LABORATORY PROCEDURES FOR PLANT CELL VIRUSES
PVC/1998/2.04/ Appendix 2
DOUBLE ANTIBODY SANDWICH ELISA (DAS-ELISA)
1. Materials used in ELISA
1.1 Microtiter plates
1.2.1 Coating buffer (pH 9.6)
Dissolve in 900 ml H2O, adjust pH to 9.6 with HCl and make up to 1 l.
1.2.2 PBS (pH 7.4) phosphate buffer saline
Dissolve in 900 ml H2O, adjust pH to 7.4 with NaOH or HCl and make up to 1 l.
1.2.3 PBS-Tween (PBST)
1.2.4 Sample extraction buffer (pH 7.4)
1.2.5 Conjugate buffer
1.2.6 Substrate buffer
Adjust to pH 9.8 with HCl and make up to 1 liter with H2O
Buffers can be stored at 4 -10°C for at least 2 months. Warm to room temperature before use.
2.1 Add 200 ml of purified IgG diluted in coating buffer (most common dilutions are 1-2 mg/ml) to each well of a microtitre plate.
2.2 Incubate at 37°C for 2-4 h.
2.3 Wash plate with PBS-Tween using wash bottle, soak for a few minutes and repeat washing two times. Blot plates by tapping upside down on tissue paper.
2.4 Add 200 ml aliquots of the test sample (extracted in sample extraction buffer) to duplicate wells.
2.5 Incubate overnight at 4 °C.
2.6 Wash three times as in step 3.
2.7 Add 200 ml anti-virus conjugate diluted 1:1000 in conjugate buffer (dilution depending on antiserum quality) to each well.
2.8 Incubate at 37 °C for 4 hours.
2.9 Wash three times as in step 3.
2.10 Add 200 ml aliquots of freshly prepared substrate (10 mg p-nitrophenyl phosphate [Sigma 104-105] dissolved in 10 ml of substrate buffer) to each well. Incubate at room temperature for 30-60 min, or as long as necessary to obtain clear reactions.
2.11 Assess results by:
a) Visual observation
b) Spectrophotometric measurement of absorbance at 405 nm
Guidelines prepared for CABRI by DSMZ, 3 Feb. 1998
© The CABRI Consortium 1999-2013.