When testing sputum samples with ELISA, instead of using DTT, which will destroy the antibody when the sample is applied to a antibody-coated ELISA-plate, is a solution to reduce the sputum samples irreversibly with TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, e.g. from Alfa Aeser: H51864) and then to remove all of the surplus reagent by dialysis or gel filtration?
The DTT will destroy any disulphide bridge, also the bridges of your antibodies. The onyl way to overcome this item is the oxidation of any free DTT. Add Dicystein in equimolar concentrations.
There's no such thing as irreversible reduction of cysteines. TCEP is a strong reductant, but once it is removed the cysteines will be as prone to re-oxidation as if they had been reduced with another reducing agent such as DTT.
Maybe the use of reduction, with DTT, and alkylation, with IAA, of your proteins. Then removal of the reagents prior to ELISA tersting.
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