The IOTest Beta Mark Kit is a multi parametric analysis tool designed for quantitative determination of the TCR Vβ repertoire of human T lymphocytes by flow cytometry. Taking advantage of the fact that Vβ specificities may be grouped into mutually exclusive combinations, the detection of 3 Vβ expressions in the same tube is possible with the use of an innovative staining strategy combining three monoclonal antibodies (mAb) with only two fluorophores. A first mAb is FITC-conjugated, a second one is PE-conjugated and a third one is a carefully balanced mixture of a PE- and a FITC-conjugated form. This kit simplifies the TCR Vβ repertoire analysis by reducing the number of tubes to analyze (8 instead of 24) and consequently the time required for obtaining the results. Moreover, this kit makes it possible to study the repertoire on T cell subsets, using additional T-cell markers, such as CD3, CD4, and CD8, conjugated to a third fluorophore. The kit is composed of 8 vials containing mixtures of conjugated TCR Vβ antibodies corresponding to 24 different specificities (about 70% coverage of normal human TCR Vβ repertoire). The test is intended for use on whole blood samples. The staining protocol includes a one-step procedure with directly conjugated antibody mixes (test volume of 20 μL) and a wash step after staining. A third color T-cell marker (preferentially as PC5-conjugate; not included) is used to gate the specific population. The kit can be used in a multi parametric environment employing at least three-color flow cytometric analysis. The test is compatible with different methods of red blood cell lysis and can be run on Beckman Coulter as well as BD Biosciences cytometers. The IOTest Beta Mark kit includes a control for PMT setting and fluorescence compensation adjustments. Since the reagent in vial A is used as to prepare this control, the kit contains 50 tests of reagent A instead of 25.
Kit antibody composition and associated Vbeta according to Wei, et al. and IMGT nomenclature
The extent of the human germline T-cell receptor Vβ gene segment repertoire. Wei et al. Immunogenetics, 40, 27-36. 1994
|Author: Brice Ezzouaouy, Caroline Scifo | Beckman Coulter Immunotech S.A.S, Marseille France Nicole Weit | Beckman Coulter GmbH, Krefeld, Germany|
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