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. 2021 Mar 16;12:636703. doi: 10.3389/fmicb.2021.636703

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Copyright © 2021 Wu, Cheney, Huang, Hazuda, Howell and Zuck.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Optimization of the immunoprecipitation (IP) Simoa assay. (A) Selection of IP capture antibody. Recovery of p24 using antibodies from various sources, conjugated to magnetic beads, showed the Capricorn antibody to yield the best capture and was similar to the input concentration after release and volume adjustment. (B) Optimization of bead concentration for p24 IP. Using a 10 mg/mL stock, adding >10 μL of antibody-conjugated beads to a lysate is sufficient for p24 capture. (C) Elution buffer selection. Comparing various elution buffers, 0.1% TFA had no significant difference from input solution after assay, indicating it efficiently releases p24 without destroying crucial epitopes. Other elution buffers resulted in poorer p24 recovery. (D) Time required for p24 IP capture. Recovery of p24 with 2- or 4-h incubation with antibody-conjugated beads is markedly lower than that of input solution, whereas recovery with 24 or 48 h gave recovery similar to that of the input solution (no IP). See Materials and Methods for statistical analysis.