Table 1. Major differences (and similarities) in ELISpot protocols submitted by 4 participant laboratories.

PROTOCOL STEP	LABORATORY A	LABORATORY B	LABORATORY C	LABORATORY D
Pre-incubation step	Yes. Overnight +/− antigen in polypropylene tubes	Yes. Overnight +/− antigen in 48 well tissue culture plates	No	No
Amount of anti-IFNγ coating antibody used	100µl at 15µg/ml	100µl at 5µg/ml	50µl at 15µg/ml	50µl at 15µg/ml
Number of cells per ELISpot well (used for test antigens)	2.5×105 or 0.625×105 for Ag mixture or 0.31×105 for positive control (calculated from proportion of original culture transferred)	2.5×105 (calculated from proportion of original culture transferred)	1.0×105	3.0×105
Time in ELISPOT plates	48 hours	18–24 hours	18–24 hours	18–24 hours
Culture medium used	RPMI 1640+10% HI-FCS	RPMI 1640+10% pooled HI- AB human serum	RPMI 1640+5% autologous plasma	RPMI 1640+10% HI-FCS
ELISpot Plates	PVDF-backed (Millipore, MAIPS4510)	As for Laboratory A	As for Laboratory A	As for Laboratory A
Capture Antibody	Anti-IFNγ (Mabtech 1-D1K)	“	“	“
Detection Antibody	Biotinylated anti-IFNγ (Mabtech 7-B6-1)	“	“	“
ELISpot Plate Pre-treatment	70% Ethanol	“	“	“