Left image: Serial sections of human kidney (FFPE) showing strong, specific staining using human anti-cytokeratin primary antibody detected with HOH-3000 (brown). Right image: Negative control showing absence of staining. Both have Hematoxylin nuclear counterstain
The need for determining specific binding of human or humanised antibodies on human tissues has been a troublesome problem for researchers, and increasingly important as validation of target binding is crucial for humanised antibodies and antibody-drug conjugates to achieve therapeutic status.
Traditional methods of immunohistochemistry often give rise to unwanted non-specific staining as the anti-human antibody can not determine between the test antibody and any endogenous Ig that might be present. Bioconjugation of the antibody to a reporter molecule, such as FITC, can be a way to circumvent this, however it is important to demonstrate that the bioconjugation itself has not affected the binding properties and behaviour of the antibody.
Reproducible bioconjugation is possible with reagents such as the SoluLink range, however this process is time intensive when there may be a range of different primary antibodies that require testing. Faster methods of bioconjugation can be much harder to reproduce. Bioconjugation levels need to be controlled, as side effects can be inactivation of the antibody, or causing proteins to fall out of solution.
Vector Laboratories has designed the H.O.H (Human on Human) Immunodetection Kit to enable detection of unconjugated primary antibodies of human origin with excellent signal to noise ratios across the spectrum of isotypes and antibody configurations, including bivalent antibodies without Fc regions. Combining an antibody pre-complex approach with a novel quenching solution has offered outstanding tissue staining results and a favourably abbreviated workflow.
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