Kinetic Studies on the Immobilization of Antibodies to High-Performance Liquid Chromatographic Supports

Abstract

Several factors can potentially affect the rate of immobilization of proteins onto solid supports, such as those used in affinity-based high-performance liquid chromatography. This study examined several of these factors and their influence on the coupling of periodate-treated rabbit immunoglobulin G antibodies to dihydrazide-activated silica. Items considered included the number of potential coupling sites on the antibodies, the density of activated sites on the support, the relative amount of antibody combined with the support, and the density of the overall reaction slurry. In each case, the rate of change in the solution-phase antibody concentration gave biphasic behavior which could be described by two competing pseudo-first-order reactions. The overall immobilization rate was essentially independent of the density of the support's activated sites (when present at a coverage of 0.1-0.4 micromol/m2) but was strongly influenced by the number of available coupling groups on the antibodies. Increasing the slurry density had no appreciable effect on the immobilization rate, and the reaction rate showed only a small change when using different types of reagents for support activation (e.g., adipic vs oxalic dihydrazide). These results are consistent with a mechanism in which the rate-limiting step during immobilization is the covalent attachment of antibodies to the support and not mass transfer of antibodies to the support's surface.

Solid supports for microarray immunoassays

“…Although oriented molecule layers result in a kinetically more homogeneous behaviour of the antibodies, this does not correlate with higher specificity. Frequently, a lower density of attached proteins is observed (Vijayendran and Leckband, 2001) since immobilization efficiency depends on the number of available coupling groups (Oates et al, 1998). In addition, attachment by means of protein A or G can be applied to certain immunoglobulin subtypes only, which are bound by these proteins with high affinity (Anderson et al, 1997;Turkova, 1999).…”

Sequential robust design methodology and X‐ray photoelectron spectroscopy to analyze the grafting of hyaluronic acid to glass substrates

“…The binding of bioactive molecules [e.g., enzymes, extracellular matrix (ECM) components, antibodies, growth factors, and peptides] to solid substrates has been analyzed extensively for use in a number of diverse applications, including biomaterials,1–5 biosensors,6, 7 and chromatography 8, 9. Although it is possible to physisorb biomolecules to solid supports, numerous reports have stressed the disadvantages with such an approach 3, 10, 11…”

Optimization of antibody immobilization for on‐line or off‐line immunoaffinity chromatography

“…An overview of the methods is shown in Table 1. Methods 1 and 2 use a reducing agent for the formation of stable alkylamine bonds [26][27][28]. Methods 1 and 3 are simple to perform but may be detrimental as immobilization is nonoriented [22,28] and covalent bonds thus may be formed in the antigen-binding parts of the antibody (cf.…”