7.1.3. Indirect ELISA
The reagents needed to perform the ELISA are:
- Bicarbonate / carbonate coating buffer, 100 mM, pH 9.6.
- Phosphate buffered saline, PBS, pH 7.4.
- Blocking solution (PBS with 1-2 % BSA).
- Washing buffer (PBS with 0.05 % Tween 20).
- Primary antibody (rabbit, chicken, mouse).
- Peroxidase or alkaline phosphatase-conjugated secondary antibody (anti-rabbit, anti-chicken; anti-mouse).
- Substrate for peroxidase alkaline phosphatase-conjugated secondary antibody (e.g. TMB (3,3´,5,5´-tetramethylbenzidine)).
- Stop solution (0.5 M H2SO4).
- Microtiter plates.
- Microtiter plate reader.
Many different types of enzymes can be used for detection. Peroxidase-conjugated secondary antibodies and TMB (3,3´,5,5´-tetramethylbenzidine) are commonly used and accessible.
- Dilute the bee homogenates in coating
The total protein concentration should not exceed 20 µg per ml.
- Coat the wells of a microtiter plate with 100 µl per well of the antigen dilution.
- Cover the plate using an adhesive plastic.
- Incubate for 2 hours at room temperature or at 4°C over night.
- Remove the coating buffer.
- Wash the plates two times filling the wells with washing buffer.
- Block the remaining protein-binding sites by adding 200 µl blocking solution to the wells.
- Incubate for 2 h at room temperature or at 4°C over night.
- Wash the plate two times with washing solution.
- Add 100 µl of the M. plutonius specific antibody diluted in blocking solution.
- The optimal dilution should be determined using a dilution assay.
- Cover the plate and incubate for 2 hours at room temperature.
- Wash the plate three times with PBS.
- Add 100 µl of the secondary, conjugated antibody diluted according to the manufacturer´s instruction.
- Cover the plate.
- Incubate for 1 hour at room temperature.
- Wash four times with washing solution.
- Dispense 100 µl per well of the substrate solution.
- Incubate for 15 min in room temperature (dark).
- Add equal volume of the stop solution (2 M H2SO4).
- Read the optical density at 450 nm using a
Compare the density reads of unknown samples against standards (e.g. suspensions of known concentrations of M. plutonius). To ensure accuracy, include standards and at least one blank sample to each plate.