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LABORATORY PROCEDURES FOR PLANT CELL VIRUSES

Appendix

PVC/1998/2.04/ Appendix 2

DOUBLE ANTIBODY SANDWICH ELISA (DAS-ELISA)

 

1. Materials used in ELISA

 1.1 Microtiter plates

 1.2 Buffers

1.2.1 Coating buffer (pH 9.6)

  • 1.59 g sodium carbonate (Na2CO3)

  • 2.93 g sodium bicarbonate (NaHCO3)

  • 0.20 g sodium azide (NaN3)

    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

  • 8.0 g sodium chloride (NaCl)

  • 0.2 g monobasic potassium phosphate (KH2PO4)

  • 1.15 g dibasic sodium phosphate (Na2HPO4)

  • 0.2 g potassium chloride (KCl)

  • 0.2 g sodium azide (NaN3)

    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)

  • PBS + 0.5 ml Tween 20 per liter

    1.2.4 Sample extraction buffer (pH 7.4)

  • PBST + 2% PVP (Sigma PVP-40 polyvinyl pyrrolidone)

    1.2.5 Conjugate buffer

  • PBST + 2% PVP + 0.2% egg albumin (Sigma A-5253)

    1.2.6 Substrate buffer

  • 97 ml diethanolamine

  • 600 ml H2O

  • 0.2 g sodium azide (NaN3)

    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.

    1.3 Substrate

    p-Nitrophenyl phosphate

     

    2. Procedure

    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

     

    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.

    Guidelines prepared for CABRI by DSMZ, 3 Feb. 1998
    Page Layout by CERDIC
    Copyright CABRI, 1998  

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