Protein immunoassaying and purification

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Protein immunoassay or protein purification using an antibody raised to an antigen comprising a polypeptide corresponding to a fragment (but not all) of the protein being assayed; the antibody is capable of binding separately both to the protein as a whole and to the polypeptide; that antibody is used to purify or (with a known concentration of the labeled polypeptide serving as a tracer) to assay the protein.

The invention described herein was made in the course of work under a grant or award from the Department of Health and Human Services.

The invention relates to immunoassaying for, and purification of, proteins--e.g., erythropoietin, a hormone involved in regulation of red blood cell development.

Protein immunoassays are useful, e.g., to diagnose human disease states. Abnormal levels of erythropoietin, for example, may signify many forms of anemia, certain heart and lung diseases, tumors, renal failure, and other diseases. Purified proteins and antibodies thereto are used to treat various disease states. For example, purified erythropoietin may be used to treat anemia associated with renal failure.

In protein immunoassays, a sample having an unknown concentration of the protein is mixed with a known concentration of the protein which has been labeled to form a tracer compound. The tracer and sample protein compete to bind to an antibody which has been raised to the protein. The extent to which the labeled protein binds to the antibody can be measured (either by measuring the bound or the unbound tracer) and will be inversely affected by the protein concentration in the sample, according to known relationships which may be used to derive that concentration. Thus, in the conventional immunoassay, the protein being assayed serves both as the antigen used to raise the antibody and as the labeled tracer used to determine the sample protein concentration.

Li U.S. Pat. No. 4,096,237 discloses such an immunoassay for a human protein hormone known as β-endorphin, using radioactively labeled β-endorphin as a tracer and antibodies raised to β-endorphin. Li also discloses that the above tracer and antibodies may be used to assay a related protein, such as camel β-endorphin or a specific peptide fragment thereof which "exhibited a parallel inhibition curve, but had 40% immunoreactivity as compared to the full sequence of camel β-endorphin" (2:18-20).

In one aspect, the invention features, generally, in a protein immunoassay, an antibody raised to an antigen which comprises a polypeptide corresponding to a fragment (but not all) of the protein being assayed. The antibody is capable of binding separately both to the protein as a whole and to the polypeptide; that antibody is used to assay the protein, and a known concentration of the labeled polypeptide serves as a tracer.

In another aspect, the invention features, generally, the use of the above-described antibody to purify the protein by introducing the antibody into a mixture containing the protein, separating the bound protein/antibody from the mixture, and then separating the protein from the antibody.

In preferred embodiments, the polypeptide comprises an epitope of the protein; most preferably, the protein is erythropoietin, and the polypeptide corresponds to at least a fragment of the twenty-six amino-acid sequence at the N terminus of erythropoietin.

The invention enables a readily available, reliable assay for proteins, particularly those which are available, if at all, in limited supply and purity. The use of a pure polypeptide, rather than relatively impure supplies of the entire protein, yields antibodies of greater specificity that provide more consistent assay results. Often, the polypeptide is more easily handled, stored, and synthesized and cheaper than the full protein. The invention provides similar benefits in methods of immunological purification of proteins.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.