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Bifunctional molecules

Inactive Publication Date: 2006-02-02
ATWELL JOHN +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] In one aspect of the present invention, the bifunctional molecule is a chimeric antibody conjugate comprising a first region which binds a specific antigen and a second region comprising at least one constant domain sequence derived from a class specific immunoglobulin. This conjugate, which may be used directly as a positive control reagent, avoids the inconvenience of manipulating full length or naturally occurring Fc fragments. Furthermore, the conjugate may be readily produced by recombinant DNA technology.

Problems solved by technology

It is becoming increasingly difficult to source sufficient quantities of immune human sera or plasma, particularly as diagnostic tests for rarer diseases become available.
Collection of blood for IgM controls from patients in early stages of infection when clinical symptoms are generally most severe poses significant ethical problems, particularly if the disease primarily affects juveniles.
Other drawbacks include the requirement for consistent collections from remote locations, the need to standardise each batch and to check for contamination with infectious agents such as HIV, hepatitis B and hepatitis C. There are also problems in obtaining control sera for specific endemic diseases in communities where the donation of blood or blood products is socially unacceptable.

Method used

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Examples

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Effect test

example 1

Production of a C-Domain (IgM) Extended scFv

[0100] The gene sequences of the four constant domains (C domains) of human IgM heavy chain were separately amplified from cDNA prepared from mRNA isolated from human peripheral blood lymphocytes using polymerase chain reaction techniques. The design of the oligonucleotide primers used in the amplifications was based upon the 5′ and 3′ base sequence of each of the four IgM heavy chain exons, obtained through GENBANK accession X14940 (Dorai and Gillies, 1989).

[0101] In the primers, specific restriction enzyme recognition sequences were added (NotI at the 5′ end and SacII at the 3′ end) to each exon sequence to facilitate the introduction of the C domain sequence at a specific site in a previously constructed plasmid expression vector. The expression cassette of this E. coli plasmid vector (pGC; Coia et al., 1996) contained VH and VL sequences from the mouse monoclonal antibody cell line 1C3, (Rylatt et al., 1990, WO91 / 04492) with binding...

example 2

Construction of Extended scFv (13C11 Antidengue) Linked to a Human IgM C Domain

[0105] The reagent was produced from a DNA construct in which the coding region for a mouse scFv directed against dengue virus was genetically linked to that of the third constant domain of human IgM heavy chain (CH3μ), cDNA was prepared from mRNA isolated from the mouse monoclonal antibody cell line 13C11, which specificity for Dengue virus surface antigens (Queensland University of Technology and PanBio Pty Ltd.). Immunoglobulin VH and VL domain DNA sequences were amplified from the cDNA using polymerase chain reaction and oligonucleotide primer sets according to Zhou et al. (1994). These were linked in the scFv format VH-linker-VL, where the linker was a 45 bp nucleic acid sequence coding for the protein sequence GGGGSGGGGSGGGGSGGGGS. The resultant fragment was digested with restriction endonucleases Nco I and Not I and purified by agarose gel electrophoresis. The expression vector as described in Exa...

example 3

Construction of Extended scFv (13C11 Anti Dengue) Linked to a Human IgG C-Domain

[0109] The gene sequences of human IgG constant domains 2 and 3 were separately amplified from cDNA from mRNA isolated from human peripheral blood lymphocytes using polymerase chain reaction techniques. The design of the oligonucleotide primers used in the amplifications were based upon the 5′ and 3′ sequences for each of the heavy chain exons obtained through Genbank accession no E06998.

[0110] Sequences coding for NotI and SacII restriction sites were added to the 5′ and 3′ end respectively of the CH2γ and CH3γ sequences to enable the insertion into pGC 13C11-CH3μ from which the CH3μ sequence had been removed as a NotI-SacIh fragment.

[0111] Expression in E. coli and purification of product was performed as described in Example 2. The presence of product in the periplasmic fraction was confirmed by analysis of samples by polyacrylamide gel electrophoresis and Western blotting, probing the FLAG® tag us...

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Abstract

A chimeric antibody conjugate comprising an antigen binding region of a non-human antibody and an immunoglobulin constant region which comprises at least one CH domain or epitope thereof, with the proviso that the constant region is not a naturally occurring FC fragment. A bifunctional molecule for use in labelling an antibody derived from a first species, the bifunctional molecule comprising a binding-region which binds to the antibody of the first species or to one or more groups provided thereon, and a constant region derived from an antibody of a second species, the constant region comprising at least one CH domain or an epitope thereof. The present invention relates to bifunctional molecules and complexes which are useful as positive control reagents in antibody based diagnostic tests. The present invention also relates to polynucleotides encoding these bifunctional molecules, and to diagnostic assays involving the use of these molecules.

Description

FIELD OF THE INVENTION [0001] The present invention relates to bifunctional molecules and complexes which are useful as a positive control reagents in antibody based diagnostic tests. The present invention also relates to polynucleotides encoding these bifunctional molecules, and to diagnostic assays involving the use of these molecules. BACKGROUND OF THE INVENTION [0002] Infection of humans by many micro-organisms leads to the initiation of a humoral immune response that can be used in the diagnosis of the disease. In the early acute phase of the infection, specific IgM class antibodies are the first to appear in serum 1-4 weeks after the onset of symptoms and last for up to three months. IgG class antibodies appear later and remain elevated throughout the patient's life. Detection of an IgM response is indicative of a recent or current infection, while the presence of an elevated IgG response is a marker for past exposure to the causative agent. Specific IgM or IgG responses to a ...

Claims

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Application Information

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IPC IPC(8): G01N33/554G01N33/569C07K14/31C07K16/12C12N15/09C07K14/36C07K16/08C07K16/10C07K16/18C07K19/00
CPCC07K14/31C07K14/36C07K16/10C07K16/1081C07K2317/52C07K2317/24C07K2317/622C07K2319/00C07K16/18
Inventor ATWELL, JOHNDEVINE, PETERCOIA, GREGORYKORTT, ALEXANDERPERRY, GILLIANBUNDESEN, PETER
Owner ATWELL JOHN
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