CRO Diaries: Biomarker Quantification For ELISA Assay Development
Biomarkers are crucial detection tools in the diagnosis and treatment of diseases. Besides, their accurate detection is vital for different stages of drug development. However, what is the drug development process? The ultimate goal of drug development is to identify and test novel compounds and assess their potential to treat diseases and medical conditions.
Biomarkers are present in serum, blood, tissue, and other body fluids. The primary objective of biomarker quantification is to produce a reliable, cost-effective, and robust assay for diagnosing and monitoring disease conditions. Besides, biomarkers are vital for follow-up treatment.
Since the early use of biomarkers in medical sciences, biomarker technologies have seen massive progress in terms of research studies and innovations. Today numerous biomarker detection methods are available, with ELISA being one of the most employed immunoassays. However, immunoassay services face several drawbacks and limitations during ELISA assay development and validation. Additionally, ELISA assay validation needs an appropriate assessment of accuracy, specificity, and sensitivity for a complete immunoassay validation. The current article discusses several aspects of biomarker quantification for ELISA assay development.
Biomarker quantification for ELISA assay development
In a typical ELISA assay development, antibodies are produced in animals against specific biomarkers. ELISA assays depend on antibody-antigen interaction. Specific epitopes of an antigen will attach to the primary antibody immobilized on the assay plate. The other remaining epitopes will bind to the secondary antibody forming a sandwich-like complex. These binding mechanics are detected using an enzyme that changes the conjugated substrate into a colored outcome. The generated color intensity is directly proportional to the bound antigen in the sample. Such simplified protocols have made ELISA the primary choice for detecting and quantifying biomarkers in biological samples.
There are numerous enzyme-substrate pairs used in ELISA assays. These pairs include alkaline phosphatase-PNPP, alkaline phosphatase-NBT, horseradish peroxidase-ABTS, and horseradish peroxidase-TMB. Despite such a variety of enzyme-substrate pairs, the sensitivity and selectivity of ELISA assays depend on the specific and selective binding of primary and secondary antibodies and the concerned antigen.
Moreover, the binding capacity of secondary antibodies is limited due to their conjugation with a large enzyme. These limitations have paved the way for the development of novel approaches using streptavidin-biotin complexes. This complex bridges the secondary antibody and the enzyme. Biotin is comparatively very small and thus does not significantly change the shape of the secondary antibody. Hence, the secondary antibody retains its specificity against the antigen. Today streptavidin-biotin complex is commonly employed in ELISA assays for sensitive and specific detection of biomarkers.
Besides, the specificity of ELISA assays has limitations as they largely depend on the immobilization chemistry of primary antibodies. Some basic immobilization methods include hydrophobic, ionic, and covalent interactions. Covalent linkage has the disadvantage of reduced protein activity, complicated chemistry, and needs toxic reagents. On the other hand, direct immobilization gradually reduces the activity of primary antibodies, which leads to non-specific interactions. Hence, each immobilization method is unique, and researchers must consider the advantages and limitations of these methods during ELISA assay development and validation.
