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Filter spot-ELISA for the enumeration of interferon-alpha antibody-secreting cells
O Prümmer1, B Cederblad, G Alm
1Department of Medicine III, University of Ulm, F.R.G.
A filter spot-ELISA was developed for the enumeration of interferon-alpha (IFN-alpha) antibody secretion by murine and human cells. Various human IFN-alpha subtypes were coated onto nitrocellulose membranes by means of broad specificity bovine antibodies. Membranes were blocked with milk proteins, and antibodies released by individual cells during a 3 h culture period were visualized as distinct spots using peroxidase-conjugated, species-specific anti-immunoglobulin antibodies and diaminobenzidine/hydrogen peroxide substrate solution. The spot-ELISA is rapid, easy to perform, and highly sensitive. Possible applications include frequency estimates of IFN-alpha antibody-secreting cells in blood, tissues and hybridoma cultures as well as the evaluation of specificity and immunoglobulin class of the respective antibodies.
A filter spot-ELISA was developed for the enumeration of interferon-alpha (IFN-alpha) antibody secretion by murine and human cells. Various human IFN-alpha subtypes were coated onto nitrocellulose membranes by means of broad specificity bovine antibodies. Membranes were blocked with milk proteins, and antibodies released by individual cells during a 3 h culture period were visualized as distinct spots using peroxidase-conjugated, species-specific anti-immunoglobulin antibodies and diaminobenzidine/hydrogen peroxide substrate solution. The spot-ELISA is rapid, easy to perform, and highly sensitive. Possible applications include frequency estimates of IFN-alpha antibody-secreting cells in blood, tissues and hybridoma cultures as well as the evaluation of specificity and immunoglobulin class of the respective antibodies.