A novel bioassay for B-cell activating factor (BAFF) based on expression of a BAFF-receptor ectodomain-tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2 endodomain fusion receptor in human rhabdomyosarcoma cells.
B-cell activating factor (BAFF) is a type II transmembrane glycoprotein belonging to the tumour necrosis factor ligand superfamily. Active soluble forms of BAFF are generated either by cleavage of the extracellular domain or by recombinant DNA technology. The current bioassay for measuring the activity of soluble BAFF involves stimulation of the proliferation of mouse splenic B-cells in the presence of goat anti-mouse IgMmicro chain which is rather cumbersome and lengthy and yields variable results. We have therefore developed an alternative functional assay which relies on the ability of BAFF to induce an apoptotic response in human rhabdomyosarcoma cells. For this, we constructed a chimeric receptor containing the ectodomain of the MuBAFF-R--the major cell receptor for BAFF--and the endodomain of the HuTRAIL-R2--one of the two functional receptors for TRAIL--which is known to contain a death domain and trigger apoptosis. When the chimeric receptor was expressed in the TRAIL-sensitive human rhabdomyosarcoma cell line KD4 clone 21, recombinant BAFF of either human or mouse sequence stimulated apoptosis, similar to TRAIL, in a dose-dependent manner. The transfected cell population, called FL17, expressing the MuBAFF-R/ HuTRAIL-R2 thus provided the basis of a novel functional bioassay for BAFF that is simple and relatively fast to perform. The construction of the chimeric receptor, development of the transfected cells expressing this receptor and the development of sensitive and reproducible bioassays for BAFF and anti-BAFF neutralising antibodies are described.
McClements
Michelle
M
Cytokine and Growth Factor Section, Biotherapeutics Group, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Herts, UK.
Williams
Stella
S
Ball
Christine
C
Bristow
Adrian
A
Wadhwa
Meenu
M
Meager
Anthony
A
D000818:Animals; D000906:Antibodies; D017209:Apoptosis; D053264:B-Cell Activating Factor / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D053265:B-Cell Activation Factor Receptor / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001681:Biological Assay; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004306:Dose-Response Relationship, Immunologic; D006801:Humans; D051379:Mice; D017434:Protein Structure, Tertiary; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012208:Rhabdomyosarcoma / Q000276:immunology* / Q000473:pathology; D013154:Spleen / Q000276:immunology; D014162:Transfection
D000906:Antibodies; D053264:B-Cell Activating Factor; D053265:B-Cell Activation Factor Receptor; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C505639:TNFRSF10B protein, human; C505719:TNFSF13B protein, human; C486662:Tnfrsf13c protein, mouse; C505720:Tnfsf13b protein, mouse