Editing Indirect ELISA (section)

== Home-Made TMB Solution ==

=== Working Protocol (receipe 1) ===

'''Solution A:'''

Dissolve

Citric acid monohydrate: 3.60 g

trisodium citrate dihydrate: 1.838 g

in 950 ml of double-distilled water.

Add 0.564 g Urea hydrogen peroxide while continuous stirring.

'''Solution B'''

Add 100 mg TMB to 15 ml&nbsp;&nbsp;N,N-Dimethylacetamide. Store in a dark bottle at -20 oC.

'''Working Solution'''

Just before use, add 50 ul solution B to 5 ml Solution A, then add 1 drop of 30% H2O2.

=== Receipe 2 ===

'''Phosphate-Citrate Buffer 0.1 M at pH 4.6 ± 0.2 plus perborate '''<br/>Mix 24 ml of 0.2 M dibasic sodium phosphate (dissolve 1.78 g Na2HPO4-2H2O or 3.58 g of Na2HPO4•12H2O in dH2O and fill up to 50 ml) with 26 ml 0.1 M citric acid (2.10 g citric acid monohydrate in dH2O and make to 100 ml with dH2O), check pH and dissolve 0.02 g sodium perborate. Store in polyethylene bottle at 4 °C stable 1 year.

<br/>'''TMB substrate 100 mM&nbsp;'''

<br/>Dissolve 240 mg 3,3’,5,5’–tetramethylbenzidine (m.w. 240) in 10 ml of DMSO. Store in opaque polyethylene bottle at 4 °C stable 1 year.

<br/>'''Working TMB chromogen&nbsp;'''<br/>Mix 25 µl of TMB substrate to 1.0 ml of phosphate-citrate buffer plus perborate, stable for several hours at room temperature or for few days at 4 °C.

=== Receipe 3  ===

from Patent

[https://patents.google.com/patent/US5206150 https://patents.google.com/patent/US5206150]

Dissolve

Citric acid monohydrate: 3.60 g

trisodium citrate dihydrate: 1.838 g

in 950 ml of double-distilled water.

Add 1.422 g Bacitracin&nbsp; with stirring until it dissolved.

Add 0.564 g&nbsp; &nbsp;Urea hydrogen peroxide while&nbsp; continuous stirring.

Add &nbsp;300 mg 3,3',5,5'-Tetramethylbenzidine&nbsp; (TMB)&nbsp; to 50 ml of dimethyl sulfoxide

Add&nbsp; TMB solution aqueous&nbsp; solution.

Mixing in an amber bottle

Store at 4° C.

The final concentrations of each of the above components is: bacitracin, 1.0 mM; urea hydrogen peroxide, 6 mM; TMB, 1.25 mM; DMSO, 5%; citrate, 25 mM and the final pH is 3.4.
[[Category:Antibodies]]