Galcer

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 detailed^{20 (link)}.

D-galactosyl-β-1,1′N-nervonoyl-D-erythro-sphingosine (GalCer) was purchased from Avanti Polar Lipids, Inc. Lipid tubules were prepared by adding GalCer at 50 vol% to 2 mg/ml stock lipid mixture (55 mol% DOPC, 35 mol% DOPS and 10 mol% cholesterol). Chloroform in the mixture was dried under argon and rehydrated in MSB to a final concentration of ~0.8 mg/ml by vortexing for 1 minute. The quality of tubules formed was assessed by TEM and stored at 4°C for no longer than one week.