ELISA procedure

1.Prepare Protein Antigen

Dilute protein antigen in Bicarbonate ELISA Coating Buffer (BECB).

Final concentration = [2µg / ml].

Make 10ml solution for 1 full ELISA plate.

Protein stocks concentration: = 10µg / µl.

Use 1µl of protein in 5ml ECB or 2µl in 10ml BECB.

Use the multichannel pipettor to add coating buffers, protein antigen (Ag), and antibody solutions.

Coat wells of high binding polystyrene microtiter plates (catalog # S2506710, Immulon
Thermoflecton Corporation, Milford, MA, USA) with Ag capture agent (protein) or control blocking
buffer (BSA) -50µl of 2µg /ml protein antigen /Coating Buffer solution per well, tap and slowly shake
to coat wells.

Cover with adhesive plastic sheet; incubate at 4°C overnight with slowly shaking.

The next morning, invert plate over sink to dump supernatant, wrap in stack of paper towels and
tap on countertop till no more solution comes off.

Wash 3 times with 300µl PBST / well (150µl 2x using 96 well washer), wrap in stack of paper towels,
tap on countertop until no more water comes off.

2.Blocking Buffer

Block with 5% BSA in PBST blocking buffer 300µl / well, cover plates with adhesive plastic sheet,
incubate for at least 2h at room temperature (RT) with slow shaking.

Invert plate over sink to dump supernatant.

Add 1% BSA in PBST: 100µl / well to the four blank wells, the first two wells of rows A and B on
each plate.

3. Add Serum (Primary Antibody)

All serum samples and negative and positive controls are stored in -70 °C.

Vortex for 30 s to a minute before diluting a sample.

Prepare fresh serum dilution (negative control, positive control, and samples) (1:1000) in 1% BSA in
PBST in 10ml plastic tubes.

You will need 100µl diluted serum / well, and each is done in duplicate, so:

3000µl total at 1:1000 = 3µl serum in 2997µl 1%BSA. Just need 600µl dilution for the negative
control, 600µl dilution for the positive control and 200µl dilution samples for test 1% BSA as a blank.

Vortex for 30 s.

Load 100µl diluted serum/well, cover plate with adhesive plastic sheet incubating for two hours at
RT, while slowly shaking (150-200 rpm).

Invert plate over sink to dump diluted serum (primary antibodies).

Wash six times with 300µl PBST using Modern plate washer; each wash has 15 s intervals.
4. Add Secondary Antibody

Dilute HRP conjugated secondary antibodies: Goat Anti-Human IgG or IgA, 1:10,000 in 1% BSA in
PBST.

You will need 100µl / well, but make a little extra to account for pipetting errors.

You will have 96 wells total ≈9,600µl, but round up to 10,000µl or 10ml diluted secondary antibodies
/ plate 1µl in 9.999ml 1:10,000 dilution.

Add 100µl diluted secondary antibodies / well, cover plates with an adhesive plastic sheet; incubate
for one hour at RT with slowly shaking (150-200 rpm).

Take peroxidase substrate out of refrigerator, and allow it to equilibrate to RT.

Invert plate over sink to dump secondary antibodies.

Wash extensively, 6 times using Modern plate washer, with 300µl PBST, as before.

5. Develop Plates Measure Optical Density

1. After the final washing develop plates with 100µl / well of RT peroxidase substrate, develop for
5 min for IgG and 30 min for IgA; protect from light at this time. Then add 50µl / well H2SO4 stop
solution.

6. Measure OD at 450nm in an ELISA plate reader

1. Use mean values of reactivity of the duplicate samples.