Comparison of three HLDA-workshopped anti-CD3 clones

HCDM (Human cell differentiation molecules) is an organisation which runs workshops on human leukocyte differentiation antigens, characterizes CD molecules and supplements their list.

Antibodies that have been submitted to HLDA workshops are well characterized and hence reliable for flow cytometry for research and mainly for diagnostic customers.

We decided to compare three workshopped anti-CD3 monoclonal antibodies TB3, UCHT1, and MEM-57 regarding their concentration-dependent signal course.

First, we conjugated them with Alexa Fluor® 647 to the same F/P ratio, and the conjugates were purified by size-exclusion chromatography. All three antibodies were then analysed in duplicates using flow cytometry on human peripheral blood, and 5-point titration curves were plotted. MFI, stain index, as well as percentage of CD3+ lymfocytes among all lymphocytes were evaluated.

Second, we analysed Pacific Orange™ conjugates of TB3 and UCHT1. The results demonstrate superiority of TB3 clone in this application.



Fig.1: Titration curves of CD3+ lymfocytes MFI of human peripheral blood stained with UCHT1, TB3, and MEM-57 anti-CD3 clones conjugated with Alexa Fluor® 647.



Fig.2: Titration curves of CD3+ lymfocytes stain index of human peripheral blood stained with UCHT1, TB3, and MEM-57 anti-CD3 clones conjugated with Alexa Fluor® 647. Stain index was calculated as the difference between MFI of positive and negative population divided by 2 SD of the negative population.



Fig.3: Titration curves of CD3+ lymfocytes percentage among total lymphocytes in human peripheral blood stained with UCHT1, TB3, and MEM-57 anti-CD3 clones conjugated with Alexa Fluor® 647.



Fig.4: Flow cytometry surface staining patterns and histograms of human peripheral blood stained with Pacific Orange™-conjugated anti-CD3 antibodies UCHT1 and TB3; working concentration 4 µg/ml.