Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1992-11-10
1995-07-04
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 2, 435 724, 435239, 4352402, 435243, 435261, 435975, 436512, 436518, 436526, 5303911, G01N 33543, G01N 33553, G01N 33569, C07K 1700
Patent
active
054299276
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method of cleaving antigen/anti-antigen and hapten/anti-hapten linkages;
In biochemistry and related fields it is frequently desirable to link two chemical or biochemical 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 by attaching them to substances assisting in their isolation and to isolate the cells 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 substances to be linked and which bind when brought into contact. Alternatively one of the binding partners may form a part of the entity requiring linkage, for example a molecule on a cell surface. A number of binding partner systems are known, for example antigen-antibody, enzyme-substrate, ligand-receptor interactions on cells and biotin-avidin binding. Antigen-antibody binding partners are however most frequently used in this regard, although the use of a hapten/anti-hapten binding pair has also recently been proposed.
Affinity binding systems are generally reversible, but it has been found, however, that particularly with antigen-antibody binding partners such linkages are difficult to reverse without destructive effects. Thus, whilst a linkage having a weak interaction strength (eg Ka=10.sup.-4 -10.sup.-6 M.sup.-1) can be broken under mild conditions for example by competition with an analogue of the "ligand" binding partner, where the linkage is stronger (eg Ka>10.sup.-8 M.sup.-1), competitions kinetics do not suffice and more drastic conditions are necessary eg pH modification, salting out, in order to modify the conformation of the binding partner(s) and thereby reduce the interaction strength. For the cleavage method to be of use, the conformational changes induced during such a cleavage step must be reversible, and the binding partners should recover their functions upon return to the original conditions. Unfortunately, in the case of antigen/antibody binding, which must be of the order of at least 10.sup.-8 M.sup.-1 to be effective in allowing specific and rapid separation, whilst the antibody molecule can usually recover its native activity following such a cleavage treatment the antigen frequently is irreversibly altered and its native properties are lost. This is particularly a problem in the separation of cells or bacteria where it is important to maintain viability. Moreover, it is often desirable to be able to liberate the entities being linked in an unmodified native form, i.e. without any foreign substances remaining attached. This is particularly true in the case of cell isolation, where any foreign substances remaining attached to the surface of the liberated cells tend to interfere in cell reproduction and viability.
Cells are commonly linked, or bound, by employing antibodies which bind to antigens or haptens on the cell surface. As mentioned above antigen/antibody linkages are particularly difficult to reverse, and methods which cleave the antigen/antibody in such a manner that a part of the antibody remains attached are not particularly suited for use in cell isolation and separation procedures. Thus there exists a need for an improved method of cleaving antigen/anti-antigen or hapten/anti-hapten linkages with minimal destructive effect and in a manner which leaves the antigen or hapten and anti-antigen or anti-hapten binding partners intact.
We have now found that the linkages in antigen/anti-antigen or hapten/anti-hapten linkage systems may readily be broken by reacting the linkage system with an antibody directed against the anti-antigen or anti-hapten binding partner, or with a fragment thereof which retains binding activity. This has the advantage that the linkage is broken under mild conditions avoiding destruction of protein or other sensitive species present, and since the bonding between the antigen or hapten and its anti-antigen or anti-hapten bindi
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Afseth John
Caignault Laurent
Funderud Steiner
Mortada Mohamad
Dynal AS
Grun James L.
Saunders David
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