In a protocol datasheet, of a particular cytokine ELISA It is suggested to start reagent diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. What is the theory behind this? Should I use heat inactivated serum?
Dear Chandrika,
It is an important question. You know we encounter to interferences generally in immunoassay e.g. ELISA method which make false results. One important interference is related to HAMA or heterophile antibodies or human anti mouse/animal antibody.
It means human serum/plasma contains human anti mouse antibody (or human anti animal) antibody.
You know in ELISA method we use mouse antibody widely (as capture and detecting Ab), so human anti mouse antibody will bind to these antibodies in the ELISA kit and prevent the intended Ag/Ab reaction and make false results.
Good problem solving: Diluting human serum/plasma with buffer containing non-immune normal animal serum (which contains a large amount animal Ab), causes reacting human anti mouse antibody with animal antibody exist in normal animal serum, and this is prevent from reaction of human anti animal antibody with ELISA kit antibodies (capture Ab/Detecting Ab). So we prevent the interference originationg from HAMA or human anti animal antibody. Yes heat activating animal serum is more useful.
Below references can help you too:
http://www.oxfordbiosystems.com/Portals/0/PDF_2/lw-IV-05-englisch.pdf
http://www.surmodics.com/assets/uploads/documents/Assay_Diluent_WhitePaper.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC371198/
http://www.origene.com/assets/documents/Assays/ELISA_Luminex_Development_Guide.pdf
http://www.clinchem.org/content/45/7/942.full
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904417/
http://www.biochemia-medica.com/content/interferences-quantitative-immunochemical-methods
http://cdn.intechopen.com/pdfs-wm/33740.pdf
Best
Mehdi
Dear Chandrika,
It is an important question. You know we encounter to interferences generally in immunoassay e.g. ELISA method which make false results. One important interference is related to HAMA or heterophile antibodies or human anti mouse/animal antibody.
It means human serum/plasma contains human anti mouse antibody (or human anti animal) antibody.
You know in ELISA method we use mouse antibody widely (as capture and detecting Ab), so human anti mouse antibody will bind to these antibodies in the ELISA kit and prevent the intended Ag/Ab reaction and make false results.
Good problem solving: Diluting human serum/plasma with buffer containing non-immune normal animal serum (which contains a large amount animal Ab), causes reacting human anti mouse antibody with animal antibody exist in normal animal serum, and this is prevent from reaction of human anti animal antibody with ELISA kit antibodies (capture Ab/Detecting Ab). So we prevent the interference originationg from HAMA or human anti animal antibody. Yes heat activating animal serum is more useful.
Below references can help you too:
http://www.oxfordbiosystems.com/Portals/0/PDF_2/lw-IV-05-englisch.pdf
http://www.surmodics.com/assets/uploads/documents/Assay_Diluent_WhitePaper.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC371198/
http://www.origene.com/assets/documents/Assays/ELISA_Luminex_Development_Guide.pdf
http://www.clinchem.org/content/45/7/942.full
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904417/
http://www.biochemia-medica.com/content/interferences-quantitative-immunochemical-methods
http://cdn.intechopen.com/pdfs-wm/33740.pdf
Best
Mehdi
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