Day I
1. Coat wells on 96-well plate (Immulon II-Dynatech Labs) with
100µl of purified R187 (1/40 in PBS). Incubate @ 37 C ON.

Day II
Prepare dilutions of standards and samples!!!
2. Rinse wells 2X with 250µl of PBS.
3. Block with 250µl PBS 5% FSC @ 37 C for 1 hour.
4. Wash plates 4X with 250µl of WASH buffer.
5. Add 100µl of diluted standars and samples to the wels.
Incubate @ 37 C ON.

Day III
6. Wash plates 4X with 250µl of WASH buffer.
7. Add 100µl of anti-P30 serum (1/2,000 in ELISA diluent).
Incubate @ 37 C 1 hour.
8. Wash plates 4X with 250µl of WASH buffer.
9. Add 100µl of RHP Donkey anti-Rabbit IgG
(1/700 in ELISA diluent). Incubate @ 37 C 1 hour.
10. Wash plates 6X with 250µl of WASH buffer.
11. Add 100 µl of peroxidase substrate (freshly made)
and monitor OD630. When it reaches 1.2
add 50µl of 2M H₂SO₄. (10-20 minutes).
12. Read samples at OD450 and calculate.