ELISA Procedure for Measuring Serum Antibody Titer

Immunoassays are a powerful technique for detecting and measuring antigens and antibodies. Immunoassays can be classified three ways based on the steps involved:

• antibody capture
• antigen capture
• two-antibody sandwich

Many types of immunoassays can be used to detect and quantitate both antigens and antibodies, but there are differences in the avidity requirements for the antibodies, the signal strengths of the labels, and the amount of background for each of these types of assays. Antibody capture assays are the most appropriate for measuring the titer of the antisera you have generated.

ELISA Procedure for Measuring Serum Antibody Titer In this type of ELISA (Enzyme-Linked Immunosorbant Assay), the antigen (peptide or protein) is bound to the polystyrene microtiter plate first. The antiserum containing the anti-peptide antibody is then added to the well and allowed to bind. Finally, a second antibody, specific for the first antibody and labeled for detection, is added to the well and allowed to bind. The second antibody usually has an enzyme conjugated to it. This enzyme catalyzes the formation of colored substance, e.g., p-nitrophenol, from a colorless substrate, p-nitrophenylphosphate (Figure 21). This colored substance is then quantified and the amount of antibody present can be calculated.

This procedure has two parts. Part 1 applies to any detection protocol. Part 2 describes two different detection methods. To measure an antibody titer, decide on the detection method first, then complete both Parts 1 and 2.

Part 1: Antigen and Antibody

Equipment Needed

• Microtiter plates
• Pipettor, 1 mL adjustable
• Eight-channel pipettor (optional)
• UV/vis microtiter plate reader
• Pipette tips

Solutions to Prepare for the ELISA Procedure

• Sodium carbonate (Na2CO3) buffer, 50 mM, pH 9.6 with 0.02% NaN3
• PBS-T (PBS containing 0.05% Tween-20)
• 3% BSA in PBS-T
• Sodium acetate (NaOAc), 0.1 M

ELISA Procedure

Run duplicates or triplicates of each of antiserum dilution. The ELISA template in Table 3 can be used to track the experiments.

1. Choose the detection method. Note: If you choose horseradish peroxidase detection, omit the sodium azide from the buffers. 2. Prepare buffer solutions. 3. Prepare a solution of the peptide, protein or peptide/conjugate (10 µg/mL with respect to the peptide or protein) in sodium carbonate buffer. 4. Pipette 200 µL of either peptide, peptide carrier conjugate, or sodium carbonate buffer (for controls) in the individual wells of the microtiter plate.

Table 3. ELISA Template