Sushama Shama

The plate was washed thoroughly thrice with phosphate butter sline Ph. 7-2, Containing 0.05% v/v twenty- 20 (PBS-T). The plate was flooded with PBS-T and incubated for five minute. This it is wrapped with filter paper and kept inverted.

Blocking of free protein binding sites. The next step involved was blocking of free protein binding site in proper order to check the binding of anti-bodies on well. The blocking butter was made with egg albumin/ PBS-Tin mg/ml manner. 300ml of blocking batter was added into the each wells and incubated for 2h at 7 ⁰C then the plate was washed thrice with PBS-T.

Coating of antibody:- After the blocking of free protein binding sites, next was coating antibodies into the wells. The sample was diluted in deferent concentration with PBS. ph 7.2 and mixed with respective antibodies in 1:1 dilution. This was mixed well and incubated over night for 3 hours at 4 ⁰C.

Coating of secondary antibodies. Anti-rabbit got IgG fagged with Peroxides enzyme was used as secondary antibody. It was diluted with PBS (1:2500) and 300ml was added each well. The colour was developed by using o- phenylene diamine (OPD) as a substrate and reaction was terminated with IM sulphuric acid (H2SO₄). The colour developed was measured at 490nm by Elisa Reader.

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