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Sample Dilution and Standard Range

Sample Dilution and Standard Range - ELISA Assay Forum

Sample Dilution and Standard Range - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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Old 06-28-2011, 09:12 PM
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Default Sample Dilution and Standard Range



Hey everyone,

I'm doing research that involves infusing an animal with 1 ml of 43mM melatonin solution and then analyzing its serum with a melatonin(MT) ELISA that has a standard range of 12.ng/ml-400ng/ml. The solution will be diluted by the animals blood, which has mean volume of 11.6ml but I'm concerned that the MT concentration form the serum samples will not fall into the standard range of the ELISA. I tried emailing the manufacturers to get an explanation:
"The concentration of the melatonin(MT) in the mouse immediate after infusion is about 3385.8 uM, with MW of MT at 232 dalton. The mass concnetration is 785.5 ug/ml. If you do 2-fold serial dilution in a 96-well plate into PBS or your buffer, the 9th dilution will give you 383 ng/mL, 10th dilution 192 ng/mL, and 11th dilution 96 ng/mL, 12th dilution 48 ng/ml. All 9th to 12th dilution fall into the detection range."

I don't understand how they calculated 3385.8uM from a 43mM MT infusion. I tried doing my own calculations but I'm always off by a few thousand uM. If someone could please dumb down this explanation I would appreciate it greatly!!

Thanks,
Sarah
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Old 06-29-2011, 01:25 AM
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Default Re: Sample Dilution and Standard Range

Hi Sarah,

Let’s speak in micrograms/mL

1 mL of 43 millimoles/litre is 43 micromoles of melatonin

1 mole is 232 grams, so 1 micromole is 232 micrograms, so 43 micromoles is 9976 micrograms, or for sake of argument, a milligram.

Distribution volume for a mouse is about 10 mL (+ or -) so your Cmax should be about 100 micrograms/mL

a 1000 fold dilution should get you right in the middle of your curve at 100 ng/mL which is equivalent to 10 two-fold serial dilutions.

Expect a half life of about 20 minutes for subsequent time points.

The kit supplier has used a distribution volume for the mouse of 12.7 mL.
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Old 06-29-2011, 09:02 PM
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Default Re: Sample Dilution and Standard Range

Thanks Elisadeveloper,

From my understanding I willl hve to go to the 15th dilution, starting with 9976 ug/ml, to fall into the range of the kit (12.ng/ml-400ng/ml). Is this correct?
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Old 06-29-2011, 09:11 PM
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Default Re: Sample Dilution and Standard Range

Quote:
Originally Posted by moust016 View Post
Thanks Elisadeveloper,

From my understanding I willl hve to go to the 15th dilution, starting with 9976 ug/ml, to fall into the range of the kit (12.ng/ml-400ng/ml). Is this correct?
I forgot about the distribution volume of 12.7ml. So instead it would be the 11th dilution using a 2-fold serial dilution method. I think it would put me around 383.5ng/ml.
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Old 06-29-2011, 10:47 PM
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Default Re: Sample Dilution and Standard Range

your mouse has 100 ug/mL in it's blood, so we'll assume the same in the serum.

100 ng/mL (mid curve) requires a 1/1000 fold dilution whether by serial dilutions or otherwise. You might consider 3 dilutions of 10 fold, 10 fold and 10fold to minimize systematic pipetting errors over a vast number of serial dilutions.

You would have to perform 9 serial dilutions to get to the same level.
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Old 08-20-2012, 10:40 AM
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