Use of Thiol-Terminal Silanes and Heterobifunctional Crosslinkers for Immobilization of Antibodies on Silica Surfaces

American University Washington D.C., Washington, Washington, D.C., United States
Analytical Biochemistry (Impact Factor: 2.22). 06/1989; 178(2):408-13. DOI: 10.1016/0003-2697(89)90662-3
Source: PubMed


A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.

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Available from: Lisa C Shriver-Lake, Feb 13, 2015
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    • "Vitronectin has an arginine-glycine-aspartic acid (RGD) integrin recognition motif and was initially identified as " serum spreading factor " due to its ability to bind tissue culture plates and mediate cell adhesion and spreading [3]. Additionally, glass beads can be functionalized with a variety of silanes containing amine and thiol functional groups that can be used for biomolecular conjugation [4] [5]. However, development of porous polymer resins with superior flow characteristics and greater surface area have since replaced glass beads as the solid support of choice for most protein purification. "
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    ABSTRACT: Conventional protein affinity chromatography relies on highly porous resins that have large surface areas. These properties are ideal for fast flow separation of proteins from biological samples with maximum yields, but these properties can also lead to increased nonspecific protein binding. In certain applications where the purity of an isolated protein is more important than the yield, using a glass solid phase could be advantageous as glass is nonporous and hydrophilic and has a low surface area and low nonspecific protein binding. As a proof of principle, we used protein A-conjugated hollow glass microbubbles to isolate fluorescently labeled neurofilament heavy chain spiked into serum and compared them to protein A Sepharose and protein A magnetic beads (Dynabeads) using an anti-neurofilament protein antibody. As expected, a greater volume of glass bubbles was required to match the binding capacity of the magnetic beads and Sepharose resins. On the other hand, nonspecific protein binding to glass bubbles was greatly reduced compared to the other resins. Additionally, since the glass bubbles are buoyant and transparent, they are well suited for isolating cells from biological samples and staining them in situ.
    06/2013; DOI:10.1155/2013/341036
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    • "Presently, surface modification methods include physical adsorption, chemical covalent immobilization, and biologic modification. Physical adsorption is seldom used because the immobilized proteins suffer partial denaturation and tend to leach or wash off of the surface and compete with adsorption (Bhatia, et al., 1989). "
    Biosensors for Health, Environment and Biosecurity, 07/2011; , ISBN: 978-953-307-443-6
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    • "The sensitivity of a surface based biosensor is thus directly affected by the packing density of the sensing element bound to the surface. Methods for surface functionalization have included the use of thiol interactions (Park & Kim, 1998; Radke & Alocilja, 2005; Bhatia et al., 1989), avidin-biotin interactions (Costanzo et al., 2005), self-assembled monolayer coated electrodes (Wana et al., 2009), polymer coated electrodes (Livache et al., 1998) and size specific capillary flow trapping (Hamblin et al., 2010). A number of proof-of-principle studies have demonstrated that a combination of AC electrokinetics with a molecular recognition method can substantially improve the sensitivity of a biosensor (Yang, 2009; Yang et al., 2006; Yang et al., 2008). "
    Biosensors - Emerging Materials and Applications, 07/2011; , ISBN: 978-953-307-328-6
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