S-specific immunoglobulin ELISA

Consumables and Materials: 

Additional Materials Required:

Pipettors and Multi-Channel Pipettors and Pipette Tips

Reagent Reservoirs

Eppendorf Tubes

Cytation 3 Plate Reader (BioTek)

Antigen Plate Preparation:

1. Dilute antigen in PBS so the final volume in each well is 50 µl.  The final amount of antigen/well will be determined by comparing any batch of new antigen to a prior quality controlled batch in a serial dilution to identify a concentration of similar performance. Example of dilutions for batches of antigen currently being used are shown in this table.

2. Using a multichannel pipettor, transfer 50 µl of diluted antigen onto a 96-well Greiner plate (Greiner Bio-One, #655061) and allow it to adsorb overnight at 4^oC.

3. Wash 3 times with 200 µl PBS plus 0.1% Tween-20 (PBST).

4. Add 200 µl 3% w/v milk powder in PBST for 1.25 hours at room temperature.

5. Aspirate blocking solution.

Sample Intake and Preparation:

1. Serum dilutions are typically serial dilutions for 5-10 points at 2 to 5-fold dilutions between points.  For each point, the final volume is 50 µl diluted in 1% w/v milk powder in PBST.

ELISA:

1. Add 50 µl of diluted sample to Antigen Plate.

2. Incubate for 2 hours at room temperature.

3. Wash 3 times with 200 µl PBST.

4. Add 50 µl of secondary antibody (see table for dilution) in 1% w/v milk powder in PBST and incubate for 1 hour at room temperature.

5. Wash 3 times with 200 µl PBST.

6. Add 50 µl of 1-Step™Ultra TMB-ELISA Substrate Solution and incubate for 15 min at room temperature.

7. Add 50 µl of 0.16N sulphuric acid to quench the reaction.

8. Read plates in a spectrophotometer (Cytation 3) at 450 nm.