ELISA

Preparation:

  1. capture antibody:

resuspend in 1 ml PBS ---720ug/ml (180X)

aliquots and store in –80 C

working concentration: 4.0 ug/ml in PBS

  1. detection antibody:

resuspend in 1.0ml PBS---72ug/ml (180X)

aliquots and store in –80 C

working concentration: 0.4ug/ml in PBS

  1. standards

IL-4

    • reconstitute in 0.5ml of Reagent Diluent, ---

100ng/ml (100x of highest dilution)

    • aliquots and store in –80 C

    • working standard curve: 7 point 2-fold serial dilution

1000pg/ml, 500pg/ml, 250pg/ml, 125mg/ml,

62.5pg/ml, 31.25pg/ml, 15.625pg/ml, 0

IFN-gamma

    • reconstitute in 0.3ml of Reagent Diluent, ---

100ng/ml (50x of highest dilution)

    • aliquots and store in –80 C

    • working standard curve: 7 point 2-fold serial dilution

2000pg/ml, 1000pg/ml, 500pg/ml, 250mg/ml,

125pg/ml, 62.5pg/ml, 31.25/ml, 0

  1. wash buffer

0.05% Tween-20 in PBS

  1. reagent diluent

1%BSA in PBS, 0.2uM filtered

  1. substrate solution

1:1 x mixture of color reagent A and B right before use

  1. stop solution

2N H2SO4

Assay

Day 1

1. Plate Preparation

A. dilute the capture antibody to the working concentration in PBS w/o carrier protein.

B. Immediately coat 96-well microplate with 100ul/well of the diluted capture antibody

C. Seal the plate and incubate overnight at room temperature.

Day 2

1. wash plate:

aspirate each well and wash with 400ul /well of wash buffer 3X, total.

2. block plate

adding 300ul of reagent diluent /well

incubate at RT for >= 1 hour

3. wash blocked plate

aspirate each well and wash with 400ul /well of wash buffer 3X, total.

4. Assay

Standard curve: 7 point 2 fold serial x2 (duplicates)

100ul of sample, with 5 fold serial dilution, x3 (triplicates)

RT, 2 hours incubation

5. aspirate each well and wash with 400ul /well of wash buffer 3X, total.

6. Add 100ul of Detection antibody, diluted in Reagent Diluent. Incubate for 2 hours at room temperature.

7. aspirate each well and wash with 400ul /well of wash buffer 3X, total.

8. 100 ul/well of working dilution of streptavidin-HRP

RT, 20 minutes, avoid light

9. aspirate each well and wash with 400ul /well of wash buffer 3X, total.

10. 100 ul/well of Substrate Solution mixture

RT, 20 minutes, avoid light

11. 50 ul of stop solution to each, mix by tapping

12. microplate reading at 450nm, subtract reading at 540 or 570 as wavelength correction.