A background-free assay for cells forming antibody to glucose oxidase coupled with an enzyme-linked immunosorbent assay (ELISA) to quantitate antibody secretion.
We describe an immunocytochemical assay for cells forming antibody to glucose oxidase (GO). The method is specific in that only cells containing intracytoplasmic antibody capable of binding the immunogen (GO binding cells; GOBC) are stained. The method is sensitive because there is no GO activity in mammalian tissues. This lack of background readily permits detection of one GOBC among 10(6) nucleated lymphohemopoietic cells. The technique is reliable because purified chemicals are used. Although it is not possible to determine the Ig class of antibody formed by an individual cell, as can be done with the hemolytic plaque assay, the amount and class of secreted antibody to GO can be quantitated by an indirect enzyme-linked immunosorbent assay (ELISA), which is also described. GO is immunogenic and stimulates the formation of large numbers of GOBC in the popliteal lymph nodes after injection with adjuvant into the footpads of mice, but 1-mg doses injected IV or IP are lethal because of its enzymatic activity, which causes hypoglycemia and methemoglobinemia.
Hard
R C
RC
Department of Pathology, Medical College of Virginia/VCU, Richmond 23298.
Miller
W G
WG
Romagnoli
G
G
D000818:Animals; D000921:Antibody-Producing Cells / Q000166:cytology*; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005949:Glucose Oxidase / Q000276:immunology* / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice
D005949:Glucose Oxidase