Enzyme Immunosorbent Assay EIA, ELISA

50 mg/ml 3,3, 4,4 -tetraaminodiphenylether or 3,3 -diamino-benzidine (DAB)or 4-chloro-l-naphthol in DMF or precipitating TMB according to Protocol 2.5.4.1. Stock solutions are stable at RT 

Indicate the dots with a pencil in a distance of 3-5 mm on a nitrocellulose sheet. Do not touch the nitrocellulose with unprotected fingers Place the nitrocellulose onto a sheet of filter paper. 

Apply 0.1 -1 pi of antigen solution, diluted in PBS, on the marks. The diameter of the resulting dot should be not more than 3 mm. Dry at air for 10 min and block the nitrocellulose strip in Soln. A at RT for 15 min. 

Incubate the wet strip, or parts of it, in antibody dilution in Soln. A at RT for 30 min. The incubation volume should be 0.5-1 ml per cm. This volume maybe reduced if the incubation is done in a lockable tube on a roller desk. Wash the strip in a sufficient volume of PBS, at least three times for 5 min each. 

Agitate the wet nitrocellulose in 0.5 ml/cm of Soln. B at RT for 15 min and wash three times with PBS. Place the strip in a fresh container and incubate with Soln. D until the color appears. Stop the enzymatic reaction by discarding Soln. D and washing with water. 

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