Solid-Phase Binding Assay for Erythropoietin Antibodies (41366)

The affinity of phytohemagglutinin-E (PHA-E) and wheat germ agglutinin (WGA) for erythropoietin was exploited to develop a solid-phase assay for erythropoietin antibodies. Purified PHA-E or WGA was covalently coupled to polystyrene tubes with the water-soluble carbodiimide 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Coupling was highly reproducible; approximately 8 μg of lectin was bound covalently per tube. For the assay, the lectin-derivatized tubes were loaded with erythropoietin, washed, and incubated with the immune serum to be tested. The presence of bound antibody was detected by adding¹²⁵I-labeled staphylococcal protein A (SPA) and measuring residual¹²⁵I-SPA after washing. In the absence of antigen, antibody was not bound in the lectin-derivatized tubes. Furthermore, in the presence of erythropoietin, nonimmune serum from a variety of species failed to bind to the tubes. The particular immune serum used in this study could be detected with as little as 5 μg of bound antigen. Since an impure preparation of human urine erythropoietin was used both as the antigen to produce the antierythropoietin immune serum and as the antigen for the assay, the specificity of the assay for erythropoietin antibodies could not be definitively established. Nevertheless, this solid-phase assay permits rapid screening (3.0 hr) of antisera for reactivity with human urine proteins and should be useful for identifying antibody-producing clones formed by somatic cell hybridomas when a partially purified urine erythropoietin preparation is used as the antigen.