Ipla n

To test total IgG from plasma, 384‐well HB plates (Perkin Elmer) were coated with AffiniPure Goat anti‐human IgG, Fc gamma fragment antibody, at 1 μg/ml in PBS for 2 h at 37°C. To test anti‐PrP autoantibodies, plates were coated with human recPrP_23‐230. After washing and blocking in 5% Milk in PBS‐T for 90 min at RT, serial dilutions of patient plasma (starting from 1:8,000 and 1:50 dilution for total IgG and anti‐PrP autoantibody ELISA, respectively) were incubated for 2 h at 37°C. Pooled normal human plasma (Innovative Research, #IPLA‐N), IgG‐Kappa, IgG‐Lambda at a concentration of 200 ng/ml or humanized POM1, (hPOM1 at 200 ng/ml and 2 μg/ml for total IgG and anti‐PrP autoantibody ELISA, respectively) served as controls. After washing five times, the following HRP‐coupled secondary antibodies were incubated at 1:4,000 in PBS‐T for 1 h, at RT: Goat anti‐human Kappa‐HRP, goat anti‐human Lambda‐HRP or goat anti‐human IgG Fc‐gamma specific‐HRP (Jackson ImmunoResearch #109‐035‐098). The plates were developed as described above.

NIH-CoVnb-112 expressed in Pichia pastoris was diluted from a concentrated stock solution into apheresis derived pooled human plasma (#IPLA-N, Innovative Research) to a final concentration of 5 µM and incubated at 37 °C for either 24 hr or 48 hr with gentle rotation. An identical sample set was prepared at 5 µM in a solution containing 35 mg/mL recombinant human albumin (#A9731, Sigma-Aldrich) and incubated at 37 °C for either 24 hr or 48 hr with gentle rotation. A no-incubation control for each the plasma and recombinant human albumin conditions was prepared at 5 µM. The samples were prepared in a manner providing all conditions complete at the same time. The samples were diluted 1:10 with 1xPBS to yield a final nanobody concentration of 500 nM and Bio-layer Interferometry was performed using immobilized biotinylated SARS-CoV-2 S protein RBD to determine retention of binding potential.

Pooled normal human plasma, collected in EDTA vial (IPLA-N) and pooled normal human serum (IPLA-SER) were purchased from Innovative Research. Human plasma collected with sodium citrate (P9523) was purchased from Sigma. Serum samples of 10 healthy controls and 10 gastric cancer patients were purchased from Asterand Bioscience.