Beta galactosidase release as an alternative to chromium release in cytotoxic T-cell assays.
In the present report, we describe a beta galactosidase release (BGR) assay to evaluate cytotoxic T lymphocyte (CTL) activity against specific targets. Transient expression of beta galactosidase (beta gal) was obtained by infection with recombinant beta gal vaccinia virus. Incubation of target cells with effector cells resulted in the release of beta gal depending on the infection time and the effector/target cell ratio. BGR was evaluated using the chemiluminescent substrate, AMPGD (3-¿4-Methoxyspiro[1,2-dioxetane-3, 2'-tricyclo(3.3.1.1(3,7))decan]-yl¿phenyl-b-D-galactopyra nos ide), a phenylgalactose-substituted 1,2-dioxetane compound. The use of a digenic vector carrying two genes coding for the beta gal gene and the antigen, respectively, permits expression of the two proteins in the same cell. Coinfection of target cells with two different vectors, carrying beta gal and antigen genes, respectively, was demonstrated to be as efficient as digenic vector when using high multiplicity of infection (MOI). The BGR assay was compared to the standard 4 h 51chromium (51Cr) release assay both in mouse and human models and showed comparable sensitivity. The BGR assay, therefore, provides a simple, specific and responsive method for measuring cell-mediated cytotoxic activity.
Bachy
M
M
Pasteur Mérieux Connaught, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France. mbachy@fr.pmc-vacc.com
Bonnin-Rivalland
A
A
Tilliet
V
V
Trannoy
E
E
D000218:Adamantane / Q000031:analogs & derivatives; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D002460:Cell Line; D002857:Chromium / Q000378:metabolism*; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods* / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D005260:Female; D005456:Fluorescent Dyes; D005697:Galactosides; D005822:Genetic Vectors; D006801:Humans; D008163:Luminescent Measurements; D051379:Mice; D008807:Mice, Inbred BALB C; D009698:Nucleoproteins / Q000235:genetics / Q000276:immunology; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D000956:Antigens, Viral; D005456:Fluorescent Dyes; D005697:Galactosides; D009698:Nucleoproteins; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D002857:Chromium; C073981:3-(4-methoxyspiro(1,2-dioxetane-3,2'-tricyclo(3.3.1.1(3,7))decan)-4-yl)phenylgalactopyranoside; D001616:beta-Galactosidase; D000218:Adamantane