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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Wash 3 times as before.
- 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.
- Wash 3 times as before.
- 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.
- Add 100 µL/well stop solution (0.5 M Oxalic Acid).
- Read absorbance at 414 nm in an ELISA reader.
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