I'm doing an ELISA to determine the amount of corticosterone in rat serum and it looks like my research assistant made some pipetting errors and allowed a little plasma to be collected in a few of the serum samples. The samples will be diluted greatly during the ELISA, but I'm wondering if the presence of a little plasma will affect my readings.
I'm not sure how you mix serum and plasma by pipetting, since the procedures for obtaining them are different. Serum is collected by allowing the blood to clot, then spinning it down. Plasma is collected in a tube containing heparin or EDTA to prevent clotting, then spun down.
If you meant that some of the red blood cell fraction got pipetted into the tube, that can be solved by spinning again, and avoiding the red chunk or transferring to a new tube. That should not affect your measurement. If the samples were hemolysed during collection (uniform pink or red color), then you'll have to read the protocol for the kit to see if it is sensitive to hemolysis.
A quickie online search says that the ELISA kits from Crystal Chem and DRG both say that either plasma or serum can be used. I didn't see any information about hemolysis listed online, though hemolysis insensitivity is usually advertized, so if it's not there, I suspect the kit probably is sensitive.
I'm not sure how you mix serum and plasma by pipetting, since the procedures for obtaining them are different. Serum is collected by allowing the blood to clot, then spinning it down. Plasma is collected in a tube containing heparin or EDTA to prevent clotting, then spun down.
If you meant that some of the red blood cell fraction got pipetted into the tube, that can be solved by spinning again, and avoiding the red chunk or transferring to a new tube. That should not affect your measurement. If the samples were hemolysed during collection (uniform pink or red color), then you'll have to read the protocol for the kit to see if it is sensitive to hemolysis.
A quickie online search says that the ELISA kits from Crystal Chem and DRG both say that either plasma or serum can be used. I didn't see any information about hemolysis listed online, though hemolysis insensitivity is usually advertized, so if it's not there, I suspect the kit probably is sensitive.
Dear Elizabeth,
In general plasma is collected from the blood for quantitative serum protein's measurements, to avoid their loss in fibrinolysis with hemoglobin in serum clotting. It shouldn't be much difference if ever for hormones concentration in plasma and serum, for corticosterone too. But the alteration in peroxidase-staining ELISA reading results from plasma samples can be high because of red hem color which normally doesn't appear in blood serum. In case you are using luminol-staining ELISA the red color from plasma does not interfere much into your reader's emission wave length, it is simple more weak, exactly like Dr. Unger just said about existence of insensitive to plasma hemolysis kits.
- Badges
- Science method
- Similar topics
- Biological Science
- Immunology
- Immunology Techniques
- Immunoassay