A method is described for covalent attachment of dinitrofluorobenzene (DNFB) to sheep red blood cells (RBC). The solubility and ionization characteristics of DNFB are considered in this procedure and relatively large amounts of hapten are reacted with RBC. Unreacted DNFB is partially precipitated and soluble DNFB is carefully removed from DNP-RBC at the end of reaction. Haptenated erythrocytes are extremely sensitive to hemolytic antibody in the direct agarplaque assay as well as in passive hemolysis. Previously reported DNFB techniques were carried out in parallel experiments and results of such comparisons highly recommend the present method for use in detection of direct anti-DNP plaque-forming cells.

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