操作步驟：

1. 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

2. 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 then tap on absorbent paper to remove residual buffer. Repeat the wash cycle two more times.

3. Add standards, samples, and blanks to the plate (final volume in all wells is 100μL). Standards: add 180μL assay buffer into wells A1 and B1, and 100μL into remaining wells of rows A and B. Vortex the Bos d 5 standard and add 20μL to wells A1 and B1. Mix well by pipetting up and down 7-10 times and then transfer 100μL into wells A2 and B2. Mix well and continue the serial doubling dilution scheme across the plate to column 10. The assay buffer in wells A11, B11 and A12, B12 will serve as Blanks. Samples: dust extracts are routinely tested starting at 1/10 dilution and can be prepared directly on the pre-coated plate: add 20μL sample to 180μL assay buffer. Mix, then transfer 100μL into 100μL assay buffer in the next well. Continue across the plate for the desired number of dilutions. A minimum of three dilutions per sample should be tested; 6-12 dilutions are recommended. Air filter extracts, allergen extracts, food products, and other types of samples may require a different dilution scheme. Note: sample dilutions may also be prepared in tubes or on a 96-well dilution plate and transferred to the pre-coated plate.

4. Incubate the plate at room temperature (away from direct sunlight) for 1 hour.

5. Wash the plate 3x with 150μL wash buffer per well. Vortex the biotinylated NBD5-2 and prepare a 1:1,000 detection antibody/conjugate mix by adding 10μL biotinylated NBD5-2 and 10μL streptavidin-peroxidase to 10mL assay buffer. Mix thoroughly and add 100μL to each well.

6. Incubate the plate at room temperature (away from direct sunlight) for 1 hour.

7. Pour the TMB substrate and stop solution into separate basins so they are ready to use in the next step. Wash the plate 3x with 150μL wash buffer per well.

8. Use a multi-channel pipette to add 100μL TMB to each well. Gently tap the plate and monitor the reaction as the blue color develops. Once OD450 reaches 0.08-0.09 for Standard 1, use a multi-channel pipette to add 50μL stop solution to each well (the color will change to yellow).

9. Read the plate at 450nm. The OD for Standard 1 should be between 1.2 and 3.5, with an ideal range of 2.0 - 2.5