Bradbury and Plückthun (2015) Reproducibility: Standardize antibodies used in research. Hulspas (2009) Considerations for the control of background fluorescence in clinical flow cytometry. Polymer dye specific block (when using two or more BD Horizon Brilliant™ or Super Bright dyes together) Cross-reactivity can improve the utility of an antibody. Fc block and cyanine dye block (especially for monocytes and macrophages). Excess soluble antigen used to block antibody binding to target cells. Excess unlabeled antibody used to block binding of labeled version of the same antibody. Targeted knock out cell lines (or cells from transgenic animals) where the antigen expression has been specifically eliminated. Primary cells or mock transfected cell line known to not express the antigen of interest. Cell lines transfected to express the antigen of interest (fusion tags such as GFP or HA can give an independent way to confirm expression when no previously validated antibody is available for direct comparison). Primary cells known to express the antigen of interest (sometimes only after stimulation). There are several different controls that can be used to validate the specificity of your antibodies. Controls to Validate Antibody Specificity: Western blotting are raised against linear epitopes that may not be present/exposed in living cells whereas common flow cytometry sample processing steps such as enzymatic digestion and fixation/permeabilization can alter the native epitopes (or the antibodies and fluorophores themselves). As part of an integrated preclinical safety assessment package for novel therapeutic antibodies and antibody-like proteins, tissue cross-reactivity studies. Note also that many antibodies used in e.g. Typical problems in flow cytometry include unspecific binding and specific cross-reactivity with off targets (resulting in false positives) as well as lack of reactivity with the stated target antigen (resulting in false negatives). Unfortunately, majority of commercially available antibodies are poorly validated and it is up to the end user to make sure that the antibodies work as expected. Nearly 50% of the antibodies submitted to the Human Leucocyte Differentiation Antigen Workshops fail to function as intended! Samples remained consistently reactive for anti-ZIKV antibodies.Ĭompared to the single serotype 3 NS1, the mix of equipotent DENV 1-4 NS1 antigens was best at achieving reduced cross-reactivity in both pre-ZIKV and ZIKV-containing sera.Accurate flow cytometry analysis depends on the specificity of the antibodies used. No samplesremained reactive in the DABA and two were reactive in the IgG capture. 
10% of samples in the DABA and 20% in the IgG capture showed a significant reduction of reactivity with blocked conjugates – likely a direct result of reduced false-positives.Ģ0 DENV antibody-containing samples were mixed with equipotent DENV 1-4 NS1 solutions.
All others were successfully quenched.Ī subset panel from 95 of the ZIKV-positive patients was then re-tested using DENV 3 NS1, showing equivalent anti-ZIKV reactivity in the majority of samples. A panel of 32 DENV antibody-containing sera were used in a modified conjugate diluent containing the DENV 3 NS1 antigens and an equipotent solution of DENV 1-4 NS1 antigens:ģ2 DENV antibody-containing samples were mixed with DENV 3 NS1 and one sample remained reactive with the DABA. Our human anti-ZIKV NS1 IgG1 antibody was used as a positive control. A quenching method was also investigated in the DABA and IgG assays, using our DENV NS1 antigens to bind cross-reacting DENV antibodies and ablate false-positive effects.
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