Primary antibody and secondary antibody
PBS containing Tween 20
Procedures: Different steps in performing in-direct ELISA is given in the Figure 30.2. These steps are as follows:
Figure 30.2: Different Steps in performing in-direct ELISA to measure antibody level in the serum
Coating: Prepare 5 µg/ml of antigen solutions in Biocarbonate buffer (50mM, pH 9.2). Dispense 50 µl per well of microtiter plate. Put it overnight inside fridge (8 – 10 hrs is sufficient).
Blocking: Block each well with 1% BSA in Biocarbonate buffer for overnight.
Preparation of Primary Antibody dilution:
Table 30.1: Preparation of different dilutions of antibody. |
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Dilution |
Antibody |
1% BSA in PBS |
Total volume |
1:100 |
2µl |
198 |
200 |
1:1000 |
20µl |
180 |
200 |
1:10000 |
20µl |
180 |
200 |
1:20,000 |
100µl |
100 |
200 |
1:40,000 |
100µl |
100 |
200 |
1:80,000 |
100µl |
100 |
200 |
1:1,60,000 |
100µl |
100 |
200 |
1:320000 |
100µl |
100 |
200 |
Dispense 50µl of each dilution in respective well. Incubate for 45 min at 370C.