Youwillneedthefollowingsolutions:

PBS(Phosphate-BufferedSaline,Ca/Mgfree):-

Solute	1x	10x
NaCl	8g	80g
KCl	0.2g	2g
Na2PO4(anhydrous)	1.15g	11.5g
KH2PO4(anhydrous)	0.2g	2g

N.B.:Na₂HPO₄.2H₂Ocanalsobeused->add1.42gfor1xand14.2gfor10xPBS.Makeupto1litrewithddH₂O.

PBSwash:-

100ml1xPBS10ulTween(0.01%,finalconcentration)

Citrate/AcetateBufferpH6.0:-

1.500ml0.1MSodiumAcetate

Sodiumacetate-4.1g.ddH₂O-500ml.

2.100ml0.1MCitricacid

Citricacid-2.1g.ddH₂O-100ml.

Titrate1with2toafinalpHof6.0.Citrate/AcetateBuffer,pH6canbestoredfrozenat-20°C.

TMBSolution-10mg/mlinDMSO(storeat4°C).

ThefollowingdetailsthemethodforusewithTMBasthesubstrate(coverwellswithclingfilmduringincubationstopreventevaporation).

1)Add3ulH₂O₂to20mlofCitrate/Acetatebuffer.Toevery5mlofbufferadd50ulTMBsolution.N.B.:Colourchangeisfromcolourlesstoblue(yellowwhenacidisadded).

2)Coatplatewithantigenstocksolution(1ug/mlinPBS,50ul/well)foratleast24hrsorovertheweekendat4°CorO/Nat37°C.

3)Removeantigenandwash3xwith1xPBS.

4)Blockwith1%BSA(100ul/well)in1xPBSforatleast1hrat37°C.

5)RemoveBSAbyflickingplate.

6)Wash3xwith1xPBScontaining0.01%tween

7)Addanti-serum(dilutedasappropiate,1/100etc.with1xPBS)andincubatefor2hrs@37°C.N.B.:Usepositiveandnegativecontrols.

8)Wash4xwith1xPBScontaining0.01%tween.

9)Addperoxidaseconjugate(50ul/well)diluted1:1000with1%BSA(dilutedinwashingsolutionasin8))andincubatefor1hr@37°C.N.B.:UsePRAM(Peroxidase-conjugatedRabbitAnti-Mouse)forAbraisedinmiceandPSAR(Peroxidase-conjugatedSwineAntiRabbit)forAbraisedinrabbits.

10)Wash4xwith1xPBScontaining0.01%tween.

11)Addsubstrate(100ul/well).

12)Incubatefor1-5minsinthedarkatRT.Checkreactionforcolourchange.Wellsshouldturnblue.

13)Stopreactionbytheadditionof25ul0.1MH₂SO₄/well.

14)ReadOD@450nmforTMB