WO1987007726A1 - Detection de particules organiques, notamment de virus, avec technique d'enrichissement - Google Patents

Detection de particules organiques, notamment de virus, avec technique d'enrichissement Download PDF

Info

Publication number
WO1987007726A1
WO1987007726A1 PCT/SE1986/000507 SE8600507W WO8707726A1 WO 1987007726 A1 WO1987007726 A1 WO 1987007726A1 SE 8600507 W SE8600507 W SE 8600507W WO 8707726 A1 WO8707726 A1 WO 8707726A1
Authority
WO
WIPO (PCT)
Prior art keywords
particles
passage
virus
filter
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/SE1986/000507
Other languages
English (en)
Inventor
Jan Peter Andersson
Rolf Nybom
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of WO1987007726A1 publication Critical patent/WO1987007726A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • the invention relates to a method of diagnosing
  • organic particles such as virus, bacteria, fungus, and pollen, by cpncentration technique, wherein liquid or gas to be examined for determining the presence of said particles therein, if any, is passed through a passage or labyrinth system and accumulated particles, if any,
  • a conventional method for diagnosing virus is serological diagnostics. When virus attacks the
  • antibodies against the virus are generated in the blood or other body liquid of the patient.
  • the method is based on the procedure that the existence of antibodies generated in the body liquid after a virus attack is determined and such antibodies are identified
  • the serological diagnostics is an indirect test, because the generated antibodies are determined and identified. There is also a direct test which involves
  • tissue isolation of virus is still more cumbersome and time-consuming and may require culturing of tissue for three to five days but usually six to eight weeks in order to produce the result of the examination.
  • the purpose of the present invention is to provide a method for rapid diagnosing virus or other microbiological agent which is then directly identified and also quantified.
  • the invention is based on conventional concentration technique which has not, however, previously been applied to the diagnosing of virus.
  • this technique has been applied as far as the determination of the presence of bacteria is concerned as will be seen from U.S. patent specification 4,124,449.
  • the concentration is effected on a filter disc which functions in the manner characteristic for every filter; the porosity thereof is such that the particles, in this case the bacteria sought for, cannot pass through the filter disc without being collected on the surface thereof.
  • the bacteria collected on the filter disc then are stained with a proper dye, or incubation with fluorescent antibodies is applied.
  • the filter is very dense, i.e. that the apertures of the filter are very small, because virus is considerably smaller than e.g. bacteria.
  • Filters are concerned having apertures of the order of 50 nm, but there are viruses which pass also through a filter having so small apertures, e.g. hepatitis, the size of which is 20 to 40 nm.
  • a filter of this kind will be rapidly clogged, which means that only microscopical amounts of liquid can be allowed to pass through the filter before the clogging is a reality.
  • a filter which is circular having a diameter of 8 mm and having apertures of the order of 50 nm only 20 to 30 ul of a liquid having a protein concentration of 0.5 g/ml can be passed therethrough before the filter is clogged.
  • virus is present in serum in a concentration of 10 3 to 107 particles/ml, which means that very few virus particles (0-3x10 /30 >ul serum diluted to a protein concentration of 0.5 mg/ml) are available at the filter surface. They are widely scattered and moreover will be covered by a protein layer; it follows that they are not available for detection by using methods known at present.
  • the invention referred to herein is based on the knowledge that in order to apply the concentration technique to accumulated virus it is necessary to use a passage or labyrinth system having such a great permeability, e.g. a filter having large apertures, that also virus particles can pass therethrough, and that it is nevertheless necessary to have the virus particles arrested on the system.
  • a passage or labyrinth system having such a great permeability, e.g. a filter having large apertures, that also virus particles can pass therethrough, and that it is nevertheless necessary to have the virus particles arrested on the system.
  • the method of the invention has obtained the characteristics appearing from claim 1.
  • FIG. 1 is a diagrammatic picture illustrating the application of antibodies to latex spheres
  • FIG. 2 is a microphoto illustrating how the spheres form aggregates when binding existing virus particles towards which the antibodies are specifically directed
  • FIG. 3 is a further microphoto which illustrates as an enlargement the formation of aggregates
  • FIG. 4 is a microphoto which illustrates isolated latex spheres which have not bound virus
  • FIG. 5 is a diagrammatic picture which illustrates the application of radioactively marked antibodies on the latex spheres
  • FIG. 6 is a diagrammatic picture illustrating the application of antibodies on a filter surface.
  • Latex spheres 10 (Batch 17155) having the size of 4.0 yum, from Polyscience, USA, are carboxylated by an inoculation procedure induced by gamma radiation, which is described in the European patent application 8585008-7, carboxyl groups being bound on the surface of the latex sphere as is illustrated in FIG. 1.
  • Rabbit anti-mouse antibodies 11 then are bound covalently by carbodiimide binding to the carboxyl groups on the surface of the sphere, mouse-monoclonic antibodies 12 of specific kind directed towards the virus sought for, then being added.
  • the antibodies 12 are bound to the
  • the antibodies 12 in the constant region thereof (Fc), while the variable, regions of the antibodies 12 are free to bind to the relevant antigen thereof.
  • the antibodies 12 can consist of monoclonic mouse antibodies directed towards the cover
  • the antibody-linked spheres are incubated in a suitable number in the liquid to be examined (10 latex spheres/ml examination liquid).
  • This liquid can comprise e.g. serum, spinal fluid, saliva, or urine.
  • the liquid is, however, centrifugated in a table centrifuge at about 1500 rpm for ten minutes so as to settle cells or cell debris.
  • the supernatant is aspirated therefrom and is incubated with the latex spheres mentioned above for one hour at 37 C, the
  • PBS phosphate buffered saline solution
  • the examination liquid as a whole is filtered e.g. by using a sample holder with a filter of the embodiment shown and described in the Swedish patent application 8404821-4.
