Challenges of Protein Content and Validation
There are times when third-party commercially available antibody duo sets and/or purified antigens required for assay development are unavailable. In many circumstances, there may be multiple clones of commercial antibody available without access to antigen. Development of mouse monoclonal antibodies using purified native or newly developed recombinant antigen has become more affordable over the past ten years.
When working with newly developed immunoassay reagents, one must demonstrate and characterize that antibodies are capable of binding to purified as well as endogenous antigen within a complex heterogeneous mixture. Testing specific antibody binding to endogenous antigens is initially qualified through Western Blot analysis. Unless one is fortunate enough to access biological specimens from antigen-deficient cells, then you should remove endogenous antigen using immunoaffinity chromatography to separate antigen out of complex protein samples such as serum or plasma.
If successful, then the multiplex immunoassay of antigen-depleted samples should result in the absence of signal contrary to positive signal measurements observed when testing normal pooled real-world specimens. Validation for multiplex immunoassay performance ought to compare single-plex titration curves against multiplex titration curves to identify any interferences with levels of detection, precision variance, dynamic range, or slope.
There are many circumstances when matrix effects occur when testing plasma samples for some analytes, which leads to changes in the sensitivity and precision characteristics of the assay. A fivefold dilution of these samples in diluent buffer usually eliminates these interferences but sometimes may result in a lower absolute sensitivity linked to the dilution of plasma samples.
Not all antibodies perform well in multiplexed immunoassays. For example, there are antibody clones for TGF-b that require antigen denaturation using strong acid treatment. Additionally, some antibody clones derived from phospho-peptide immunization react only with the denatured phospho-specific protein target.
There are many examples where these immunoassays require a chaotropic agent to unfold and expose buried antigenic epitopes. Chemical treatments such as these typically obstruct immunoassay performance of other antigens and antibodies in the panel when using antibodies specific for the native antigen configuration.