ELISA protocol

Double antibody sandwich ELISA (DAS-ELISA)
1.	Coating.Dilute IgG (white cap) 1:1000 in coating buffer and add 200 µl to each well of a microtitre plate.
2.	Incubate at 37 °C for 2-4 h.
3.	Wash plate with PBS-Tween using wash bottle, soak for a few minutes and repeat washing two times. Blot plates dry by tapping upside down on tissue paper.
4.	Add 200 µl aliquots of the test sample (extracted in sample extraction buffer) to duplicate wells.
5.	Incubate overnight at 4 °C.*
6.	Wash three times as in step 3.
7.	Dilute anti-virus conjugate (red cap) 1:1000 in conjugate buffer and add 200 µL (dilution depending on antiserum quality) to each well.
8.	Incubate at 37 °C for 2-4 hours.
9.	Wash three times as in step 3.
10.	Add 200 µl aliquots of freshly prepared substrate (1 mg/ml of p-nitrophenyl phosphate [Sigma 104-105] in substrate buffer) to each well. Incubate at room temperature for 60-120 min or until unambiguous reactions are obtained.
11.	Assess results by:	a) Visual observation
		b) Spectrophotometric measurement of absorbance at 405 nm
* While overnight incubation at 4°C is what we recommend, a 4-hours incubation at 37 °C will reveal similar results.
Reference: Clark, M. F. and A. N. Adams. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34: 475-483.