Information for LDH assay - protocol for LDH assay (Dec/04/2006 )
I am interested to know about the LDH assay. Please let me know the protocol and other details of this assay.
Thanks n cheers
This is an indirect enzymatic spectroscopic method. LDH catalyses the lactate to pyruvate reaction at pH8.8 - 9.8 with the concomitant production of NADH. It is in fact NADH that is measured (340nm), it's production being proportional to the lactate. Provided the reactants are in excess, the activity of the LDH can be measured in either kinetic or end-point modes (i.e. measure at 15 mins or measure when there is no more change in absorbance).
Some labs use the reverse reaction (at pH 7.4 - 7.8) and measure drop of NADH. There are a few problems with standardisation of the assay, e.g. at what temp. Your "room temp" might be warmer than mine in Scotland! Most labs use either 30 degs or 37degs but that may change if your species isn't a 37degs species.
Alternative to an activity assay, you could measure LDH mass e.g by HPLC, mass spec or one of the immunological methods.
You do get LDH isoenzymes and if you wanted to measure them you would probably use electrophoresis.
Here's a paper that gives the detail: "Recommended methods for the determination of four enymes in blood." J. Clin Lab. Invest. 33: 291-306 1974
Any probs, give us a shout.
Well, thanks for your reply. I don't have acess to this article you forwarded me. Can you send me some more details or protocol for this assay. I have some disulfide compounds which I m interested to check though this assay.