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.
Protocol:
1. antigen (5-20 µg/ml) in coating buffer is added to plastic tubes or microtiter plates (currently in use are Linbro plates or Costar serocluster "U" vinyl plates #2797). 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 compley 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) 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 .
