LEAP Testing Service

ASTM D7427-08 Allergen ELISA. This assay is actually a set of four ELISAs used to quantitate the level of four NRL allergens. The assays use a set of monoclonal antibodies to: (1) capture the allergen to the plate and a second monoclonal to (2) quantitate the level of allergen present. A brief protocol is presented below.

Procedure:
1. Dispense 100 ul Assay Buffer to each well.
2. Pipet in 25 ul of standard, control and sample into appropriate wells in duplicate
3. Cover plate and incubate at RT for 1 h on a shaker.
4. Wash wells 4X with 300 ul Wash buffer.
5. Dispense 100 ul well enzyme conjugate (2Ab- conjugated to HRP)
6. Cover plate and incubate 30 min RT on shaker.
7. Wash wells 4X with 300 ul wash buffer.
8. Add 100 ul Substrate Solution.
9. Cover plate, incubate 15 min RT on shaker.
10. Stop reaction with addition on 100 ul stop solution (1% SDS)
11. Shake the plate for at least 1 -2 minutes before reading absorbance at 414 nm. Make sure there are no bubbles present in any of the wells before reading the plate.
12. The sum of the 4 allergens is reported.