Editing Indirect ELISA (section)

== Buffer ==

<br/>'''Bicarbonate/carbonate coating buffer '''(100 mM)<br/>Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:<br/>3.03 g Na2CO3<br/>6.0 g NaHCO3<br/>1000 ml distilled water<br/>pH 9.6

'''PBS'''<br/>1.16 g Na2HPO4,<br/>0.1 g KCl<br/>0.1 g K3PO4,<br/>4.0 g NaCl (500 ml distilled water) pH 7.4.

Add NaCl to 0.3 M to&nbsp;&nbsp;avoid unspecific reactions

'''Blocking solution'''<br/>Commonly used blocking agents are 1% BSA , serum, non-fat dry milk, casein, gelatin in PBS.

'''Wash solution'''<br/>Usually PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

'''Antibody dilution buffer'''<br/>Primary and secondary antibody should be diluted in 1x '''blocking solution''' to reduce non specific binding.

'''0.05 M Phosphate-Citrate Buffe'''r

Dissolve one Phosphate-Citrate Buffer Tablet (Product No. P 4809) in 100 ml of deionized water with stirring to yield a 0.05 M phosphate-citrate buffer, pH 5.0.<br/>OR<br/>Add 25.7 ml of 0.2 M dibasic sodium phosphate (Product No. S 0876), 24.3 ml of 0.1 M citric acid (Product No. C 7129), and 50 ml deionized water. Adjust pH to 5.0, if necessary.

'''TMB Substrate Solution'''

Dissolve one 3,3’,5,5’–tetra- methylbenzidine tablet in 1 ml of DMSO and add to 9 ml of 0.05 M Phosphate-Citrate Buffer, pH 5.0. Add 2 μl of fresh 30% hydrogen peroxide (Product No. H 1009) per 10 ml of substrate buffer solution, immediately prior to use.

OR

Dissolve one 3,3’,5,5’–tetramethylbenzidine tablet in 1 ml of DMSO and add to 9 ml of 0.05 M phosphate- citrate buffer, pH 5.0, containing 0.03% sodium perborate (capsules, Product No. P 4922).

'''Stop Solution&nbsp;'''

Reaction may be stopped by the addition of 50 μl of 2 M H2SO4 per 200 μl of reaction mixture, or equal vol of 0.5 N HCl.