Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-11-08
1998-11-10
Wolski, Susan
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
209213, 209214, 210695, 210222, 422101, 435 71, 435 72, 435 722, 435 732, 435 75, 435 792, 435 29, 435803, 436518, 436526, 436806, 436824, C12Q 168
Patent
active
058341978
DESCRIPTION:
BRIEF SUMMARY
This application is the national phase of international application PCT/GB95/01056 filed May 10, 1995 which designated the U.S.
The present invention relates to methods of capturing species from liquids and to assay procedures involving said species.
Whilst the present invention is of broad and general applicability, it has particular relevance to the problem of monitoring organisms in water and will be described with particular reference to that context.
Current methods for assaying the content of organisms in water such as cryptosyporidium and giardia are time consuming and labor intensive.
A major problem in such assay procedures is that the organisms may be present in very low numbers in substantial volumes of water and must first be concentrated into a sample of substantially reduced volume. Conventionally, this is done by passing large volumes of water through a cellulosic filter material which is then broken up and placed in a smaller volume of liquid in which it is agitated over a prolonged period with a view to releasing the captured organisms from the filter material. The proportions of organisms present in the liquid samples which are captured by this way and successfully released from the filter material is relatively poor and the operation is prolonged taking typically about twenty-four hours to perform. The product of this procedure is a sample in which the organisms are still very dilute.
In WO 93/16383, an assay for cryptosyporidium oocysts by an electrorotation assay technique is described, which requires direct visualization of the oocysts under a microscope. To run such an assay on water samples of the kind normally encountered requires further concentration of the organisms beyond the stage reached following the filtration procedure described above.
In unrelated assay procedures, it is known that magnetically attractable particles may be coated with selective reagents such as antibodies or oligonucleotides. Such coated magnetically attracted particles are used in assay procedures by mixing the particles in suspension with a species to be captured so as to form a complex in which the species is captured to the particles. The magnetic particles are then collected by magnetic attraction so as to concentrate and localise the captured species for further operations.
The present invention now provides a method of capturing a species from a liquid comprising attracting magnetically attractable particles to a solid support by magnetic forces, which particles have an affinity for said species, and contacting said particles on said support with said liquid to capture said species on to said particles on said solid support by reduction of said magnetic forces.
Because the particles are held on the solid support during the time in which they are being contacted with the liquid containing the species to be captured, it is possible for the volume of liquid containing the species to be much greater than the volume occupied by the particles during this operation. Large volumes of the liquid may be washed through or over the solid support bearing the magnetically attracted particles, so that the particles may capture said species in sufficient quantity for further operations to be carried out, even if the species is present at great dilutions in the liquid. For instance, the volume of the liquid contacted with the particles may be greater than the volume occupied by the solid support by a factor of at least 10, more preferably from to 100 or more.
The liquid may be passed repeatedly over the solid support, e.g. by continuous recirculation, so as to improve the capture of said species.
The particles may be assayed for the captured species whilst retained on the solid support. It will generally however be more appropriate to release the particles with the captured species. This may be done simply by vigorous washing or even air blasting whilst maintaining the magnetic attraction but is preferably accomplished by reducing the magnetic attraction.
When the particles are released from the solid support, they
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Clinica Chimica Acta,69 (1976) 387-396, Solid-Phase, Magnetic Particle Radioimmunoassay, Lynn Nye et al. (See Appln. p. 5).
Clinical Chemistry, vol. 26, No. 9, 1980, Magnetizable Solid-Phase Fluoroimmunoassay of Phenytoin in Disposable Test Tubes, pp. 1281-1284, R.S. Kamel et al. (See Appln. p. 5).
Journal of Immunological Methods 53 (1982) pp. 109-122 Albumin Magnetic Microspheres: A Novel Carrier for Myelin Basic Protein, Haim Ovadia et al. (See Appln. p. 5).
Biotechnology and Bioengineering, vol. XIX, pp. 101-124 (1977)Nonporous Magnetic Materials As Enzyme Supports:Studies with Immobilized Chymotrypsin, P.A.Munro et al.
Genera Technologies Limited
Wolski Susan
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