Trypsin pre-treatment corrects SRID over-estimation of immunologically active, pre-fusion HA caused by mixed immunoprecipitin rings

Abstract Influenza vaccines are the primary intervention to prevent the substantial health burden of seasonal and pandemic influenza. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on their content of immunologically active (capable of eliciting functional antibodies) hemagglutinin (HA). Single-radial immunodiffusion (SRID), the standard in vitro potency assay in the field, is believed to specifically detect immunologically active HA. We confirmed that, with conformationally homogeneous HA preparations, SRID specifically detected native, pre-fusion HA, which elicited influenza neutralizing and hemagglutination inhibiting (HI) antibodies in mice, and it did not detect low-pH stressed, post-fusion HA, which was selectively removed from the SRID gel during a blotting step and was significantly less immunologically active. This selective detection was due to the SRID format, not a conformational specificity of the sheep antiserum used in the SRID, as the same antiserum detected non-stressed and low-pH stressed HA similarly when used in an ELISA format. However, when low-pH stressed HA was mixed with non-stressed HA, SRID detected both forms in mixed immunoprecipitin rings, leading to over-quantification of pre-fusion HA. We previously reported that trypsin digestion of antigen samples selectively degrade stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique, reversed-phase high pressure liquid chromatography (RP-HPLC), can specifically quantify trypsin-resistant, immunologically active, pre-fusion HA. Here, we report that trypsin digestion can also improve the specificity of SRID so that it can quantify immunologically active, pre-fusion HA when it is mixed with less immunologically active, post-fusion HA.