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ANTI-HUMAN IgG1 ANTIBODY

Inactive Publication Date: 2014-02-06
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a way to detect the active form of a therapeutic antibody using a sandwich assay format. The method involves using an antibody that specifically binds to the therapeutic antibody but doesn't bind to the antibody of the experimental animal. A second antibody is used that binds to the antigen, allowing for easy detection. The method can also be used to determine the ratio of active to total therapeutic antibody, which can serve as an indicator of efficacy. Additionally, the method can be used to determine if there is any epitope overlap between two different antibodies.

Problems solved by technology

The detection of a human or humanized antibody becomes more and more difficult the closer the test animal is related to H. sapiens.

Method used

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  • ANTI-HUMAN IgG1 ANTIBODY
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  • ANTI-HUMAN IgG1 ANTIBODY

Examples

Experimental program
Comparison scheme
Effect test

specific embodiments

[0083]Embodiment 1: A monoclonal antibody characterized in that the antibody specifically binds to an antibody of human IgG1 class and does not specifically bind to the immunoglobulin of rhesus-monkey, marmoset monkey, baboon monkey and cynomolgus monkey.[0084]Embodiment 2: The antibody according to Embodiment 1, characterized in that the antibody binds to an antibody of human IgG1 class with a kappa light chain.[0085]Embodiment 3: The antibody according to any one of the preceding Embodiments, characterized in that the antibody does not bind to an antibody of human IgG1 class with a lambda light chain.[0086]Embodiment 4: The antibody according to any one of Embodiments 1 and 2, characterized in that the antibody binds to an antibody of human IgG1 class with a lambda light chain.[0087]Embodiment 5: The antibody according to any one of Embodiments 1, 2 and 4, characterized in that the antibody does not bind to an antibody of human IgG2 class.[0088]Embodiment 6: The antibody according...

example 1

Preparation of the F(ab′)2 Fragment of Human IgG (Immunogen)

[0128]The full length human antibody of the class G (human IgG) in 100 mM sodium citrate buffer, pH 3.7 was incubated with pepsin (1 μg pepsin per mg IgG). The fragmentation was analyzed by analytical gel filtration and stopped after 90 minutes by adjusting the pH value to 6.5 by the addition of potassium phosphate. After dialysis of the mixture against 10 mM sodium citrate buffer with 10 mM sodium chloride, pH 5.5, the solution was applied to an SP-sepharose chromatography column and the isolated fractions eluted in a salt gradient were analyzed individually by analytical gel filtration. The pool containing the antibody F(ab′)2 fragments were applied to an affinity matrix with immobilized polyclonal antibodies against human Fcγ to eliminate trace amounts of Fcγ fragments. The flow through was pooled, concentrated to about 16 mg / ml and finally applied to a gel filtration column (Superdex 200).

example 2

Generation of Monoclonal Anti-Human IgG Antibodies

a) Immunization of Mice

[0129]Female NMRI mice, 8-12 weeks of age, were each primarily immunized intraperitoneally with 100 μg of the antibody F(ab′)2 fragments prepared according to Example 1 mixed with CFA (Complete Freund's Adjuvant). Two further intraperitoneal immunization steps followed after 6 and 10 weeks, each with 100 μg of the antibody F(ab′)2 fragments per mouse mixed with IFA (Incomplete Freund's adjuvant). Subsequently, intravenous boost immunizations were done, each with 50 μg of antibody F(ab′)2 fragments in PBS (phosphate buffered saline) three days before the fusion.

b) Fusion and Cloning

[0130]Spleen cells of the mice immunized according to a) were fused with myeloma cells according to Galfré and Milstein (Galfré, G. and Milstein, C, Methods Enzymol. 73 (1981) 3-46). Approximately 2.1×108 splenocytes were mixed with 4.2×107 myeloma cells (P3x63-Ag8.653, ATCC CRL1580) and centrifuged (10 min. at 300×g and 4° C.). The c...

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Abstract

Herein are reported a monoclonal antibody specifically binding to a human IgG1 antibody and not specifically binding to the immunoglobulin of an experimental animal and the use of the antibody in immunoassays.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of International Application No. PCT / EP2011 / 063906 having an international filing date of Aug. 12, 2011, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C §119 to European Patent Application No. 10173090.1, filed Aug. 17, 2010.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 15, 2013, is named P4659C1SeqList.txt, and is 9,880 bytes in size.FIELD OF THE INVENTION[0003]Herein are reported antibodies specifically binding to antibodies of human IgG1 class and use thereof.BACKGROUND OF THE INVENTION[0004]Since the development of the first monoclonal antibodies by Koehler and Milstein in 1974 a lot of efforts have been dedicated to the development of antibodies which are appropriate for therapy in humans. The first mono...

Claims

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Application Information

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IPC IPC(8): G01N33/68C07K16/42
CPCC07K16/4241G01N33/686C07K16/4283C07K2317/30C07K2317/33C07K2317/54C07K2317/92G01N33/6854C07K16/18C07K16/42C12N5/12G01N33/68C12N5/163C07K2317/14
Inventor ESSIG, ULRICHKLOSTERMANN, STEFANKOWALEWSKY, FRANKSTUBENRAUCH, KAY-GUNNARVOGEL, RUDOLFWESSELS, UWE
Owner F HOFFMANN LA ROCHE & CO AG
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