Cross-adsorbed antibodies exhibit minimal cross-reactivity with those species, whether presented by the experimental antibodies or by endogenous proteins in the sample. For example, Alexa Fluor® 488 AffiniPure Donkey Anti-Mouse IgG (H+L) ( min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot) has been cross-adsorbed against bovine, chicken, goat, guinea pig, Syrian hamster, horse, human, rabbit, rat and sheep IgG and serum proteins. Sr Prot)" in the description have been adsorbed and/or tested against IgG and/or serum proteins of the species indicated in the parentheses. Mouse Anti-Rat IgG (H+L) (min X Hu, Bov, Hrs, Ms, Gt, Rb Sr Prot) minimizes background when detecting a rat primary antibody on mouse tissue. Secondary antibodies with inherent minimal cross-reactivity can be useful in limiting background staining on samples that present endogenous Ig, e.g. These antibodies are said to have inherent minimal cross-reactivity to the host species. mouse anti-rat), the diversity of paratopes in a polyclonal antiserum is limited by the small number of sequences that are unique to the target species. If the host animal is closely related to the target species (e.g. The many paratopes that are developed by the host animal are predominantly directed against antigenic epitopes that appear most foreign: the host animal tends not to produce antibodies against “self”.
Polyclonal secondary antibodies consist of diverse molecules, each with a different paratope that binds a specific epitope on the target immunoglobulin. JIR confirms that species cross-reactivity has been minimized by ELISA and IEP testing. Depending on the antibody description, the gels used for cross-adsorption may contain proteins from different species, different mouse IgG subclasses, or different antibody fragments. Cross-adsorbed secondary antibodies have been further purified on similar gels to remove those antibodies that happen to recognize shared epitopes on other proteins. This is common in closely related species such as mouse and rat, but may also occur in less obvious pairings.Ĭross-reactivity may lead to the detection of endogenous immunoglobulins from the sample (background signal) and/or detection of other experimental antibodies in a multiple labeling protocol (off-target signal).Īffinity-purified antibodies are isolated from antiserum by binding to target proteins that have been immobilized on solid-phase gels. The paratope (antigen-binding site) of an antibody that recognizes immunoglobulin from one species may detect a homologous epitope on immunoglobulin from another species. Species cross-reactivity occurs when immunoglobulins from different species share conserved sequences and similar quaternary structure.
#Antibody cross reactivity update
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