Skip directly to: content | left navigation | search

Appendix B
 

FDA PROCEDURE FOR DETECTION OF ANTIBODIES TO CRY9C

COAT ELISA PLATES
  1. 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.)
     
  2. Pipette 100 ul/well into Dynatech Immulon I plates. Include grass and cat antigen to serve as reagent controls.
     
  3. Incubate overnight at 4C.
ADD SERA TO PLATES
  1. Allow plates to equilibrate to RT.
     
  2.  Aspirate liquid from wells with 12-channel manifold. Wash plates 1 x with PBS.
     
  3. Block for two hours RT with PBS-10% HIFBS, 100 ul/well.
     
  4. Aspirate liquid from wells as above. Wash plates 2 x with PBS.
     
  5. Dilute sera 1:2 with sample diluent (PBS-5% HIFBS, 0.05% Tween 20). Dilute goat antisera 1:5000 with sample diluent.
     
  6. Add diluted sera to wells, 100 ul/well, in duplicate. Pipette know positive sera in cat and grass antigen-coated wells.
     
  7. Incubate plats at RT for two hours or overnight at 4C.
ADD CONJUGATE
  1. Allow plates to equilibrate to RT (if incubated at 4C).
     
  2. Aspirate liquid from wells. Wash plates 4X with PBS-0.1% Tween-20. Allow wash buffer to remain in wells for 1 min.
     
  3. 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.
     
  4. Incubate 2 hrs. at RT.
DEVELOP COLOR
  1. Aspirate liquid from wells and wash 4x as above with PBS-0.1% Tween-20.
     
  2. Add 100 ul/well of substrate solution (TMB-Elisa, Gibco Labs., Cat. #15980-014).
     
  3. Incubate 15 min at RT.
     
  4. Stop reaction with 100ul/well 1N H2SO4.
     
  5. Read absorbance with 96-well Elisa plate reader, 450 nm.

Other Environmental Hazards & Health Effects Topics