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Serum creatinine assay troubleshooting

Assay Creatinine Serum Troubleshooting

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#1 plm8123

plm8123

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Posted 20 January 2013 - 05:04 PM

Hi,

I recently used a serum creatinine assay kit from abcam (link below) and obtained some unexpected results. I was wondering if anyone on this forum could share their experience with measurement of serum creatinine that could possibly shed light on what's going wrong:

http://www.abcam.com...it-ab65340.html

Serum samples were used that were stored at -80 degrees Celsius, previously aliquoted, hence at the time of assay at most two or three freeze thaw cycles would've been performed.

This assay is based on conversion of creatinine to sarcosine, which is then oxidised and reacts with a probe to produce red colour measured with a spectrophotometer set at 570nm. The reaction is performed using a reaction mix containing all supplied enzymes and then again without creatininase in the reaction mix to account for background. The concentration is determined from the difference between the sample reading and its background.

I ended up with measurements of serum creatinine around 10pg/microlitre whereas the normal range is 45 to 110pg/microlitre. I tested serum samples from three subjects, and all of them had around 10pg/uL. The background reading was quite high for all of the samples, so it's definitely necessary. The serum samples had varying amounts of haemolysis, but the background reading should account for this. If anything, haemolysis should overestimate the concentration... not underestimate it.

I did not dilute the serum samples.

The standard curve was linear and covered 0 to 200pg/uL, with absorbances from 0.0052 (water + reaction mix in the well) to 1.778, which suggests I didn't dilute the standards incorrectly either (given that our spectrophotometer measures from 0.000 to 4.000).

If anyone could help, it would be much appreciated. Thanks.





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