To confirm the reactivity of colostrum mAbs to commensal bacteria WCL and C.1086 gp120 Env, SPR binding assays were performed on a Biacore 4000 (GE Healthcare) maintained at 25°C as described30 . Briefly, the colostrum mAbs were immobilized on a Series S CM5 sensor chip to 5,000 to 6,000 response units (RU) using standard amine coupling chemistry. Uninfected human stool bacterial WCL, 1086Cgp120 or C.1086V1/V2 protein was injected over the immobilized colostrum mAbs. Injection time was 150s and the dissociation activity was monitored for an additional 100s. The maximal RU of binding at 150s was reported. The dissociation constant (kd) was calculated using a 1:1 Langmuir model from 160s to 250s. For bacterial WCL, avidity scores were calculated using the formula, RU/kd. All data analysis was performed using the BIAevaluation 4.1 analysis software. (GE Healthcare).
The lipids, Lyophilized powder of D-galactosyl-β-1,1' N-octanoyl-D-erythro-sphingosine (Galcer) and chloroform stock of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), were purchased from Avanti Polar Lipids (Alabaster, AL).The Galcer liposomes were prepared at a 1:1 Galcer:POPC molar ratio and used in the Galcer blocking assay as detailed20 (link).
The lipids, Lyophilized powder of D-galactosyl-β-1,1' N-octanoyl-D-erythro-sphingosine (Galcer) and chloroform stock of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), were purchased from Avanti Polar Lipids (Alabaster, AL).The Galcer liposomes were prepared at a 1:1 Galcer:POPC molar ratio and used in the Galcer blocking assay as detailed20 (link).