ELISA PROTOCOL 【Yale University 】

Materials:
1) TS Buffer: 10 mM Tris pH 8.0
150 mM NaCl
2) TST Buffer: TS Buffer with 0.05% Tween-20
3) Alkaline Phosphatase Buffer: 50 mM NaCarbonate
1 mM MgCl2
(Bring to pH 9.8 with 1 N HCl)
4) AP Substrate: p-nitrophenyl phosphate (5 mg/tablet). Sigma
Cat.# N9389
5) AP-conjugated secondary Ab: AP-Rabbit anti-mouse IgG
(Zymed). Reconstitute in ddH2O as prescribed by manufacturer and
aliquot and store at -70 °C.
6) 100 mM EDTA pH 8.0
Protocol:
1) Add 50 µl of antigen to each well of the ELISA plate. I use a 1
µg/ml of purified Sec4p most antigens will work in the 1-10 µg/ml
range, but you need to determine this emperically. Allow the
antigen to bind to the wells at room temperature for 1-2 hours or
overnight at 4 °C.
2) Discard antigen solution by flicking into sink and then placing
upside-down onto Kimwipes. Wash 1X with 100 µl TST Buffer. Add
200 µl TST with 2% BSA to block. Incubate at room temperature for
1 hour.
3) Discard block solution and add 50 µl monoclonal cell culture
supernatants directly or add the same volume of sera diluted in TST
w/ 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 Alkaline
Phosphatase. I use a 1:500 dilution of rabbit anti-mouse IgG
(Zymed). Incubate at room temperature for 1 hour. About 15 min
before the end of the incubation start dissolving the p-nitrophenyl
phosphate substrate tablets. For each ELISA plate dissolve 1 tablet
in 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 and
incubate at room temperature for 10-30 min. Stop the reactions by
adding 50 µl of 0.1 M EDTA pH8.0. For screening monoclonals it is
best to let them go until all of the positives are clearly visible,
however if you want to be quantitative it is important not to let
them get intensely yellow or they will be off scale--the plate reader
is most accurate when the wells have just begun to clearly turn
yellow.

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