Antigen specific T-Cell ELISPOT - (Nov/15/2011 )

Hi everybody,

I have one simple question:

When you measure the antigen specific cellular immune response, as I understood, you have to quantify interferon gamma secreting epitope specific T-cell, so from what I gather here is you have two parameters to check; First, the production of Interferon gamma and next epitope specificity.. Does that mean you have to do ELISPOT twice?

Thanks in advance.
It's Me

ELISPOT PROTOCOL

STEP 1:- PREWETTING WITH 70% ETHANOL FOR EACH WELL 50ul KEEP FOR 5 MIN.
STEP 2:-WASH WITH PBS 250ul FOR 3 TIMES.
STEP 3:- 100ul CAPTURE ANTIBODY 5ug/ml,(5ul/ml).STOCK CONC.1mg/ml.
STEP 4:- OVERNIGHT INCUBATION AT 4 DEGREE.APPROX .18 HRS.
WASH WITH PBS FOR 6 TIMES (300 ul/well).
STEP 5:- BLOCKING WITH FBS FOR EACH WELL 250ul FILTER WITH( .45 MILIPORE FILTER).
STEP 6:- 4 HRS INCUBATION.
INBETWEEN PREPARE EFFECTOR CELLS IN RPMI MEDIUM.
STEP 7:- SEED CELLS INTO WELL WITH 1 MILLION CELLS /ml .(100ul PER WELL).
FOR POSITIVE CONTROL
STEP 8:-ADD MITOGEN---(PHA) 50ul PER WELL.(+1)
STEP 9 :- OR ANTIGEN 50 ul PER WELL.{(-2)(-) control (-3)}
STEP 10:-INCUBATE FOR 18-24HRS IN 37 DEGREE CO2 INCUBATOR.
STEP 11:- WASH WITH 2 TIMES PBS AND 4 TIMES PBST APPROX 250-300ul.
ADD SECONDARY ANTIBODY(BIOTINYLATED).
20ul SEC ANTIBODY + 10 ml 7.5 % BSA in DPBS.—ADD 100UL PER WELL.
STEP 12:-INCUBATE FOR A ATLEAST 3HRS AT ROOM TEMP.
STEP 13:- DISCARD CONTENT OF PLATE..
STEP 14:- PREPARE BEFORE HALF HOUR 10 Ml DPBS IN PETRIDISH + 1 DROP A+ B REAGENT OF VICTASTAIN + 10 Ul T-20
STEP 15:- WASH 2 TIMES PBS & 4 TIMES WITH PBST
STEP 17:- ADD 100ul PER WELL & INCUBATE FOR 1 HR AT ROOM TEMP.
PREPARATION OF SUBSTRATE :-
A:- 2.5 ml OF DMF TAKEN IN 5 ml TUBE COVER WITH ALMINIUM FOIL + 1 TABLET OF AEC.
B:- 47.5 ml DW SIGMA TAKEN IN 50 ml TUBE + 280 ul SODIUM ACETATE + 180 ul ACETIC ACID
STEP 18:- MIX A+ B and ADD 25ul H202.
STEP 19:-10 ML MIXTURE WAS FILTERD BY 0.45 ul.
STEP 20:-AVIDIN –ENZYME COMPLEX DISCARD .
STEP 21:-WAS PLATE WITH 3 TIMES PBST THEN 3 TIMES WITH PBS.
STEP 22:-100ul/well AEC SUSTRATE ADDED.
STEP 23:-INCUBATE FOR 5 MIN IN DARK.
STEP 24:-STOP DEVELOPMENT UNDER TAP WATER.
STEP 25:-DRY WELL WITH PAPER TOWEL.
STEP 26 :- OVERNITE DRY PLATE IN DARKNESS.

Dear Chinmaya Mahapatra,

Thank you for your detailed protocol.