FDA PROCEDURE FOR DETECTION OF ANTIBODIES TO CRY9C
COAT ELISA PLATES
Suspend purified Cry9C solution to a concentration of 2 ug/ml in
carbonate/bicarbonate buffer, pH 9.6. Suspend crude grass antigen to a
concentration of 40 ug/well, and crude cat antigen to a concentration of
0.2 units/ml. (Optimum concentration of antigen previously determined by
block titration with known positive and negative sera.)
Pipette 100 ul/well into Dynatech Immulon I plates. Include grass
and cat antigen to serve as reagent controls.
Incubate overnight at 4C.
ADD SERA TO PLATES
Allow plates to equilibrate to RT.
Aspirate liquid from wells with 12-channel manifold. Wash
plates 1 x with PBS.
Block for two hours RT with PBS-10% HIFBS, 100 ul/well.
Aspirate liquid from wells as above. Wash plates 2 x with PBS.
Dilute sera 1:2 with sample diluent (PBS-5% HIFBS, 0.05% Tween 20).
Dilute goat antisera 1:5000 with sample diluent.
Add diluted sera to wells, 100 ul/well, in duplicate. Pipette know
positive sera in cat and grass antigen-coated wells.
Incubate plats at RT for two hours or overnight at 4C.
Allow plates to equilibrate to RT (if incubated at 4C).
Aspirate liquid from wells. Wash plates 4X with PBS-0.1% Tween-20.
Allow wash buffer to remain in wells for 1 min.
Add affinity purified peroxidase-conjugated goat anti-human IgE (KPL,
Cat #074-1002) to wells, 100 ul/well. For wells with goat serum, add
affinity purified peroxidase-conjugated donkey anti-goat IgG (Jackson
Labs., Cat. #705-035-147). Appropriate dilution of conjugate must be
determined for each new lot. Conjugate is diluted in PBS-10% HIFBS.
Incubate 2 hrs. at RT.
Aspirate liquid from wells and wash 4x as above with