Indirect ELISA

The indirect ELISA is used primarily to determine the strength and/or amount of antibody response in a sample, whether it is from the serum of an immunized animal or the cell supernatant from growing hybridoma clones.

Procedure

1. All incubations are done as follows: Cover with plate sealer tape or place in a sealed box containing a wet paper towel and incubate for 2 hours at room temperature. For the four controls: 2 are negative controls using pre-bleeds of the same concentration as your staring dilution of 1° antibody; the third is a negative control where no 1° antibody is added, just blocking buffer at this step; and the fourth is a positive control, either from a previously positive bleed or cell supernatant, or you can lay down 1° antibody as antigen.
2. In a 96-well ELISA plate (Nunc MaxiSorp is best), add 100 ng of antigen in 50 µL in each well you will be using for your test, as well as four control wells. The perimeter wells on the plate are generally not used, as they tend to give poor results. Incubate.
3. Dump out the antigen solution and add 100 µL of blocking buffer (1% BSA, 0.1 M KPi, 0.1% Tween-20, 0.02% thimerisol, pH 7). If your carrier protein for injection was BSA, then substitute 1% non-fat dry milk for the BSA. Incubate. The blocking step incubation can be also done at 4 °C overnight.
4. Dump out the blocking buffer and bang the plate upside down on some paper towels to remove all the liquid. Wash 3 times with wash buffer (0.1 M KPi, 0.05% Tween-20, pH 7), shaking the wash out vigorously each time. Again bang out the residual wash buffer.
5. Add 50 µL/well of your 1° antibody. For screening hybridomas, this will be cell supernatant. For obtaining a titer on serum from an immunized animal, you will need to perform serial dilutions of the serum in blocking buffer in the plate. 1:1 serial dilutions are done by placing 100 µL in the first column of your plate of your starting dilution of serum (1:499 in blocking buffer is usually a good starting point). Then place 50 µL/well of blocking buffer down all the wells remaining in the rows you are using. Now pipet out 50 µL from the first well (with your starting dilution) and place in the next well in the row. Mix by pipeting the solution up an down, and then transfer 50 µL of this solution to the next well and again mix. Continue these dilutions down the row until the last well, where you remove 50 µL and throw away. Incubate.
6. Wash 3 times as before.
7. Add 50 µL/well of a 1:1999 dilution in blocking buffer of HRP-labelled 2° antibody that is directed against the species of your primary antibody (anti-mouse for monoclonals and mouse serum, anti-rabbit for polyclonal antibodies raised in rabbits). Incubate.
8. Wash 3 times as before.
9. Add 100 µL/well of ABTS horseradish peroxidase substrate. Incubate at room temperature for 5-20 minutes, depending on the rate of color development. Keep the time identical for subsequent comparisons of titer, and for hybridoma screening go the full 20 minutes.
10. Add 100 µL/well stop solution (0.5 M Oxalic Acid).
11. Read absorbance at 414 nm in an ELISA reader.