Figure 9

Binding profile of human serum IgM and IgG, and NP-specific mAbs on LASV VLP and recombinant nucleoprotein. Human sera collected from household contacts of patient G676, individuals hospitalized at the KGH at the time of analysis, or from supposedly LASV naive controls were diluted 1:100 in a proprietary sample diluent buffer containing 0.05% Tween 20 (Corgenix Medical Corp.) and assayed by ELISA on plates coated with 2 μg/mL total VLP protein (Figure 9A, 9C) or 2 μg/mL rNP (Figure 9B, 9D) per well. Detection of bound human IgM (Figure 9A, 9B) or IgG (Figure 9C, 9D) was performed as outlined in methods. LASV VLP captured IgM from three samples (G676-M, G676-Q, G688-1), all of which were also detected by rNP ELISA (Figure 9A, 9B), but did not result in binding by IgM from 14 additional samples that also tested positive on rNP (Figure 9A, 9B), including the G652-3 positive control. Similarly, VLP detected LASV-specific IgG in 2 samples (G679-2, G679-3), but did not identify 24 others detected in rNP ELISA (Figure 9C, 9D). For analysis of mAb binding profiles LASV VLP were coated in high protein binding ELISA plates at the same concentration as above. The indicated NP-specific mAbs were then used in a binding assay, at 1 μg/mL, alongside mouse IgG as a negative control (Figure 9E). For capture and detection of NP in solution, each NP-specific mAb was coated on ELISA plates at 5 μg/mL, followed by incubation with serial dilutions of nucleoprotein in sample diluent (Figure 9F). Captured NP was detected with a polyclonal Goat α-NP-HRP conjugate.