Fig 2.

Isolating IgM mAbs that mediate complement-mediated neutralization in an immunologically naïve individual. (A) Schematic outline of the EBOV-GFP neutralization screening strategy. Graph created with BioRender.com. (B) Normalized infectivity data from the screen with the preliminary hit wells (>5 standard deviations below the mean normalized signal) in color. (C) Example images from the screen depicting the strongest hit (9-K21) and a control well. (D) Data from the hit verification round. Raw supernatants of the nine hit wells were tested for neutralization activity at seven dilutions and for toxicity at one dilution. The heat map represents infectivity (viability) normalized against control wells that received no B-cell supernatant. (E) Summary of sequencing results from the hit wells. For each hit well (9-K21, 14-E14, and 14-H15), the CDR sequences of the top contigs (by number of reads) are written out. The neutralizing combinations are depicted by a gray background. The colors of the vertical bars represent the relative read abundancies. (F) Neutralization experiment identifying the functional heavy and light chain combinations. Each kappa and lambda light chain was combined with each heavy chain of a hit well, and the IgM was expressed in small scale. The raw supernatants were tested for neutralization activity against EBOV-GFP at three dilutions and toxicity against Vero-E6 cells.