Suppose the coating antibody (may be against some pathogen affecting chicken, for instance Newcastle disease virus i.e anti-NDV IgG from chicken) used in the sandwich ELISA is raised in chickens, then the primary antibody (again anti-NDV IgG) should be raised from chickens or any other species? The reason for this question obviously would be the secondary antibody viz., anti-chicken IgG can bind to both coating antibody and primary antibody if they are raised in chickens. In such cases, there will be false positive signal. Can anybody explain this?
Such a sandwich ELISA can be performed with antibodies from the same species, but only if the detection antibody is directly conjugated to enzyme or biotin. In any of these cases, you would be able to detect it without recognizing the coating antibody. Otherwise, you can use coating and primary antibodies from different species and a conjugated secondary antibody that does not crossreact with the coating antibody species. Be careful with that, as many anti-IgG antibodies crossreact, but some of them have minimal reactivity with immunoglobulins from other species (you can find that info in the datasheet).
Dear Gururaj, I am not sure if I understood your problem correctly but here is what I think;
anti-NDV IgG from chicken seems to be your capture antibody! So what is that you want to capture by this antibody ----- NDV or IgG! Why do you want to capture primary IgG and how can you capture chicken IgG by a chicken IgG? You need to use preferably Fab anti-chicken-Fcg (raised in cow/sheep/goat/mouse) to capture anti-NDV IgG from chicken and then detect binding with anti-chicken IgG Fcg (conjugated with HRP/PO) which is again raised in cow/sheep/goat/mouse. Else you can use NDV to capture specific IgG against NDV. Let me know if I was closer to your problem or not.
Thanks for the answer Dr. Abhishek Saxena, I want to capture New castle disease virus (i.e. Antigen) using known coating and primary antibody.
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