Testing Rabbit Bleeds By Elisa
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Testing Rabbit Bleeds By Elisa
Claire Walczak
January 1996
Protocol:
A. Coupling Peptide to BSA
- 1 ml of 2 mg/ml BSA in 0.1 M NaHCO3.
- 1 ml of 0.2% glutaraldehyde in 0.1 M NaHCO3.
- 0.5 ml peptide (1 mg/ml in DMSO). Peptide can be added as a solid if soluble.
- Mix, adding glutaraldehyde last. Peptide and BSA turn a little yellow even before adding glutaraldehyde.
- Incubate 90' at 37 deg C.
- Add 0.1 volumes of 0.1 M NaBH4 in 0.1 M NaHCO3. There will be some bubbling. Add same amount of NaBH4 after 15'. Do a quick microfuge spin if there are too many bubbles.
B. ELISA with Peptide conjugated to BSA
- Coat 10 ug/ml antigen (peptide conjugated to BSA) diluted in TBS (50 ul/well) ON at 4 deg C.
- Remove antigen and rinse wells 2Xs with TBST.
- Block 2 hr with 200 ul of 5% NFDM in TBST.
- Remove blocking reagent and rinse wells 2Xs with TBST.
- Incubate in primary antibody diluted in Blocking Buffer for 2 hr at RT. I do tripling dilutions beginning at 1/10 (50 ul/well).
- Remove primary antibody and rinse wells 4Xs with TBST.
- Incubate in secondary antibody (1/5000 Goat anti-rabbit conjugated to AP) diluted in Blocking Buffer for 1 hr at RT (50 ul/well).
- Remove secondary antibody and rinse wells 4Xs with TBST.
- Rinse wells 2Xs with 50 mM HCO3; 0.5 mM MgCl2, pH 10.
- Develop in 1 mg/ml p-Nitrophenyl phosphate in 50 mM HCO3; 0.5 mM MgCl2, pH 10 (50 ul/well).
- Read A410 in ELISA reader.
