Abstract
To assay serum antibodies by indirect ELISA, it is critical to eliminate a variety of false positive and negative reactions attributed to the principle. These include 1) the background (BG) noise reaction caused by hydrophobic binding of immunoglobulin components in sample specimens to solid surfaces, 2) false positive reaction caused by non-specific binding of immunoglobulins to target-antigens by protein-protein interactions, and 3) other false positive and negative reactions caused by buffer components. No current blocking agents can prevent these false positive and negative reactions, and antibody assay results vary significantly depending on the buffer system used. To address these fundamental problems, we investigated all types of non-specific reactions involved in indirect ELISAs, and the blocking efficacy of current buffer systems and a newly developed ELISA buffer, ChonBlock™. The accuracy and reliability of these assay results were examined in detail by inhibition tests in individual buffer systems. Based on these studies, we are providing a definitive ELISA protocol for all users to improve ELISA technique and obtain accurate, reliable, and reproducible assay data against a variety of antigens.
Highlights
To assay serum antibodies by indirect enzyme-linked immunosorbent assay (ELISA), it is critical to eliminate a variety of false positive and negative reactions attributed to the principle
In an indirect ELISA for antibody assays, various types of false positive and negative reactions are involved, regardless of antigens. Of these non-specific reactions, the most intense false positive reaction is BG noise reaction caused by hydrophobic binding of immunoglobulin components in sample specimens to solid surfaces
BG noise optical density (OD) values were significantly reduced by ChonBlockTM in both IgG and IgA anti-LPS antibody assays, and the antigen-antibody reaction was clearly distinguished from non-specific false positive reaction
Summary
ABSTRACT To assay serum antibodies by indirect ELISA, it is critical to eliminate a variety of false positive and negative reactions attributed to the principle. These include 1) the background (BG) noise reaction caused by hydrophobic binding of immunoglobulin components in sample specimens to solid surfaces, 2) false positive reaction caused by non-specific binding of immunoglobulins to target-antigens by protein-protein interactions, and 3) other false positive and negative reactions caused by buffer components. No current blocking agents can prevent these false positive and negative reactions, and antibody assay results vary significantly depending on the buffer system used To address these fundamental problems, we investigated all types of non-specific reactions involved in indirect ELISAs, and the blocking efficacy of current buffer systems and a newly developed ELISA buffer, ChonBlockTM. ARTICLE INFO Article history: Received 19 December 2016; Accepted 23 March 2017; Available online 30 March 2017
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