ELISA PROTOCOL 【Yale University 】

Materials:1) TS Buffer: 10 mM Tris pH 8.0150 mM NaCl2) TST Buffer: TS Buffer with 0.05% Tween-203) Alkaline Phosphatase Buffer: 50 mM NaCarbonate1 mM MgCl2(Bring to pH 9.8 with 1 N HCl)4) AP Substrate: p-nitrophenyl phosphate (5 mg/tablet). SigmaCat.# N93895) AP-conjugated secondary Ab: AP-Rabbit anti-mouse IgG(Zymed). Reconstitute in ddH2O as prescribed by manufacturer andaliquot and store at -70 °C.6) 100 mM EDTA pH 8.0Protocol:1) Add 50 µl of antigen to each well of the ELISA plate. I use a 1µg/ml of purified Sec4p most antigens will work in the 1-10 µg/mlrange, but you need to determine this emperically. Allow theantigen to bind to the wells at room temperature for 1-2 hours orovernight at 4 °C.2) Discard antigen solution by flicking into sink and then placingupside-down onto Kimwipes. Wash 1X with 100 µl TST Buffer. Add200 µl TST with 2% BSA to block. Incubate at room temperature for1 hour.3) Discard block solution and add 50 µl monoclonal cell culturesupernatants directly or add the same volume of sera diluted in TSTw/ 1% BSA. Incubate 1 hour at room temperature.4) Discard the primary antibody solution and wash 3X with TST.5) Add 50 µl of diluted secondary antibody conjugated to AlkalinePhosphatase. I use a 1:500 dilution of rabbit anti-mouse IgG(Zymed). Incubate at room temperature for 1 hour. About 15 minbefore the end of the incubation start dissolving the p-nitrophenylphosphate substrate tablets. For each ELISA plate dissolve 1 tabletin 5 ml of AP Buffer.6) Discard the secondary antibody solution and wash 3X with TST.7) Add 50 µl per well of the p-nitrophenyl phosphate solution andincubate at room temperature for 10-30 min. Stop the reactions byadding 50 µl of 0.1 M EDTA pH8.0. For screening monoclonals it isbest to let them go until all of the positives are clearly visible,however if you want to be quantitative it is important not to letthem get intensely yellow or they will be off scale--the plate readeris most accurate when the wells have just begun to clearly turnyellow.

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