Blockers Practical Guide

Blocking the Solid Phase

Solid phase quantitative immunoassays such as ELISA, lateral flow and Western blot all involve the immobilization of antibodies to a surface. Non-specific binding to this surface by other proteins or biomolecules can reduce an assay’s specificity and sensitivity. Solid phase blocking agents are specifically designed to saturate these unoccupied binding sites to prevent non-specific binding, while enhancing assay sensitivity.

When developing a new immunoassay, the first step is to optimize the antigen or capture antibody coating conditions on the solid phase in order to maximize the amount of protein coated. After coating, any remaining unoccupied binding sites must be blocked in order to prevent non- specific binding of subsequent reactants. An ideal blocking agent is typically protein-based as it is able to block both hydrophobic and hydrophilic sites on the solid phase. In addition, it can serve as a stabilizing agent and prevent denaturation while proteins react at the surface of the solid phase. The concentration of the blocker and the amount of blocking time must be optimized for each assay. Using inadequate amounts of blocker will result in excessive background and a reduced signal-to-noise ratio. Using excessive concentrations of blocker may mask antibody- antigen interactions causing less sensitivity. ELISA without blocking buffer Unbound debris such as proteins, interfering molecules in the patient sample, and assay reactant bind to the solid phase and compete with the specific antigen-antibody reaction causing background noise and reduction in specific assay signal.

IDEAL BLOCKING AGENTS HAVE THE FOLLOWING CHARACTERISTICS: • Effectively block nonspecific binding of assay reactants to the surface of the well • Do not disrupt the binding of assay components that have been adsorbed to the well • Act as a stabilizer (prevent denaturation) of assay reactants on the solid phase • Do not cross-react with other assay reactants • Do not possess enzymatic activity that might contribute to signal generation of the substrate or degradation of the reactants • Perform consistently across various lots

ELISA with blocking buffer Blocking buffer is designed to bind to open sites on the solid phase, preventing unbound debris from non-specifically binding.

Detector Label

Detection Antibody Capture Antibody Antigen

Unbound Debris

Blocking Buffer

GENERAL BLOCKING PROTOCOL

1. After coating the solid-phase with primary antibody, add the blocking solution directly to the wells, beads, blotting membrane or nitrocellulose membrane. 2. Determine the best concentration of blocker for your assay. Blockers can be used at 1x concentration or diluted. 3. Determine the optimum incubation temperature and time for proper absorption of the blockers. Longer times and higher temperature increase the rate of blocking. Typical temperatures are 25°C- 30°C incubated for 30 minutes to 2 hours. 4. Proceed to wash steps.

NOTE: In addition to blocking, it is essential to perform thorough washes between each step. Washing steps are necessary to remove unbound reagents and decrease background noise. Insufficient washing will allow high background. A common technique is to use a diluted solution of the blocking buffer along with some added high-purity detergent.