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. 2021 Jul 21;10(8):1843. doi: 10.3390/cells10081843

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Improved sensitivity of CA/09 rHA-specific ASC through affinity capture of coating antigen. PBMC were stimulated in vitro and evaluated for antibody-secreting (ASC) reactivity against recombinant hemagglutinin protein representing the A/California/04/2009 (CA/09) H1N1 vaccine strain. (A) Representative well images depicting antigen-specific IgG⁺ ASC in wells coated directly with 10 μg/mL of CA/09 rHA or through affinity capture using the genetically encoded hexahistidine (6XHis) tag. Magnification and contrast enhancements were uniformly performed on all images to aid their visualization in publication. (B) CA/09 rHA-specific FluoroSpots were merged into FCS files and visualized as bivariate plots. FluoroSpots originating from assay wells in which CA/09 rHA was directly captured on the membrane (black dots) or through affinity capture (red) are shown as overlays. The combined number of FluoroSpots detected in replicate wells for each of the respective donors is indicated in the inset using the same red/black color code.