Hi Larissa, Samples with trace hemolysis (light-red color) may have little or no effect on ELISA results, samples that are heavily hemolyzed (dark-red color) should be avoided. Immunoassays are mostly unaffected by hemolysis unlike other analytes measured by spectral or chemical means. However, hemolysis may be unacceptable for immunoassays of relatively labile analytes like insulin, glucagon, calcitonin, parathyroid hormone, ACTH and gastrin, due to the release of proteolytic enzymes from erythrocytes that degrade these analytes. Samples with any sign of hemolysis are not acceptable for such assays. Because hemolysis may also interfere with some signal generation steps of different types of immunoassays, grossly hemolysed specimens should not be used.
regards
Thank you very much Mr. Ehab El-Nahas. I will evaluate cytokines by Elisa and Immunochemioluminescence... Best regards
In high concentrations of free hemoglobin (as in hemolysed samples) the hemoglobin can bind unspecifically at the solid phase. Hemoglobin has a peroxidase (like) activity so that you can get a false positive signal.
How to get this solved: Validation with s small set of samples by adding different defined concentrations of free hemoglobin (from washed and lyzed RBC's. Digitonin can be used for the lysis of RBS or freezing+thawing, ...
... and in liquid samples there will be some volume added from the red blood cells. This will dilute your sample as well.
Hi, I am facing the same problem as you. But I am using serum samples. My sample is grossly hemolysed. I am looking the level of specific antibody produced as a result of immunization experiments. Will the hemolysis affects my ELISA results?
there is very little in the published literature on this, however please see https://www.ncbi.nlm.nih.gov/pubmed/16312237 Here they show that specific Abs can be detected from whole blood samples kept for 118 days, which are hemolysed.
However, it's best practice to avoid hemolysed serum samples as you can never be sure of the effect of hemolysis on your specific ELISA.
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