Indirect ELISA
The indirect ELISA is used primarily to determine the strengthand/or amount of antibody response in a sample, whether it is fromthe serum of an immunized animal or the cell supernatant fromgrowing hybridoma clones.
Procedure
1. All incubations are done as follows: Cover with platesealer tape or place in a sealed box containing a wet paper toweland incubate for 2 hours at room temperature. For the fourcontrols: 2 are negative controls using pre-bleeds of the sameconcentration as your staring dilution of 1° antibody; the third isa negative control where no 1° antibody is added, just blockingbuffer at this step; and the fourth is a positive control, eitherfrom a previously positive bleed or cell supernatant, or you canlay down 1° antibody as antigen.
2. In a 96-well ELISA plate (Nunc MaxiSorp is best), add100 ng of antigen in 50 µL in each well you will be using for yourtest, as well as four control wells. The perimeter wells on theplate are generally not used, as they tend to give poor results.Incubate.
3. Dump out the antigen solution and add 100 µL of blockingbuffer (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 stepincubation can be also done at 4 °C overnight.
4. Dump out the blocking buffer and bang the plate upsidedown on some paper towels to remove all the liquid. Wash 3 timeswith wash buffer (0.1 M KPi, 0.05% Tween-20, pH 7), shaking thewash out vigorously each time. Again bang out the residual washbuffer.
5. Add 50 µL/well of your 1° antibody. For screeninghybridomas, this will be cell supernatant. For obtaining a titer onserum from an immunized animal, you will need to perform serialdilutions of the serum in blocking buffer in the plate. 1:1 serialdilutions are done by placing 100 µL in the first column of yourplate of your starting dilution of serum (1:499 in blocking bufferis usually a good starting point). Then place 50 µL/well ofblocking buffer down all the wells remaining in the rows you areusing. Now pipet out 50 µL from the first well (with your startingdilution) and place in the next well in the row. Mix by pipetingthe solution up an down, and then transfer 50 µL of this solutionto the next well and again mix. Continue these dilutions down therow 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 bufferof HRP-labelled 2° antibody that is directed against the species ofyour 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 peroxidasesubstrate. Incubate at room temperature for 5-20 minutes, dependingon the rate of color development. Keep the time identical forsubsequent comparisons of titer, and for hybridoma screening go thefull 20 minutes.
10. Add 100 µL/well stop solution (0.5 M Oxalic Acid).
11. Read absorbance at 414 nm in an ELISA reader.