Reagents and instruments for immunology, cell biology and molecular biology.

Increase ELISA Accuracy: The Role of Blocking Buffers

Boost your ELISA accuracy with blocking buffers—key to reducing noise, enhancing sensitivity, and ensuring reliable results in protein detection.

Introduction

Enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting and quantifying proteins, antibodies, and hormones. The accuracy and reliability of ELISA results are significantly influenced by the choice of blocking buffers, which prevent non-specific binding and reduce background noise.

Blocking buffers are essential for preventing non-specific interactions between antibodies and the microplate surface. Non-specific binding can lead to elevated background signals, resulting in false positives or inaccurate quantification of target analytes. The primary role of a blocking buffer is to cover unoccupied binding sites on the plate, thereby reducing background noise and improving the signal-to-noise ratio of the assay (Huber et al., 2009).

Mechanism of Action

The primary mechanism by which blocking buffers function is through the saturation of unoccupied binding sites on the solid phase (e.g., microplate wells). After the immobilization of capture antibodies or antigens, free binding sites remain available for non-specific interactions. Blocking buffers occupy these sites, thereby preventing other proteins or antibodies from binding non-specifically.

Blocking buffers not only prevent non-specific binding but also stabilize biomolecules bound to the well surface. This stabilization is crucial for maintaining the integrity and functionality of the immobilized proteins during subsequent assay steps. Research has shown that blocking agents can reduce conformational changes in immobilized proteins, thereby preserving their activity and ensuring accurate detection (Xiao et al., 2012).

Advantages of Using Blocking Buffers in ELISA

Enhanced Sensitivity		Reduction of Non-Specific Binding

Stabilization of Adsorbed Proteins		Reduced Cross-Reactivity

The use of effective blocking buffers in ELISA protocols offers numerous advantages, including reduced non-specific binding, enhanced sensitivity, protein stabilization, flexibility across assay formats, minimized steric hindrance, reduced cross-reactivity, and improved reproducibility. A comprehensive range of blocking buffers is offered to support ELISA workflows, ensuring optimal performance, reduced background interference, and reliable results.

References

Huber, D., Rudolf, J., Ansari, P., Galler, B., Führer, M., Hasenhindl, C., and Baumgartner, S., 2009. Effectiveness of natural and synthetic blocking reagents and their application for detecting food allergens in enzyme-linked immunosorbent assays. Analytical and bioanalytical chemistry, 394, pp.539-548.

Xiao, Y., and Isaacs, S.N., 2012. Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine serum albumin (BSA)—not all BSAs are alike. Journal of immunological methods, 384(1-2), pp.148-151.