Microbiome-Specific Antibody Detection Assay

Human gut commensal bacteria, comprising 33 commensal bacteria strains (RePOOPulate)^{56 (link)}, double-stranded DNA (UltraPure Salmon Sperm DNA, Thermo Fisher) and bovine serum albumin (BSA, Sigma-Aldrich) were coated on a MaxiSorp ELISA plate (Nunc) in PBS in triplicates and incubated overnight at 4 °C as recently reported^{56 (link)}. Plates were washed and blocked with 3% BSA in PBS for 2 h at RT before incubation with plasma (1:100) or CSF (1:5) for 1 h. After incubation with anti-human IgG or IgA horseradish peroxidase (Jackson ImmunoResearch) for 1 h, the assay was developed with TMB peroxidase substrate (Seracare). A polyclonal polyreactive IgG antibody (ED-38; undiluted transfection supernatant) was used as a positive assay control^{67 (link)}. Triplicates with a coefficient of variation (CV) greater than 15% were corrected for by excluding one value. Corrected duplicates with a CV above 15% were excluded from the analysis (n = 4). Negative control signals (secondary antibodies only) were subtracted in a plate-specific manner.