Optimizing the ratios of these antibodies is complex and background can be tricky to eliminate. The second method involves pre-complexing the human antibody with anti-human IgG secondary antibody, and then using human IgG to block unbound secondary antibody. Indirect detection of the hapten label requires covalent modification of the therapeutic antibody, which can affect the target binding characteristics. There are two main methods currently used to get around this issue: indirect detection using a molecular tag (hapten)-usually biotin or fluorescein isothiocyanate (FITC)-or precomplexing the human antibody with anti-human IgG secondary antibodies. Although background can be reduced by lowering primary antibody concentration or shortening incubation times, high assay sensitivity is essential to avoid missing real off-target effects. Indirect detection by a secondary antibody directed against anti-human IgG cannot distinguish between the therapeutic antibody and endogenous IgG, resulting in background staining.
Most therapeutic antibodies are either fully human or humanized making standard IHC methods impractical due to high concentrations of endogenous human immunoglobulins. Chromogenic IHC is currently a preferred technology for TCR testing. The US Food and Drug Administration (FDA) recommends that antibody-drug candidates undergo tissue-cross reactivity (TCR) tests against 42 human tissue to confirm on-target binding and assess any off-target binding that could cause adverse effects (1,2). Tissue-Cross Reactivity Tests: Recommendations from the FDA IHC helps scientists confirm that a therapeutic antibody will bind only to an intended target, not other non-target epitopes. In these studies, human or humanized antibodies are often used to treat different cancers and autoimmune conditions. Moving beyond studying basic biology, IHC is an important tool in drug development and disease research, used to detect changes in disease marker expression post-treatment, as well as in therapeutic antibody development. Although less quantitative than immunoassays like Western blotting and ELISA, the appeal of IHC is that it preserves the tissue’s spatial information, allowing researchers to visualize the pattern of expression of a protein in relation to other molecules and structures that surround it. For researchers wishing to know not only "if" but "where" a protein or molecule of interest is expressed within a tissue, immunohistochemistry (IHC) is an invaluable and accessible technique.
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