researchers for its high sensitivity, excellent reproducibility, and wide dynamic range. Our new ultrasensitive assay format has the capability to detect proteins in the fg/mL range. We have demonstrated detection limits of less than 1 fg/ml for several cytokines (Glezer 2014), and recently developed an assay for free and complexed prostate specific antigen (PSA) with sufficient sensitivity to detect native levels in women (Nikolenko 2015). We have now applied this technology to the early detection of HIV infection (Stengelin 2015).
The detection limit for our HIV p24 immunoassay is approximately 1 fg/mL - over 1,000 fold more sensitive than current 5th generation p24 immunoassays. A sensitivity of 1 fg/mL is sufficient to detect one virus particle per 25 μL of sample volume. p24 was undetectable in the serum or plasma of 32 apparently healthy donors. Two seroconversion panels were tested: SeraCare PRB948 (days 0 and 18, PCR negative; days 22 and 23, PCR positive) and PRB962 (days 0 and 2, PCR negative; days 7, 9, 14, and 17, PCR positive). In both cases, the MSD p24 assay result was negative for all PCR-negative samples and positive for all PCR-positive samples, and infection was detected well before conventional p24 immunoassays.
In conclusion, we developed a new-generation p24 immunoassay that is comparable in sensitivity to PCR assays.
- D.J. Westreich, M.G. Hudgens, S.A. Fiscus, C.D. Pilcher. Optimizing screening for acute human immunodeficiency virus infection with pooled nucleic acid amplification tests. Journal of Clinical Microbiology, 46, 1785 (2008).
- E.N. Glezer, M. Stengelin, A. Aghvanyan, G. N. Nikolenko, D. Roy, M. Higgins, J. Kenten, G. B. Sigal, and J. N. Wohlstadter (2014, November). Cytokine Immunoassays with Sub-fg/mL Detection Limits. Poster presented at the American Association of Pharmaceutical Sciences Annual Meeting, San Diego, CA.
- G. N. Nikolenko, M. Stengelin, L. Sardesai, E. N. Glezer and J. N. Wohlstadter (2015, April). Accurate measurement of free and complexed PSA concentrations in serum of women using a novel technology with fg/mL sensitivity. Poster presented at the American Association for Cancer Research Annual Meeting, Philadelphia, PA.
- M. Stengelin, D. Roy, A. Aghvanyan, J. Kenten, G. B. Sigal, E. N. Glezer, and J. N. Wohlstadter (2015, July). HIV p24 Immunoassay with the Sensitivity of PCR Methods. Poster presented at the American Association for Clinical Chemistry Meeting, Atlanta, GA.