Complement is a critical reagent in serum bactericidal activity (SBA) assays, but human complement sources can be difficult to identify and standardize. For assays against serogroup B Neisseria meningitidis (MenB), separate complement sources must be identified and qualified for each strain tested. IgG/IgM-depleted serum has the potential to be a universal human complement SBA (hSBA) source if function of the complement cascade can be preserved and adequate performance in assays can be demonstrated.
To determine if large-scale antibody-depleted serum is suitable for use in MenB hSBA assays, we screened 40 donor sera to determine intrinsic killing against five MenB strains. We created one pool of intrinsically negative sera, representing a standard hSBA pooled complement, and a pool of broadly bactericidal sera [C’(-) and C’(+), respectively]. One half of each pool was aliquoted as unprocessed complement and the other half underwent IgG/IgM depletion [dC’(-) and dC’(+), respectively]. We compared these four C’ pools in MenB hSBA assays using six different strains and at least eight heat-inactivated test sera with negative, low, medium, or high hSBA. To further characterize the IgG/IgM depleted complement, a comparative quantitative proteomics study was conducted using data-independent acquisition (DIA) Mass Spectrometry.
Overall, 95% of titers determined with C’(-), dC’(-) or dC’(+) were within expected assay variation of ± 2-fold dilution of the median titer (343 of 359 titers), including all test strains and sera assayed (n=52 strain-serum pairs). Comparative quantitative proteomic results confirmed depletion of immunoglobulin proteins. Fifteen other proteins were significantly more abundant in the pools prior to depletion using a cutoff of 1.5-fold difference, including galectin-3-binding protein, fibulin, complement C1q subcomponent subunit B, and C4b-binding protein alpha chain, which were ≥2-fold more abundant prior to IgG/IgM depletion.
IgG/IgM depletion removed bactericidal activity from the C’(+) pool, and both the positive and negative depleted pooled complement sources were similar to the negative complement pool when used as complement sources in MenB hSBA assays. Proteomic characterization of the effects of IgG/IgM depletion on pooled serum may be relevant for additional applications of this source of active human complement.