Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Patent
1993-08-06
1996-07-02
Ceperley, Mary E.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
436525, 436532, 436533, 436823, 5303911, G01N 33531
Patent
active
055321704
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method for the preparation of functionalised particles for use in assay techniques.
A number of different instrumental techniques have been suggested for the determination of chemical and biochemical analytes in solution. Many of these have involved the immobilisation of a binding partner for the analyte on a surface, eg of glass or metal, and then bringing the solution containing the analyte into contact with the surface. Some change in a property of that surface, eg refractive index, due to binding of the analyte with the immobilised binding partner is then monitored, eg by evanescent wave or surface plasmon resonance techniques. The magnitude of this change gives a qualitative and/or quantitative indication of the presence of analyte in the sample.
Especially where the analyte is a relatively small molecule, eg a hapten, such methods may be unreliable since the change in surface property brought about by binding of the analyte may be relatively minor. Also, these methods may not achieve the sensitivity of conventional detection techniques.
One approach to this problem which is well known is to conjugate analyte molecules or specific binding partners of the analyte (or analogues of either thereof) with a larger particle and to use these conjugates in competition or displacement assays. Broadly speaking the change in surface property occurring on association or disassociation of a conjugate with the surface is greater than that which would occur with non-conjugated species and the sensitivity of the device is thereby increased.
In the preparation of biomolecule conjugates to particles, it is generally necessary to limit the number of biomolecules attached to each particle. This is certainly the case in sensitivity enhancement for sensor techniques which detect changes in refractive index and thickness using enhancer probes, such as particles to which biomolecules are attached, for a displacement or competition assay. If there is more than one biomolecule attached to each probe then multiple attachment points may occur and displacement can be difficult due to the high affinity. This is illustrated in FIG. 1. FIG. 1(a) shows particles P to which DNA molecules D are attached. The particles P are bound to a surface S by means of (i) single and (ii) multiple binding of the DNA molecules D to oligonucleotides N immobilised on the surface S. FIG. 1 (b) shows analogous binding of particles P via biomolecules B attached to the particles P and antibodies A immobilised on the surface S.
Similarly in a competition assay, if more than one biomolecule is attached to an enhancer probe then the sensitivity of the assay is significantly reduced. Adsorption at low concentrations of the biomolecules can be used to limit the number attached to the particle; however, this is difficult to control, is not reproducible and attachment via adsorption may inactivate the biomolecule.
We have now devised a method of preparing functionalised particles for use in assay techniques which overcomes or substantially mitigates the above disadvantages.
According to the invention, there is provided a method of preparing functionalised particles, which comprises containing a cleavable bond so as to immobilise the cross-linking agent, of 5-1000 nm with the immobilised cross-linking agent, and substrate.
The method according to the invention is advantageous inter alia in that reducing preparation times considerably. separations. modified particles will be obtained. similar prepacked columns.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1A shows particles P to which DNA molecules D are attached, wherein the particles P are bound to a surface S by means of (i) single and (ii) multiple binding of the DNA molecules D to oligonucleotides N immobilized on the surface S;
FIG. 1B shows analogous binding of particles P via biomolecules B attached to the particles P and antibodies A immobilized on the surface S;
FIG. 2 is a schematic diagram of the preparation of thiol-modified particles;
FIG. 3 is a schematic diagram of the prepar
REFERENCES:
patent: 4529712 (1985-07-01), Jou et al.
"Facile preparation and some applications of an affinity matrix with a cleavable connector arm containing a disulfide bond," Preparative Biochemistry, 17(2), 121-141 (1987).
Buckle Philip E.
Pollard-Knight Denise V.
Ceperley Mary E.
Fisons plc
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