Molecular Diagnostics, Inc.

Miles Inc.

Vincent T Marchesi

William J Knowles

Wallace Haigh

Nanibhushan Dattagupta

Donald M Crothers

Monoclonal antibodies specific for the glycosylated lysine residue at position 525 in glycoalbumin and a method for producing such antibodies. The monoclonal antibodies are useful as reagents in immunoassays for the specific determination of glycoalbumin in human blood samples which is indicative of the severity of the diabetic condition. The monoclonal antibodies are secreted by hybridomas obtained by fusing a myeloma cell with a lymphocyte that has been taken from an animal, usually a mouse, immunized with a peptide immunogen and which produces antibody to the lysine 525 residue in glycoalbumin. The synthetic peptide immunogen comprises a peptide residue which includes an .epsilon.-amino glucosylated lysine and an adjacent amino acid sequence in which at least one of the amino acid units is in a position corresponding to the peptide sequence of human albumin adjacent to lysine 525, the glycosylated peptide residue being linked to an immunogenic carrier.

Monoclonal antibodies specific for human glycoalbumin

A protein is covalently coupled to a 3'terminal end of a nucleic acid which carries several labels. In an assay the protein will specifically recognize some component of a test system; in an immunoassay the protein can be Protein A which will recognize the FC portion of IgG which is bound to an unknown antigen if present in the test sample.

Nucleic acid-protein conjugate used in immunoassay

Monoclonal antibodies specific for the glucosylated N-terminal peptide residue in Hb A.sub.1c, a method for producing such antibodies, hybridoma cell lines secreting such antibodies and a method for their production, and immunoassay methods and reagent systems using such antibodies for the determination of Hb A.sub.1c in human blood samples. The monoclonal antibodies are secreted by hybridomas obtained from the fusion of a myeloma cell and a lymphocyte which has been taken from an animal, preferably a mouse, immunized with a synthetic peptide immunogen and which produces antibody specific for the glucosylated N-terminal peptide residue in Hb A.sub.1c. The synthetic peptide immunogen comprises an N-terminal plucosylated peptide residue having at least 2 amino acid units corresponding to the N-terminus of the beta-subunit of human hemoglobin and an immunogenic carrier to which the glucosylated peptide residue is linked. The immunoassay involves treatment of the blood sample to expose the glucosylated N-terminal peptide epitope and detection thereof by binding of the specific monoclonal antibody or a fragment thereof.

Antibodies for use in determining hemoglobin A.sub.1c

Binding of a particular protein by an antibody reagent involving denaturation of the protein and use of an antibody reagent specific for binding a linear peptide epitope therein. Denaturation by chemical or physical means effectively exposes or enhances the exposure of the linear peptide epitope for binding by the antibody reagent which is preferably raised against a synthetic peptide immunogen. The technique is particularly useful in performing immunoassays for protein analytes, such as a glycosylated protein, in aqueous test samples.

Binding of antibody reagents to denatured protein analytes

Monoclonal antibodies specific for the glucosylated N-terminal peptide residue in Hb A.sub.1c, a method for producing such antibodies, hybridoma cell lines secreting such antibodies and a method for their production, and immunoassay methods and reagent systems using such antibodies for the determination of Hb A.sub.1c in human blood samples. The monoclonal antibodies are secreted by hybridomas obtained from the fusion of a myeloma cell and a lymphocyte which has been taken from an animal, preferably a mouse, immunized with a synthetic peptide immunogen and which produces antibody specific for the glucosylated N-terminal peptide residue in Hb A.sub.1c. The synthetic peptide immunogen comprises an N-terminal glucosylated peptide residue having at least 2 amino acid units corresponding to the N-terminus of the beta-subunit of human hemoglobin and an immunogenic carrier to which the glucosylated peptide residue is linked. The immunoassay involves treatment of the blood sample to expose the glucosylated N-terminal peptide epitope and detection thereof by binding of the specific monoclonal antibody or a fragment thereof.

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Guilford, CT, United States of America

Vincent T Marchesi