Preparation and Characterization of Reconstructed Small Intestinal Brush Border Membranes for Surface Plasmon Resonance Analysis

Abstract

Purpose. To prepare the surface generated by small intestinal brush border membrane vesicles (BBMVs) for the surface plasmon resonance (SPR) analysis, which allows the real-time measurement of binding events occurring on the intestinal membrane. Methods. BBMVs were isolated from Sprague-Dawley rats, suspended in HEPES-buffered saline, and flowed over the surface of a SPR sensor chip composed of dextran derivatives modified with lipophilic residues. The surface coverage was determined from binding of bovine serum albumin to BBMV-immobilized sensor chip. The performance of BBMVs immobilized was evaluated by their interaction with otilonium bromide and bile salts. Results. The stable BBMV surface was achieved when BBMV suspension was flowed over the sensor chip for 8 h at a rate of 2 l/min. The flow of otilonium bromide resulted in an increased SPR signal because of its binding to calcium channel, which is known to be distributed over the gastrointestinal tact. When bile salts were flowed over ileal and duodenal BBMV surfaces, respectively, a slightly higher SPR signal was observed in the ileal BBMV surface, indicating the specific interaction of bile salts with bile acid transporters. Conclusions. BBMV surfaces may be useful for the estimation of binding events on the intestinal membrane by SPR analysis, especially for the drugs that are orally administrated.KEY WORDS: surface plasmon resonance; intestinal brush border membrane; otilonium bromide; bile salts.

Class II MHC/Peptide Interaction in Leishmania donovani Infection: Implications in Vaccine Design

“…Conditions for the reproducible isolation of plasma membranes from MFs were adapted from those described by other investigators (35,36). Membrane was prepared from N-MF and infected MF cell types.…”

“…Membrane was immobilized on the L1 sensor chip for SPR studies, as described previously (35,36). The isolated membranes were immobilized on the surface of the L1 chip to achieve the desired response unit.…”

“…p-MHC-II complex formation was analyzed on the isolated membrane in real time, as described in other system (35,36,46). Increasing concentrations of OVA [323][324][325][326][327][328][329][330][331][332][333][334][335][336][337][338][339] were flown over the immobilized membrane on the L1 chip, and k on and k off were determined using a , respectively, for 6-h I-MF membrane (Fig.…”

Evaluation of absorption of heparin-DOCA conjugates on the intestinal wall using a surface plasmon resonance

“…4. It is known that this sensorgram with a fast steadystate binding level is useful for assaying the binding level of orally absorbed drugs on the lipid-based SPR system [19][20][21][22][23]. In the case of duodenal BBM surface, the SPR signals might have resulted from non-specific physical interactions, such as lipophilicity, hydrogen bonding and ionic strength, because the bile acid transporters are absent in the duodenum.…”

“…We previously studied a new surface plasmon resonance (SPR) system to evaluate drug adhesion on the intestinal surface using biosensor chips on which BBM vesicles were immobilized [20,21]. Surface plasmon resonance technique has been in use recently for an in-real time assay that allows a direct analysis of drug adhesion on the phospholipid surfaces immobilized on biosensor chips in a continuous flow system [22,23].…”