assay protocol

ELISA Principle:

Enzyme-linked immunosorbent assay (ELISA) has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-controlcheck in various industries. Attempting to detect (and quantify) the presence of the antigen in the sample proceeds as follows: Antigens from the sample are attached to a surface. Then a further specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.

Firstly the indirect ELISA is the conventional ELISA. The competitive ELISA and the sandwich ELISA are the two prevalent ELISA subtypes. Being differentiated by different principles, they are prefered by different people. Moreover, there is a more sensitive type of ELISA, the ELISpot.

As for the indirect ELISA, following the detection antibody is added, it forms a complex with the antigen. The detection antibody can be covalently linked to anenzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visiblesignal, which indicates the quantity of antigen in the sample.

Materials:

  • 96-Well Microtiter Plates
  • Eppendorf Tubes
  • Twelve-Channel Pipettor
  • 1mL Adjustable Pipettor
  • Humid Chamber
  • Wash Bottle or ELISA Plate Washer
  • ELISA Plate Reader
  • TMB

Reagents:
PBS (pH 7.3)
Coating Solution: 0.1 M Sodium Carbonate (pH9.6) with 0.02% Sodium Azide
Wash Solution: 0.9% NaCl-0.05% Tween 20 (NaCl/T)
PBS-TA: PBS-Tween 20 (0.05%) - sodium azide (0.02%) pH 7.3
AP Substrate Buffer: 0.05 M sodium carbonate-0.001 M MgCl2 (pH 9.3)
HRP Substrate: Mix equal parts TMB peroxidase substrate and TMB peroxidase soln B.
Notes: 1. Sodium Azide cannot be used in conjunction with HRP conjugates.
2. Wash cycles are critical steps to eliminate background.

ELISA Protocol:

1. Antigen (5-20 µg/ml) in coating buffer is added to plastic tubes or microtiter plates. Incubate for 4 hours at 37°C and then store at 4°C until use (if less than two weeks). For the vinyl plates, wash three times with washing solution, dispel liquid by slapping on paper towels and then cover and store at -70°C. These plates are good for at least 6 months.

2. Wash tubes or plates with washing solution three times, waiting 10 minutes between washes. Make sure all wells are filled with wash solution during each wash cycle.

3. Block tubes or plates with 0.5% BSA for 1 hr by completely filling tubes or all wells.

4. Wash tubes or plates with washing solution four times, waiting 10-15 minutes.

5. Add 100µl of antisera diluted in PBS-TA to each well, Incubate for 5 hours at RT or overnight in refrigerator. Normally a 1:100 dilution is sufficient. Monoclonal antibodies should be used undiluted.

6. Wash the plate as before and then add conjugate diluted with PBS-T(A). Incubate for 4 hours at RT for AP conjugates; 1.5 hours at RT or 1 hr at 37 C for HRP conjugates. Normally a 1:1000 dilution is sufficient.

7. Wash four times as before and then add 100 µl substrate to each well.

For AP conjugates: Substrate solution is p - nitrophenyl phosphate (Sigma) at 1 mg/ml in substrate buffer. Allow the color to develop for 100 minutes and stop reaction by adding 10 µl of 1 N NaOH. Read absorbance at 405nm.

For HRP conjugates: Mix equal parts of the TMB system, place in wells, allow to develop until desired intensity (app. 5 min) and stop reaction by adding 100 µl of 1M Phosphoric Acid. Read at 450 nm.

Conjugate Preparation

1. Centrifuge 0.3ml enzyme (alkaline phosphatase, Sigma type VII, sp act. 1,140 U/ml) at 8,000 x g for 2 mins.

2. Add to the pellet 0.1 ml specific antibody (animal specific, pathogen specific, to choose an antibody,please click here ) affinity purified, resulting in a 3:1 ratio of enzyme to antibody.

3. Dialize overnight against PBS.

4. Add 10 µl of gluteraldehyde to a final concentration of 0.2%.

5. Incubate at RT for 2 H, dilute to 1ml, and dialize overnight against PBS.

6. Dilute to 10ml with 0.05 M Tris-5% BSA-1mM MgCl2-0.02% NaN3 .

7. Store at 4°C.