操作步驟：

1. Allow the pre-coated plate to reach room temperature while in the sealed pouch.

2. Prepare 1x working dilutions of the 10x wash and assay buffers in clean containers using 18.2MΩ de-ionized water or Type I ultrapure water. For one plate: Wash buffer: add 15mL concentrate to 135mL water Assay buffer: add 2.5mL concentrate to 22.5mL water Adjust volumes accordingly for multi-plate assays. *Diluted buffers may be stored at 4OC for up to 1 week

3. Remove the plate from the foil pouch and wash by adding 150μL wash buffer to each well. Empty the wells by inverting the plate and tapping on absorbent paper. Repeat the wash 2x.

4. Add standards, samples and blanks to the plate (final volume in all wells should be 100μL). Standards: pipette 180μL assay buffer into wells A1 and B1 and 100μL into remaining wells of rows A and B. Add 20μL Bla g 2 standard to wells A1 and B1. Mix well and transfer 100μl into wells A2 and B2. Continue across the plate to wells A10 and B10 to make 10 serial doubling dilutions. Samples: dust extracts for Bla g 2 analysis are routinely diluted two-fold starting at 1/10. Other types of samples, like air filter extracts and allergen extracts, may require different dilutions. It is recommended to test each sample at a minimum of three dilutions; 6-12 are recommended. Blanks: add assay buffer to wells A11, B11 and A12, B12.

5. Return the plate to the foil pouch or cover with plate sealer and incubate for 1 hour at room temperature.

6. Wash wells 3x with 150μL wash buffer. Prepare a 1:1,000 detection antibody/ conjugate mix by adding 10μL Rabbit polyclonal antiserum and 10μL Peroxidase Conjugated Goat Anti-Rabbit to 10mL assay buffer. Mix well and add 100μl to each well. Cover and incubate for 1 hour at room temperature.

7. Wash wells 3x with 150μL wash buffer. Add 100μl TMB substrate to each well and monitor the reaction as the blue color develops. When OD450nm reaches 0.08 for the first standard in wells A1 and B1 (generally within 1-5 minutes), add 50μL stop solution (the color will change to yellow).

8. Read the plate at 450nm. The ideal OD for standard 1 is 2.0-2.5.