The neonatal Fc receptor (FcRn) in intestinal epithelium may be the primary mechanism for transfer of maternal immunoglobulin G (IgG) from suckled dairy to serum; however the factors adding to the rapid uptake of IgG are poorly understood. expression by reverse transcription polymerase chain reaction, and mAb epithelial localization was visualized by immunohistochemistry. After duodenal administration, serum levels of mAb variants correlated with their rFcRn off-rate at pH 7.4, but not their affinity at pH 6.0. The greatest serum levels of IgG were measured when mAb was administered in the duodenum where rFcRn mRNA expression is greatest, and was increased further by duodenal administration in pH 6.0 buffer. More intense human IgG immunostaining was detected in epithelium than the same variant administered at higher pH. These data suggest an increased contribution for cell surface receptor. We conclude that, in the neonate duodenum, receptor off-rates are as important as affinities for Kv2.1 (phospho-Ser805) antibody FcRn mediated uptake, and cell surface binding of IgG to rFcRn plays contributes to IgG uptake alongside pinocytosis; both of which responsible for increased IgG uptake. model for IgG transcytosis (Rodewald and Kraehenbuhl, 1984; Benlounes et al., 1995; Martin et al., 1997). We previously showed that Tg276 transgenic mice that express human FcRn in the intestinal mucosa do not Pimasertib functionally transport human IgG after intestinal administration (Kliwinski et al., 2013). When human IgG was administered to the small intestine of 2-week-old suckling rat pups, approximately 80% of the uptake was FcRn-dependent while the remaining 20% was FcRn-independent and non-receptor mediated (Kliwinski et al., 2013). The present study aims to further characterize the pharmacological interactions between IgG and FcRn that contribute to the rapid uptake of IgG in the neonatal rat, including FcRn affinity and off-rates, pH-dependence, the effect Pimasertib of differential intestinal administration sites, and aims to increase evidence that cell surface receptors have a role. MATERIALS AND METHODS DETERMINING THE AFFINITY AND OFF-RATES OF HUMAN IgG VARIANTS TO RAT FcRn The mAbs used in this study were a recombinant chimeric human IgG1 monoclonal antibody specific for human respiratory syncytial virus (RSV), also known as B21M. The WT and FcRn binding Pimasertib affinity variants (H435A, N434A, and N434Y) with mutations at the 434 and 435 amino acid positions (EU numbering; Firan et al., 2001; Yeung et al., 2009; Deng et al., 2012) were tested for binding affinity to rat FcRn. In addition to determining the affinity of human mAbs to rat FcRn at pH 6.0 and 7.4 we developed a method to determine the off-rate of pre-bound (at pH 6.0) mAb after the buffer conditions were changed to pH 7.4. GLC biosensor chip conditioning and Rat FcRn immobilization A ProteOn XPR36 GLC biosensor chip (Bio-Rad, Hercules, CA, USA) was preconditioned with 0.5% sodium dodecyl sulfate (SDS), 50 mM NaOH and 100 mM HCl in both the vertical (ligand) and horizontal (analyte) channels. Following equilibration with PBS-TE running buffer (20 mM Na-phosphate, 150 mM NaCl, 0.005% Tween-20, 3 mM EDTA, pH 6.0), FcRn was immobilized using an amine coupling kit at a temperature of 25C and a flow rate of 30 l/min. All channels were activated with a mixture of EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide] (0.2 M) and sulfo-NHS (0.05 M) at 30 l/min for 4 min, followed immediately by immobilization of rat FcRn (3 g/mL in 10 mM sodium acetate pH 4.5) over channels 1 and 2 at 30 L/min for 5 min. The reference channel was treated identically without injection of FcRn. All channels were then blocked for 5 min with an injection of 1 1 M ethanomine-HCl (pH 8.5). This method resulted in rat FcRn coupled at response levels of 193 and 161 RU (1 RU = 1 pg protein/mm2) in channels 1 and 2, respectively. Analysis of affinity between rat FcRn and IgG variants at pH 6.0 Following immobilization, the four IgG1 (WT, N434A, N434Y, and H435A) mAb variants were diluted with running buffer formulated at pH 6.0. Each mAb was tested at five concentrations in duplicate using a fivefold dilution series. The five concentrations of each analyte were injected simultaneously at a flow rate of 60 L/min for a 1 min association phase which was followed by a 3 min dissociation phase. The surface was regenerated prior to each subsequent mAb and tested with two injections of sodium phosphate (pH 8.0) followed by one injection of running buffer (pH 6.0). Affinity analysis and kinetic constants were calculated from the sensorgrams using the bivalent analyte model of the ProteOn XPR36 software program. Evaluation of off-rate between rat IgG and FcRn variations in pH 7.4 using the ProteOn co-injection setting Pursuing immobilization, the four IgG1 mAb variations had been diluted with working buffer formulated at pH 6.0. Each mAb was examined at five concentrations in duplicate utilizing a.