過(guò)敏原標準品:Recombinant Phl p 5a prepared in 1% BSA/50%glycerol/PBS, pH 7.4.

濃度:2500 ng/mL (The rPhl p 5a was from E. coli and purified by conventional biochemical methods. Validity of the rPhl p 5 standard was confirmed by comparison with the European Pharmacopoeia reference CRS (Y0001566), recombinant major allergen rPhl p 5 containing 8.56 μg of rPhl p 5 per vial.)

操作步驟：

1. Prepare a 1x dilution of the 10x wash and assay buffer concentrates 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 3mL concentrate to 27mL 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. ?Extracts of dust samples are routinely started at 1/10 dilution. Air filter extracts, allergen extracts, and other types of samples may require a different dilution scheme ?Standard and sample dilutions can be prepared directly on the plate ?Pre-dilutions of samples can be made in tubes or on a dilution plate if necessary. A minimum of three dilutions per sample is recommended ?The example below is for testing six samples starting at 1/10 dilution Add 100μL assay buffer to all wells, plus an additional 80μL to wells in column 1. Standard: gently vortex the Phl p 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 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: add 20μL sample to wells C1 through H1. Mix, then transfer 100μL into 100μL assay buffer in the next well. Continue across the plate for the desired number of dilutions. *Remove and discard 100μL from the last wells for the standard and sample dilutions (final volume in all wells should be 100μL).

4. Cover the plate and incubate at room temperature (away from direct sunlight) for 1 hour. *Gentle agitation on a plate shaker during incubations may reduce variability.

5. Wash the plate 3x with 150μL wash buffer per well. Gently vortex the biotinylated Bo1 and prepare a 1:1,000 detection antibody/conjugate mix by adding 11μL biotinylated Bo1 and 11μL streptavidin-peroxidase to 11mL 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 reagent reservoirs 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 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 within 30 minutes (absorbance values begin to decrease after 30 minutes). The OD for Standard 1 should be between 1.2 and 3.5.