Division Material and Reagents:X1

Appropriate ELISA microtitre plates.

Antigen: supplied as a cell extracted concentrate, prepared as described below, and stored at either – 20 °C or – 70 °C.

Blocking buffer: phosphate buffered saline (PBS) containing 0,3 % BTV negative adult bovine serum, 0,1 % (v/v) Tween-20 (supplied as polyoxyethylene sorbiton monolaurate syrup) in PBS.

Monoclonal antibody: 3-17-A3 (supplied as hybridoma tissue-culture supernatant) directed against the group-specific polypeptide VP7, stored at - 20 °C or freeze-dried and diluted 1/100 with blocking buffer before use.

Conjugate: rabbit anti-mouse globulin (adsorbed and eluted) conjugated to horseradish peroxidase and kept in the dark at 4 °C.

Chromogen and substrate: Orthophenylene diamine (OPD-chromogen) at a final concentration of 0,4 mg/ml in sterile distilled water. Hydrogen peroxide (30 %w/v-substrate) 0,05 % v/v added immediately before use (5μl H ₂ O ₂ per 10 ml OPD). ( Handle OPD with care - wear rubber gloves - suspected mutagen ).

1 Molar sulphuric acid: 26,6 ml of acid added to 473,4 ml of distilled water. ( Remember Acid must be added to water, never water to acid .)

Orbital shaker.

ELISA plate reader ( the test may be read visually ).