Highly Sensitive Immunoprecipitation Method for Extracting and Concentrating Low-Abundance Proteins from Human Serum

Abstract

Purification and identification of small, low-abundance proteins from complex samples such as serum for use in Western blotting experiments has long been a challenge. Unfortunately, commercially available methods are not always suitable for small sample sizes and low-abundance proteins such as cardiac troponin T (cTnT). In this report we present a highly sensitive immunoprecipitation method we have developed for extracting and concentrating low-abundance proteins from serum. We compared the performance of our method with that of the commercially available ImmunoPure® Protein-A IgG orientation Kit (Pierce), using antibodies directed against cTnT and serum of a patient with acute myocardial infarction (AMI). Both methods are based on the binding of antibodies to Sepharose beads and subsequent cross-linking with a diimido ester. The formation of amidines between imido esters and amines from protein side chains was first described in 1962 by Hunter et al. (1), who suggested this for cross-linking purposes. Levy et al. (2) described the use of diimido esters for covalent coupling of antibodies to immobilized antigens. Later studies reported similar methods for antibody immobilization with cross-linkers such as dimethyl pimelimidate and dimethyl suberimidate (3)(4). These methods are based on the fact that an amine is able to react with an imido ester only if it is deprotonated, which occurs at pH > p K a. At these pH values, the nucleophilic attack of an R-NH2 on one of the two outer backbone carbon atoms of dimethyl pimelimidate produces a tetrahedral intermediate that splits into the amidine link and methanol. Hunter et al. (1) showed that the right choice of pH even allows a distinction to be made between α- and e-amines. They showed that a pH between 7 and 8 favors reaction with α-amines, whereas a pH between 9 and 10 favors reaction with e-amines. …