SCREENING FOR CHARACTERIZATION OF MURINE MONOCLONAL ANTIBODIES AGAINST HUMAN LIMPHOCYTES T ANTIGENS USING JURKAT CELLS

GOLIM M. A.(1), BITTENCOURT R. A. C.(1), MURADOR P.(1), ROSSI-FERREIRA R.(1), MACHADO P. E. A.(1), DEFFUNE E.(1)

(1)Botucatu Blood Center, UNESP, Botucatu, SP, Brazil;

The hybridoma technology, developed in the mid-1970s by Köhler and Milstein (1975), made it possible to produce virtually limitless quantities of highly specific murine monoclonal antibodies (mAbs) that recognize distinct epitopes of cellular antigens. There is currently available a large number of such reagents which detect different molecules on both normal and neoplasic cells from various lineages. The process of immunization in Balb/C was done with human T-cell leukaemia for production of mAbs allows the isolation of reagents with a unique, chosen specifity. Jurkat cell is a cell line derived from human T-cell leukaemia derived from vigorously proliferating leukemic T-cells, is a widely used model of human T lymphocytes and used to determine the screening to characterizes mAbs produced in our laboratory. The aim of this study was make a screening to characterize the specificity mAbs against antigens of T cells using Dot Blot technique. Whole lysate Jurkat cells had been used like antigen for electrophoresis in SDS-PAGE and Dot Blot techniques. In electrophoresis load up to 10µL of lysate per 1.0mm of well width for gels 0.45mm thickness, gradient 8-25% and rainbow molecular weight standards. In Dot Blot the nitrocellulose membrane had been incubated with whole lysate Jurkat cells overnight for coating. After incubate the membrane in primary antibody (mAbs produced) diluted in Blotto at room temperature. The membrane was incubated for with horseradish peroxidase (HRP) conjugated secondary antibody, diluted to 1:500 in Blotto and the reaction had been revealed. From 20 mAbs tested, 12 presented positive reactions in Dot Blot, showing that this mAbs have specificity against Jurkat antigens cells. Techniques like Western Blotting will be use to identify the mAbs specificity to a determinate protein through molecular weight and flow citometry tests will comparing the fluorescence intensity media expressed in our mAbs versus commercial mAbs.

KEY WORDS: screening for mAbs, jurkat cells, dot blot

CORRESPONDENCE TO:

Marjorie de Assis Golim, Hemocentro - Distrito Rubião Júnior, s/nº - campus da UNESP - Botucatu - SP CEP: 18.618-000. Email: marjorie_unesp@yahoo.com.br