Radioimmunoassay for erythropoietin
AssigneeResearch Corporation
InventorsJames W. Fisher, Arvind B. Rege
Life of this patent
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Abstract
A kit and method for the radioimmunoassay (RIA) for erythropoietin. Also, a method for purifying erythropoietin and purified labelled erythropoietin-conjugate for use as a tracer in the RIA.
Claims53
In a radioimmunoassay of erythropoietin comprising
- (a) reacting binder containing a fixed number of antibody binding sites with a mixture of labelled and unlabelled erythropoietin;
- (b) separating bound erythropoietin from free erythropoietin;
- and
- (c) determining the percent labelled erythropoietin bound;
- the improvement comprising the use of labelled erythropoietin prepared by the conjugation labelling technique wherein the labelled erythropoietin has been purified by immunosorption with anti-erythropoietin antiserum that has been preadsorbed with human proteins that are low in erythropoietin content.
An erythropoietin-conjugate having the structure. ##STR1## wherein ESF is erythropoietin, lys is lysine and .sup.125 I is iodine-125, being substantially free of urinary proteins, having a specific activity of between 40 and 55 microcuri/microgram and an immunoreactivity of between 75 and 90 percent.
An antigen-antibody complex comprising
- (1) purified erythropoietin that has been conjugated with .sup.125 Iodinated p-hydroxyphenylpropionic acid N-hydroxysuccinimidyl ester and
- (2) antiserum to erythropoietin that has been adsorbed with human proteins that are low in erythropoietin content.
A process for purifying a erythropoietin-conjugate having the formula ##STR2## wherein ESF is erythropoietin, lys is lysine and .sup.125 I is iodine -125, which comprises adsorption and dissorption of said conjugate with erythropoietin antiserum selected from the group consisting of whole erythropoietin anti-serum and the immunoglobulin fraction of erythropoietin antiserum, said antiserum having been absorbed with human proteins that are low in erythropoietin content.
An antigen-binder complex comprising
- (a) binder-labelled erythropoietin and
- (b) binder-unlabelled erythropoietin.
- wherein the binder-labelled erythropoietin has been purified by immunosorption with antiserum that has been preadsorbed with human proteins that are low in erythropoietin content.
A kit for the radioimmunoassay of erythropoietin comprising a container having within the container a multiplicity of smaller containers and contents said contents comprising
- (a) lyophillized assay buffer concentrate;
- (b) lyophillized rabbit anti-erythropoietin antiserum;
- (c) goat anti-rabbit gamma-globulin;
- (d) erythropoietin standards;
- and
- (e) erythropoietin that has been conjugated with a moniodinated succimidyl ester of 3-(4-hydroxyphenyl)-propionic acid
- wherein the erythropoietin conjugate has been purified by immunosorption with antiserum that has been preadsorbed with human proteins that are low in erythropoietin content and with the proviso that each item is of such quantity that when all the items are brought together at a proper pH, dilution and temperature in the presence of an erythropoietin sample of human blood serum or plasma, antibodies in the antiserum bind to both labelled erythropoietin and erythropoietin in the sample.
Dependencies are inferred from the claim text.