Fig 2: Some older individuals’ sera deposit disproportionately little ficolin-2 on serotype 11A bacteria.Sera were evaluated for ficolin-2 deposition on serotype 11A pneumococcus by flow cytometry and for ficolin-2 content by ELISA. NHS, normal human serum collected from a healthy young adult volunteer.

Fig 3: Some older adults’ sera have heat-resistant ficolin-2 inhibition.Sera were inactivated at 56 °C (solid symbols) or held at room temperature (open symbols) for 45 minutes prior to serial dilution, mixing with recombinant ficolin-2, and incubation with serotype 11A pneumococci. The elevated signal at lower dilutions of the noninhibiting serum sample without heat inactivation is accounted for by the innate ficolin-2 within this sample.

Fig 4: Freshly-collected sera from UAB Study of Aging participants whose archived samples inhibit ficolin-2 have significantly more ficolin-2 than their archived counterparts.Sera were assayed for ficolin-2 content by a commercial ELISA. *, P < 0.05 by paired t-test; connecting lines show matched samples. The mean ficolin-2 concentration (±SEM) was 2.21 ± 0.41 µg/ml for archived sera and 3.89 ± 0.73 µg/ml for matched freshly-collected sera.

Fig 5: Inhibitors in archived sera interfere with an ELISA to quantify ficolin-2.(A) Ten inhibiting sera and ten noninhibiting sera were selected at random from UAB Study of Aging archived sera and assayed for ficolin-2 by a commercial ELISA. Each data point is shown along with mean and standard deviation. NS, not significant; *, P < 0.05; ***, P < 0.001 by one-way ANOVA with Tukey’s post-hoc test. Values for young, healthy controls were previously reported by our laboratory (Brady et al., 2014a). (B) Inhibitors in archived sera interfere with a commercial ficolin-2 ELISA. Three normal human sera (NHS1-3) and four inhibiting sera (IS1-4) were assayed by a commercial ELISA alone or in mixture. Mixtures were designed so that observed ficolin-2 would represent the sum of the samples (see Materials and Methods). Open bars represent the sum of the samples and thus the expected value of the assay. (C) Ficolin-2 immunoblot of sera tested in B suggests that IS1 and IS2 have comparable levels of ficolin-2 to NHS1. Ficolin-2 appears as a band of ~37 kDa under these conditions.