Unfortunately, this BG noise reaction is not taken into consideration and not determined as a negative control in antigen non-coated wells. In the indirect ELISA system for serological antibody assays, the inherent high binding affinity of serum immunoglobulins to solid surfaces creates strong false positive BG noise reaction. The enzyme-linked immunosorbent assay (ELISA) system is widely used to assay antibodies and antigens without fully comprehending the numerous vexing phenomena attributed to the principle, which utilizes the high binding affinity of proteins to solid surfaces such as micro-titer plates and latex beads. 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. The accuracy and reliability of these assay results were examined in detail by inhibition tests in individual buffer systems. 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™.
No current blocking agents can prevent these false positive and negative reactions, and antibody assay results vary significantly depending on the buffer system used. 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.
To assay serum antibodies by indirect ELISA, it is critical to eliminate a variety of false positive and negative reactions attributed to the principle.
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