Elsevier

Biosensors and Bioelectronics

Volume 12, Issue 11, December 1997, Pages 1101-1106
Biosensors and Bioelectronics

Antibody immobilization using heterobifunctional crosslinkers

https://doi.org/10.1016/S0956-5663(97)00070-5Get rights and content

Abstract

Covalent attachment of functional proteins to a solid support is important for biosensors. One method employs thiol-terminal silanes and heterobifunctional crosslinkers such as N-succinimidyl 4-maleimidobutyrate (GMBS) to immobilize proteins through amino groups onto glass, silica, silicon or platinum surfaces. In this report, several heterobifunctional crosslinkers are compared to GMBS for their ability to immobilize active antibodies onto glass cover slips at a high density. Antibodies were immobilized at densities of 74–220 ng/cm2 with high levels of specific antigen binding. Carbohydrate-reactive crosslinkers were also compared to GMBS using a fiber optic biosensor to detect fluorescently-labeled antigen. At the concentrations tested, the antibodies immobilized with carbohydrate-reactive crosslinkers bound more antigen than GMBS immobilized antibodies as indicated by the fluorescence signal.

Introduction

Proteins are attached to solid supports for biosensing, bioprocessing, affinity chromatography and assay purposes. Covalent immobilization of the protein is preferred when the coated substrate is subjected to flow or extended times in solution to prevent leaching of the protein from the substrate. Covalent immobilization chemistries have demonstrated preserved functional capability of the immobilized protein for up to 2 years (Klibanov, 1979; Ligler et al., 1990).

Bhatia et al. (1989)and Ligler et al. (1991)demonstrated the use of a thiol-terminal silane and a heterobifunctional crosslinker for the attachment of antibodies to glass. The thiol-terminal silane immobilization procedure has been thoroughly examined for demonstration of specific covalent attachment. This protocol has also been used successfully by other groups for the immobilization of antibodies (Feldman et al., 1995; Choa et al., 1996) for use in biosensors and for the immobilization of enzymes (Bhatia et al., 1991).

Since the initial studies with the thiol-terminal silane, a wider variety of crosslinkers has become commercially available. One concern about the original protocol was that the lack of control over antibody orientation might limit the proportion of binding sites available to bind antigen. With the availability of crosslinkers reactive with the carbohydrate moiety of the antibody which is located in the Fc region, it is now possible to immobilize the antibody via this region. Crosslinkers with longer bridge regions also offer the possibility that the binding capability of the antibody might be enhanced by spacing it further from the silane film. This study was undertaken to determine if other crosslinkers, with either extended bridges or carbohydrate-reactive moieties, enhance the density or functionality of immobilized antibodies compared to the crosslinker most extensively used with the thiol silanes, N-succinimidyl 4-maleimidobutyrate (GMBS). Functionality was also tested using a fiber optic biosensor.

Section snippets

Materials

Cover slips (2·2×4·0 cm, Premium Cover Glass, Fisher Scientific, Fairlawn, NJ) were cut in half (1·1×4·0 cm, total surface area 8·80 cm2) prior to use. The silane, 3-mercaptopropyltrimethoxysilane (MTS) and the heterobifunctional crosslinkers N-succinimidyl 4-maleimidobutyrate (GMBS), N-succinimidyl 3-maleimidobenzoate (MBS), N-succinimidyl 6-maleimidocaproate (EMCS) and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) were obtained from Fluka (Hauppauge, NY). Succinimidyl

Results

A total of nine different heterobifunctional crosslinkers were evaluated in this study to determine their ability to immobilize antibodies at a high density while retaining maximum activity. Since the chemistry using GMBS has been defined extensively, GMBS was used as the reference to which all other data were compared. The crosslinkers vary in reactive groups, length and rigidity of the bridging region. The majority of the crosslinkers contained a succinimide moiety which reacted with the

Discussion

Overall, the antibodies were immobilized at similar densities and activities with the various crosslinkers tested. The density of the immobilized antibody is partly influenced by the solid support. Antibody immobilization using the thiol-terminal silane chemistry has been performed on glass slips, fused silica and platinum with the densities highest for the platinum followed by fused silica, then glass (Ligler et al., 1991). For this study, glass slips were employed for the radioactive

Acknowledgements

The authors thank Paul Charles for assistance in preparing the radiolabeled proteins and Keeley King for initial work on the fiber optic biosensor. The authors thank Linda Chrisey and Jim Whelan for their careful review of this paper. This work was funded by an ONR/NRL Accelerated Research Initiative. The views expressed here are those of the authors and do not represent those of the US Navy, Department of Defense, or the US Government.

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