Solid phase binding assay

Details Solid phase binding assay Solid phase binding assay

1993-04-01

1998-02-10

Chin, Christopher L.

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

436501, 436524, 436532, 435 792, 435 793, 435 794, 435 795, G01N 33543

Patent

active

057168547

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to an improvement in assay methods comprising the specific binding of a second substance to a first substance already bound to a solid phase surface.
In our WO 90/05303 there are disclosed sensing surfaces capable of selective biomolecular interactions and designed to be used in biosensor systems, particularly systems based upon surface plasmon resonance (SPR). In this type of optical biosensors changes in the refractive index in a layer close to a thin metal film are detected by the consequential changes of the intensity of a totally reflected light beam. For a more detailed description of such a biosensor, see our WO 90/05295 relating to an optical biosensor system, and our WO 90/05305 relating to a sensor unit and its use in biosensor systems.
The above mentioned sensing surfaces comprise a film of a free electron metal, preferably silver or gold, having one of its faces coated with a densely packed monolayer of specific organic molecules. To this monolayer a biocompatible porous matrix, e.g., a hydrogel, is bound, which matrix is employed for immobilizing a suitable ligand for a target biomolecule to be determined by the particular biosensor.
To make the matrix bind a desired ligand it is activated by the introduction of suitable functional groups, the matrix thereby normally obtaining a positive or negative net charge. Provided that the ligand to be bound, typically a protein, is of the opposite charge, the electrostatic interaction between the ligand and the matrix will concentrate the ligand at the surface and thereby provide for efficient binding of the ligand to the surface. Such binding to electrically charged surfaces has been well-known in the art for a long time, such as in enzyme immobilization and affinity chromatography (see, e.g., U.S. Pat. No. 4,829,009, WO 83/02954, and J. Immunol. Methods 1988, vol. 111(2), p. 157-66).
Although such an electrostatic effect is advantageous for the binding of ligands, the possible presence of a residual charge at the solid surface after the ligand binding has, however, hitherto been believed to be a possible source of problems in the subsequent analytical steps because of undesired ionic interactions with the sample.
It has now, in accordance with the present invention, surprisingly been found that the provision of a residual charge at the solid surface remaining after the binding of a ligand thereto may be favourably utilized to facilitate and optimize the binding of an analytical species in the subsequent steps of a particular assay to be performed at the solid phase surface. In the present context the term "analytical species" means any reactive species used in the assay except the initially bound ligand. Thus, for example, in a sandwich type assay, the so-called secondary antibody (in SPR-assay serving as an "enhancement agent") may be efficiently concentrated to the matrix by the created electrostatic interaction resulting in a substantially complete binding to the primary analyte already bound to the ligand, provided that, on one hand, the matrix has an original charge sufficient to leave a residual charge after the immobilization of the ligand thereto, and, on the other hand, that the reaction conditions are selected properly in terms of ionic strength and pH.
Thus, for the purposes of the present invention the reaction conditions involve low ionic strength and an appropriate pH of the reaction medium to ensure a negative or positive charge of the secondary antibody opposite to the charge of the solid phase surface, in contrast to the physiological conditions used in the prior art, i.e., medium ionic strength and about neutral pH. For instance, in the case of a residual negative charge of the matrix, the pH should be selected below the isoelectric point of the antibody to make the latter positively charged, and vice versa. Under these conditions the electrostatic interactions between such opposite charges of the matrix and the secondary antibody will be utilized without too many screening ions being present. It is also

REFERENCES:
patent: 4287300 (1981-09-01), Gibbons et al.
patent: 4829009 (1989-05-01), Graves
patent: 5244630 (1993-09-01), Khalil et al.
patent: 5459078 (1995-10-01), Kline et al.
patent: 5459080 (1995-10-01), Adamczyk et al.
Graves, Howard C.B., Journal of Immunological Methods, 111 (1988) pp. 157-166.

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