Blockers Practical Guide

Why Use Blockers?

Blockers are used in ELISA and LF assays to reduce interference from proteins in patient samples that could produce false results and an incorrect diagnosis.

Choosing the best assay format depends on the intended application of the assay, the type of samples to be analyzed, the availability of reagents and whether the assay is intended for a single analysis in one lab, or is intended to be used in many laboratories by various technicians. Generally, an immunoassay will fall into one of the following categories: • Sandwich Antigen Detection ELISA - designed to measure the amount of target antigen/analyte • Antibody Capture ELISA - used to screen for antibodies (e.g: lgG, lgM, lgA & lgE) to a specific target • Competitive ELISA - detects antigen/analyte present in a sample and is commonly used when the antigen is small with only 1-2 epitopes Several parameters that are critical to assay performance are common among all of these formats: (1) the choice of solid phase (2) the choice of antibodies/antigen and (3) the choice of blocking agents. In order to produce an assay with high sensitivity and specificity, the most critical element is selecting antibodies or antigens that have a highly specific interaction with the target molecule. However, it is possible to improve assay sensitivity and specificity with the use of blocking agents. These blockers work by reducing non-specific binding resulting in an increase in signal-to-noise ratio. Non-specific interactions in an assay can occur (1) between the solid phase and non-target proteins, which can absorb to the surface of the solid substrate and (2) between antibodies within the assay itself and endogenous antibodies present within a patient’s sample. To prevent non-specific binding, blocking buffers are used after the solid-phase coating step to block any remaining open binding sites. Other blocking agents are used in the sample preparation to prevent interfering antibodies from binding to the assay antibody components.

TYPES OF IMMUNOASSAY SOLID PHASES

Binding Capacity

Material

Type of Interaction

Nitrocellulose

High High High

Hydrophobic, Hydrophilic

PVDF Nylon

Hydrophobic Hydrophobic

Plates &Tubes Polystyrene

Low Low

Hydrophobic Hydrophobic

Polyvinyl

Derivatized microtiter plates

Covalent, Hydrophobic, Hydrophilic

Low

Beads

Polystyrene Denvatized Polystyrene

Moderate

Hydrophobic

Covalent, Hydrophobic, Hydrophilic Covalent and Hydrophobic

High

Microparticles

High

A solid surface which has a high binding capacity but does not cause damage to the native protein conformation of the immobilized antigen or antibody is ideal.

KEY PERFORMANCE DIFFERENCES BETWEEN MONOCLONAL AND POLYCLONAL ANTIBODIES

In general, a MAb is often chosen as the primary antibody to establish the highest level of specificity in an assay, and a PAb is chosen as the secondary antibody, to amplify the signal via multiple binding events. However, any combination can be used. All candidate antibodies must be tested together with the intended sample type in order to select the best performers.

Monoclonal antibodies (MAb) • Generally produced in mice or recombinantly, and recognize a single epitope. • Since only one antibody molecule can bind to the antigen, the interaction is highly specific but can lack sensitivity, depending on the affinity constant.

Polyclonal antibodies (PAb) • Produced in goats, sheep, chicken, rabbits and other animals. • Polyclonal sera is a heterogeneous composite of antibodies with unique specificities. The concentration of specific antibody (PAb) is typically 50-200mg/mL. • PAbs are able to recognize multiple epitopes on any one antigen which makes them less sensitive to antigen mutational changes.

• PAbs are useful when the nature of the antigen is not well known. However, their quantity is limited by the lifespan of the host animal.