Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1994-04-12
2001-08-14
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S069100, C435S069700, C435S070300, C435S070210, C435S071100, C435S173300, C435S173300, C436S501000, C436S512000, C436S518000, C436S536000, C530S378000, C530S387300, C530S810000, C530S866000, C530S867000
Reexamination Certificate
active
06274324
ABSTRACT:
This invention relates to reagents having specific binding properties. The invention relates in particular to reagents comprising a specific binding agent linked to a solid surface or linked to a tracer.
BACKGROUND OF THE INVENTION
Natural antibodies, either polyclonal or monoclonal, have been used widely as specific binding agents. When immobilised on solid phases, such as pegs, dip-sticks, wells and moisture-permeable membranes such as filters and strips, or when linked to various tracers (otherwise known as labels or markers), they can be used in assays.
Antibodies are large complex multi-chain proteinaceous structures. Although it has been appreciated for some while that substantial portions of these structures seem unrelated to the specific binding properties, the minimum portion necessary to provide adequate specific binding has been a matter of debate. It has already been shown that so-called Fv fragments, ie. an antibody fragment essentially comprising only a single heavy-chain variable region and its corresponding light chain variable region, can exhibit specific binding activity. Very recently it has also been shown by Ward et al (
Nature
, 1989, Vol. 341, p.544-546) that a single variable domain from an antibody can exhibit significant specific binding activity. The production of single variable domain antibodies (Dabs), as described by Ward et al, is also described in detail in EP 0368684 A1 (Medical Research Council) published on May 16, 1990.
To be of practical use in immunoassays, specific binding activity alone is not sufficient. The specific binding agent must also be capable of being linked to other material, for example a label such as an enzyme or a particle, or to a solid phase. This linkage must be achievable without any significant adverse effect on the specific binding activity. Such adverse effects can easily arise through chemical or conformational changes in the specific binding region, or simply by physical (stearic) hindrance of access to the specific binding region. In the case of conventional specific binding reagents, ie. whole antibody molecules or large portions of such molecules such as Fab fragments, the specific binding region or regions comprise only a minor proportion of the total molecule. The comparatively vast residual bulk of the molecule, which is apparently not directly involved in the specific binding activity, provides abundant scope for the existence of locations which can participate in chemical or physical linkage with other materials such as labels and solid phases. These regions can be relatively remote from the essential specific binding regions, and the resulting linkages need not interfere with the specific binding activity.
However, in the case of a specific binding entity essentially comprising only one or more variable domains unassociated with any substantial portion of the originating antibody or antibodies, eg. a Fv fragment or a single variable domain (Dab), the relative proportion of the molecule which participates in the essential specific binding activity is very much higher. Indeed, it would be expected that any attempt to link the small specific binding entity to another material will entail a very high risk that the essential specific binding activity will be adversely affected.
SUMMARY OF THE INVENTION
An objective of the present invention is to facilitate the linking of such small specific binding entities to other useful materials with less risk of damage to their essential specific binding properties.
DESCRIPTION
The invention provides a specific binding reagent, comprising:
i) one or more variable domain proteins (V
H
and/or V
L
unassociated with any substantial portion of originating antibody or antibodies;
ii) a linking group, which does not contribute to the specific binding properties of the reagent, comprising at least 5 amino acid residues, and which is hydrophobic and/or includes at least one lysine residue, the coupling properties of the linking group thereby being enhanced; and
iii) a solid surface or a tracer, coupled via the linking group to the variable domain protein(s).
DESCRIPTION OF PREFERRED EMBODIMENTS
For the purposes of this specification, the “reagent” of the invention may be a water-soluble or water-dispersible material, or may be a solid device such as a bead, peg, dip-stick, or well or other container, having a surface on which the variable domain protein(s) are immobilised by means of the linking group.
Preferably the linking group comprises not more than 20 amino acid residues. Preferably the linking group is hydrophobic and includes at least one lysine residue. The presence of a lysine residue provides a very convenient site for covalent attachment to proteinaceous tracers, such as enzymes.
To provide the linking group with sufficient hydrophobicity to achieve the purposes of the invention, the polypeptide chain comprising the linking group should is contain a sufficient number (which may be as few as two, if the residues are adjacent) of amino acid residues selected from the group consisting of valine, leucine, iso-leucine, phenylalanine, tyrosine, tryptophan, proline and alanine. We have found that even if the majority of the amino acid residues in the polypeptide are other, relatively polar (and hence relatively hydrophylic), amino acid residues, the presence of merely a low proportion of residues from the above group can confer effective hydrophobicity on the polypeptide. The hydrophobic region or regions can be adjacent to regions of high charge density, ie. the peptide chain is of mixed character, without the essential hydrophobicity of the linking group as a whole being lost.
An important embodiment of the invention is a single variable domain protein (Dab) attached to a proteinaceous ‘tail’ which acts as the linking group as defined above, the ‘tail’ being coupled to a solid surface or to a tracer without significant loss of specific binding activity.
A particularly preferred linking group, especially for use in coupling to a solid plastics surface, comprises the “Myc” amino acid sequence(SEQ ID NO:1):
GLU-GLN-LYS-LEU-ILE-SER-GLU-GLU-ASP-LEU-ASN
The linking group will normally be attached at or near one end of a variable domain protein. Normally, the point of attachment will be the amino terminus of the peptide linking group. This is the left hand end of the sequences A and B as seen in
FIG. 2
of the accompanying drawings. Preferably, the variable domain protein(s) and the linking group have been produced together by expression in a genetically modified organism. The is polypeptide linking group may, for example, be synthesised (cloned) together with a variable domain protein and comprise a proteinaceous tail on one end of the domain sequence. The linking group will comprise at least about 5 amino acid residues, to confer sufficient length to “distance” the variable domain from the surface or tracer to which it is linked.
Actual coupling can be achieved, for example, by means of conventional bifunctional chemical cross-linking agents. Preferably, such a chemical coupling site is sufficiently remote, within the linking group, from the variable domain sequence itself that any molecule which becomes coupled to the linking group is held at a distance from the variable domain sequence.
Where the tracer is a protein, such as an enzyme, it is preferably covalently coupled to the linking group via the e-amino group of a lysine residue in the linking group. Examples of suitable enzymes are horse raddish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase and urease.
In one embodiment of the invention, in which the variable domain is attached via the linking group to a solid surface, the surface is a surface of a solid structure formed from plastics material, such as polystyrene, polyvinylchloride (PVC) or polyethylene teraphthalate glycol (PETG). Examples of surfaces to which it would be extremely useful to immobilise variable domains are so-called “latex” particles (which are minute solid particles of plastics material such as polystyrene, generally used i
Davis Paul James
De Winter Ronald Frank Jacobus
Verhoeyen Martine Elisa
Celsa Bennett
Pillsbury & Winthrop LLP
Unilever Patent Holdings B.V.
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