![]() ![]() These principles apply to all immunological procedures including ELISA, Western blots, flow cytometry, immunohistochemistry, and immunocytochemistry.Ĭross-reactivity of labeled secondary antibodies with endogenous immunoglobulins on the tissues or cellsĬhoose a labeled secondary antibody cross-adsorbed against the species of the experimental tissue, if possible. A labeled anti-goat secondary antibody will significantly cross-react with bovine IgG since goat, sheep and cow are closely related species. However, most commercially available BSA and dry milk products are contaminated with bovine IgG, which can cause a problem when goat or sheep primary antibodies are used. Two commonly used blocking reagents are bovine serum albumin (BSA) and dry milk. For example, if a primary antibody is made in mouse and normal mouse serum were used for blocking, the mouse IgG would bind to the sticky sites and be recognized by labeled anti-mouse IgG. Never block the tissue or cells with normal serum from the same host species as the primary antibody. The IgG in serum should occupy sticky sites on the tissue or cells to prevent non-specific binding of the labeled IgG antibody. It is most effective to block tissue or cells with normal serum (5% v/v) from the same host species as the labeled secondary or tertiary antibody. Improper blocking of the tissues or cells Not diluting the secondary antibodies far enough.Reactivity of the labeled secondary antibody with immunoglobulins in the diluent.Cross-reactivity of the labeled secondary antibodies with endogenous immunoglobulins on the tissues or cells.Improper blocking of the tissues or cells.Perform a control experiment excluding the primary antibody to isolate the secondary antibody as the cause of background. Background may be caused by the primary antibody.When comparing the immunogen sequence and another protein sequence, an aligment score of 85 % or higher indicates the antibody may cross react the percentage alignment should ideally be much lower than this.Technical Service: What are common causes of background? There are several websites that provide tools for calculating the percentage alignment, which are accessible from the EMBL-EBI website. If you have the immunogen sequence of your antibody, you can check the sequence alignment of the immunogen with other proteins the antibody could react with. Using F(ab) and F(ab') 2 antibody fragments rather than full immunoglobulins can further minimize potential cross-reactivity between antibodies and cell-surface receptors.Ĭhecking potential cross-reactivity for similar proteins.This increases specificity and minimizes cross-reactivity. If a secondary antibody has been pre-adsorbed against potentially cross-reactivie proteins, it will not react with those proteins.a mouse primary might require a donkey anti-mouse secondary.Īvoiding cross-reactivity with pre-adsorption and fragments If chosing a secondary antibody, make sure it's raised against the primary antibody in a different species, e.g.Check the 'overview' and 'target' sections of the datasheet. Make sure it's validated to bind to the target.Checking potential cross-reactivity for similar proteins Find out what you need to know to minimize cross-reactivity.Īvoiding cross-reactivity with pre-adsorption or antibody fragments When selecting an antibody, it's important to ensure it is specific to the target and does not cross react. ![]()
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