ELISA PROTOCOL（Yale University）

1. 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

2. Protocol

(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/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 placingupside-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 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 turn yellow.