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ELISA --- horseradish peroxidase detection

1-6. Code plates, add primary antibody, and wash.

7. Add 100 l of anti-mouse IgG-horseradish peroxidase (HRP) conjugate (1:4,000 in PBS/0.05% Tween-20). and incubate for 30 minutes at room temperature or 37 C.

8. Wash the plate 5 times with the PBS/0.05% Tween-20.

9. Add 100 l of substrate (0.1% TMB (3,3',5,5'-tetramethylbenzidine), 0.01% hydrogen peroxide, in 0.1 M sodium acetate.).

10. Incubate for 10-60 minutes at room temperature. Observe the blue color change.

11. Stop the reaction with 50 l 1 M H2SO4.

12. Read absorbance at 450 nm for the stopped reaction and at 650 nm for the upstopped raction.