Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1985-09-23
1989-04-18
Marantz, Sidney
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 7, 436501, 436518, 436531, 436807, G01N 33543, G01N 33569
Patent
active
048227323
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method and a means for concentrating and detecting biological material.
Previously known methods for assaying biological and biochemical materials have been unsatisfactory as it has often been difficult to concentrate said material for assay. Different techniques, such as spin-drying and ultrafiltration, to obtain an increased concentration of the biological and biochemical material to be analysed have been used. However, it appeared that several molecules could not be concentrated in such a way and examples thereof are antigens, nucleic acids, hormones, enzymes, ligands, etc. A serious drawback with said techniques is that also contaminating molecules are concentrated and interfere with the detection of the desired material.
The present invention relates to a method for separately concentrating biological and biochemical materials having affinity properties. This is achieved by letting, in a flow, a fluid sample of the material to be detected and which material has affinity to another material, such as antigen-antibody, DNA-DNA, DNA-RNA, RNA-RNA, lectin-receptor, receptor-ligand as well as other bio-specific pairs, e.g. phages and viruses, pass a solid surface, at which surface the other material to which the material to be detected has affinity is attached, so that in relation to the volume of the fluid a many times larger volume passes the surface. During the passage of the flow, the material to be detected adheres to the other material to which it has affinity and forms a complex, which can be read off by different markers such as enzymes, radio activity, laser, etc.
The detection process according to this invention can be automatized and the sensitivity increased at least ten times as compared to previously known methods such as "enzyme-linked immunosorbent assay" (ELISA) with microplates or by radioimmunoanalysis (RIA) techniques, immunofluorescence, etc. (Voller, A., Bartlett, A., and Bidwell, D. E., "Enzyme immunoassays with special reference to ELISA techniques", J. Clin. Pathol. 31, 507-520 (1978); Overby, L. R., and Mushahwar, J. K., "Radioimmune assays", p. 39-70, in M. W. Rytel (Ed.), Rapid diagnosis in infectious disease, CRC Press, Boca, Fla. (1979); Dahle, A. B., and Laake, M., "Diversity dynamics of marine bacteria studies by immunofluorescent staining on membrane filter", Appl. Environ. Microbiol. 43, 169-176 (1982).
Depending on the flow ratio and the flow time, a considerable lower limit for the detectable level is obtained. The concentration of bacteria and the amounts of the bound substances are other variables. The extent of bound substance is increased with increased specificity.
The amount of flow must be tested each time and depends inter alia on the size of the molecules to be detected.
The solid surface can be any solid surface which is immobilized, but is preferably hydrophobic and consists of polymeric substances such as glass, plastic material, metal, silicon, etc. The binding to said surface is obtained (1) by passive adsorption to the hydrophobic surface, or (2) by immobilizing the specific molecule, e.g. antibody, enzyme, antigen, to a solid phase by adsorption or covalent binding.
The fluid sample can consist of water, water-based systems, e.g. buffer, body fluids, in cyclon media impacted air samples, in suitable buffer systems re-suspended solid samples containing the substance to be detected.
In bacteria assay it has been shown to be advantageous if the sample contains at least 10.sup.3 bacteria per milliliter suspended therein, but also as small amounts as 10.sup.2 bacteria per milliliter could be detected.
The concentration of the desired substance can be decreased if the volume of the sample is increased and yet give detectable results.
A non-limiting example of a means used according to the present invention is shown in the enclosed schematical view of the concentration process by means of immunological technique (FIG. 1).
A sample (1) containing for example antigen is pumped by a pump (2) into a duct (3) to the inner surfaces of which a
REFERENCES:
patent: 3950134 (1976-04-01), Miles
patent: 4153675 (1979-05-01), Kleinerman
patent: 4693985 (1987-09-01), Degen
"Molecular Cell Biology" by James Darnell et al., pp. 643-646, Scientific American Books, W. H. Freeman and Company, New York, 1986.
Australian Patent Abstract, AV-A-90139/82, "Acetylcholine Receptors or Antibodies or Anti-Antibodies Bound to Carrier".
Gochman, N. et al., Anal. Chem., vol. 49, No. 13, 1183A-1187A (1977).
Chemical Abstracts, 91:189319d (1979).
Sandstrom Gunnar
Wolf-Watz Hans
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