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 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.

     Figure 21. Antibody detection in an Enzyme- Linked                     Immunosorbant Assay (ELISA)


 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





























5. Cover the plate and incubate for three hours at room temperature or overnight at 2-6°C.
6. Remove unbound antigen by washing three times with the PBS-T buffer. A squirt bottle is a   handy way to add the buffer to the wells. Remove the antigen solution and washes by inverting   the plate quickly and tapping the bottom on paper towels to remove any drops. This ensures that   there is no cross contamination or dilution.
7. Block the remaining adsorption sites by adding 300 µL of 3% BSA/PBS-T to each well. Incubate   for one hour at room temperature.
8. Wash the plates twice with PBS-T.
9. Prepare a 1:50 dilution of the sera by pipetting 60 µL of the sera into 2940 µL of PBS-T. Use   microcentrifuge tubes with flip-top caps. Store all dilutions on ice.
10. Prepare serial dilutions using the dilution schedule in Table 4.

Table 4. Serial Dilution Schedule

Dilution of
Volume of 1%
Volume of Previous
Dilution (µL)
1000 of   1:50
1000 of 1:100