Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-09-18
1998-06-09
Ceperley, Mary E.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 2, 435 723, 435 725, 436519, 436526, 5303911, G01N 33553, C07K 1628
Patent
active
057632030
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the immobilisation and separation of cells and other particles, and in particular to an immobilisation system based on a hydroxyboryl/cis-diol linkage.
In biochemistry and related fields it is frequently desirable to link together, and then subsequently to dissociate, two chemical/biochemical or biological entities for example in isolation or purification or in the immobilisation of substances on solid supports. In particular it is often required to isolate cells or subcellular particles by attaching them to substances assisting in their isolation and to isolate the cells etc. subsequently in viable form.
Such linkage is often accomplished using affinity binding, that is by means of a pair of binding partners which are, for example, separately attached to the entities to be linked and which bind when brought into contact. Such binding partners may be exogenously added to the entities requiring linkage, or may form part of an entity requiring linkage, e.g. a molecule on a cell surface. A number of binding partner systems are known for example antigen-antibody, enzyme-substrate, receptor-ligand, but generally speaking selective linkage or capture is most commonly achieved using an antibody as a target specific binding partner, recognising an antigen on the surface of the target.
Many methods are known for attaching binding partners such as antibodies to supports to provide affinity capture matrices. The supports may be provided with a range of functional groups which may be activated to give a covalent bond between the support and the antibody by reaction of the activated group with amino or SH groups in the antibody. Examples of the most common methods are: 1. Coupling of antibodies to supports containing --CH.sub.2 --OH groups by activating with sulphonyl chlorides which gives sulphonyl esters on the supports which in turn react with amino groups or --SH groups on the antibody to give covalently coupled antibody with --CH.sub.2 --NH-- and --CH.sub.2 --S-- bonding respectively. 2. Coupling of antibodies to supports containing COOH groups by activation of the carboxylic groups with carbodiimide and N-hydroxysulphosuccinimide whereby amide bonds are formed between the support and antibody. 3. Coupling of antibodies to supports containing amino groups which have been activated with glutaraldehyde and thereby react to form covalent bonds with the amino groups of the antibody. 4. Coupling of antibodies to supports containing epoxy groups takes place without further activation as the epoxy groups react directly with amino groups and --SH groups on the antibody.
A problem frequently observed with such attachment methods, and consequently with immobilisation or separation systems based upon them, is that the efficiency of binding attainable of the binding partner to the support is often low, leading in turn to poor binding efficiencies of the target substance; in many cases it is difficult either to achieve a sufficient "density" of binding of the target-specific binding partner to the support, or to attach the binding partner to the support in the correct orientation to bind the target effectively. Commonly used covalent attachment methods are usually indiscriminate and it is not unusual to observe as low as 20% immobilisation. This is largely thought to be due to binding of the binding partner on the support in incorrect orientation, and is a significant problem, limiting the utility of such separation systems.
Depending on the target substance, and the application, it may or may not be desirable to liberate the target, for example following separation from a mixture. In some cases it may be necessary to remove the support, for example in the isolation of pure cell fractions for clinical use or for functional studies, whereas in other cases there may be no need to do so, e.g. in the negative selection of unwanted substances. A further problem which may therefore be observed with affinity-based binding systems in the cases where it is desired to release the target substance, is the
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Berge Arvid
Kilaas Lars
Stenstad Per
Ugelstad John
Ceperley Mary E.
Sinvent AS
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