Abstract: A method for determining the specificity of one or more antibodies, or one or more alternative affinity binders, for a target antigen can include the following steps: (i) analyzing binding of the antibody, or the alternative affinity binder, to a target antigen expressed by an antigen-expressing cell; (ii) analyzing binding of the antibody, or the alternative affinity binder, to a target antigen expressed by an equivalent antigen-expressing cell, in which cell expression of the target antigen has been silenced and/or the gene of the target antigen has been deleted; and (iii) comparing the binding reactions of the antibody, or the alternative affinity binder, in steps (i) and (ii). In various embodiments, the method further features that a reduction or loss of binding in step (ii), compared to step (i), indicates that the antibody, or the alternative affinity binder, has affinity and/or specificity for the target antigen.