I have used agapppe diagnostic kit for the estimation of serum LDH. In the procedure, 1ml of reaction mixture (4 ml of R1 + 1ml of R2) mixed with 10 micro litre of serum and incubated for 1 minutes at 37 degree celsius. But i didn't get the results. If any body using the kit for estimating LDH, could you please tell the exact procedure to be done.
According to the kit insert you have to measure the change in absorbance per minute ( OD/min) during 3 minutes with 1 minute preincubation. How exactly are you doing?
Hai Jukka, i added 1ml of reaction mixture (4 ml of R1 + 1ml of R2) to 10 micro litre of serum and incubated for 1 minutes at 37 degree celsius. But i didn't get color reaction even after 10 minutes of incubation.
What kind of control are you using? Have you verified that you actually have some kind of enzyme activity in your samples?
I do not know this kit, but I assume it uses the conventional reaction NAD/NADH 2, so no way to make visible reaction. What is your source of light? If by chance it does not emit sufficiently light in the UV or if the instrument does not select the correct wavelength, you can observe any change in absorbance. I suppose you have tested different sera. It is also possible that by chance you are working with a serum with very high LDH activity and that all the substrate of the reagent has been consumed when you begin to make this measurement
Mr. Jukka Salminen, i have not done this assay before. My serum sample is of control animals not the treated ones. Still any biological samples usually have an optimum level of enzyme activity.
Miss Nicole, The principal of this reaction is based on NAD/NADH2 and 340 is the wavelength. I hope i may not get visible color reaction.
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