  • the filter preferably comprises a hydrophilic polycarbonate filter
  • the size of the filter apertures is chosen according to the detection system used in the final determination of virus. With such a filter a large volume of liquid or
  • 35 gas can be filtered without clogging of the filter.
  • a suitable filter is available under the registered trademark NUCLEOPORE.
  • the latex spheres which have bound virus in the example CMV, will aggregate, i.e. they will form lumps on the surface of the filter by complex formation of several latex spheres and bound virus particles (FIGS. 2 and 3) while latex spheres to which no virus is bound, will lay isolated on the filter surface (FIG. 4). Virus that has not been bound then will pass through the filter, because the virus particles are considerably smaller than the filter apertures. If the filter apertures are larger than the latex spheres also the latex spheres will pass through the filter if they have not bound virus and formed complexes.
  • Detection of the aggregates of virus and latex spheres on the filter can take place in different manners.
  • One manner comprises accumulation of the sample on the filter surface in the sample holder mentioned above according to the Swedish patent application 8404821-4 and coating of the sample with an ionized layer of gold or platinum having a thickness of 0.5 to 5 nm, by evaporation in vacuum in a so-called sputter, the filter surface then being analyzed in a scanning electron microscope. Then, a filter is used having an aperture size of 0.8 yum mounted in an electrically conducting holder, which is a prerequisite for the operation of this detection system.
  • the picture that will be seen can have the appearance illustrated in FIGS. 2 to 4. According to FIGS.
  • the latex spheres are coated with specific antibodies from body liquid, which have bound to the surface thereof virus particles and formed complexes of multiple aggregates of virus and latex spheres.
  • the binding between the latex spheres is particularly clearly seen in FIG. 3.
  • the latex spheres coated with specific antibodies on the contrary are in lack of virus.
  • the surface thereof is completely smooth and the latex spheres lie isolated on the filter surface.
  • latex spheres which have bound virus and together with virus form aggregates which cannot pass through the filter can be determined by marking of the latex spheres with peroxidase, the filter when substrate has been supplied, obtaining a yellow-green colour due to the fact that the accumulated latex spheres on the filter surface will obtain a yellow colour.
  • the colour reaction can be measured spectrophotometrically or visually by systems similar to those used for ELIZA (enzyme-linked immunosorbent assay) .
  • this reaction method requires a diameter of the filter apertures which is larger than the diameter of the latex spheres such that latex spheres pass through the filter if they have not formed aggregates by binding to the specific virus thereof.
  • a filter is used having an aperture size of the order of 10 Aim.
  • Detection of a more advanced type can also be applied by coating the latex spheres which are coated with virus and have accumulated on the filter surface, with a second specific antibody radioactively marked, which is made by supplying said second antibodies to the filter where the latex spheres have collected. If there is virus on the latex spheres, the specific second antibody radioactively marked will bind to the virus particles and then cannot be washed away from the filter surface. However, if there is no virus on the latex spheres, said second radioactive antibody will be washed away from the filter. This is shown in FIG.
  • virus designated 13 is bound to the radioactively marked antibodies 14 which can comprise I ⁇ 25 * A ⁇ t ⁇ r washing three times, the remaining radioactivity on the filter surface is measured by so-called RIA (radioimmunoassay) in a ga mameter.
  • RIA radioimmunoassay
  • a further alternative detection method comprises marking of the second antibody 14 directed towards specific virus, with peroxidase, a yellow-green colour reaction being obtained after the addition of substrate if this antibody binds and will not be washed away, and can be measured in the same manner as mentioned above.
  • Example 2 Instead of binding rabbit anti-mouse antibodies to latex spheres in the manner described in Example 1 the binding of these antibodies is effected directly to a polycarbonate filter disc which shall be treated in the same manner according to the European patent application 8585008-7, the filter surface being carboxylated by an inoculation method induced by gamma radiation, which makes possible to link the Fc-part of the antibody by means of a covalent binding to the filter surface.
  • FIG. 6, where 15 designates a filter holder and 16 designates a filter disc. After washing the filter surface four times, the examination liquid is filtrated through the filter. Then, the specific mouse-monoclonic antibody directed towards the cover of the virus, is added.
  • the aperture size of the filter surface is chosen such that the aperture size is about ten times larger than the diameter of the virus particles. Detection can take place in the same manner as described above in Example 1 (in a scanning electron microscope, by radioimmunoassay or by ELIZA) . For the filtration there is used a filter holder of the type described in the Swedish patent application 8404821-4.
  • the filter can be arranged with different levels for the accumulation of latex spheres and virus, respectively, of different sizes or different kinds, respectively., on the surface of the different levels such that it is possible to determine by a single filtration the presence of different viruses.
  • the filter can also comprise a plate having a number of mutually spaced depressions the bottoms of which are provided with a filter so as to receive therein different samples.
  • the filter can be formed by the latex spheres proper which are supported by a perforated bottom.
  • the filter can be replaced by another passage or labyrinth system for concentration of virus and latex spheres, respectively.
  • the detection can take place by emitting light therethrough.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Dans un procédé servant à diagnostiquer des particules organiques, notamment des virus, des bactéries, des champignons et des pollens, par une technique de concentration, le liquide ou le gaz à examiner pour déterminer la présence éventuelle desdites particules dans du liquide organique traverse un système de passages ou de labyrinthe et les éventuelles particules accumulées sont ensuite détectées. Un système de passages ou de labyrinthe présentant une perméablilité permettant le passage desdites particules est utilisé pour la concentration. La surface du système de passages ou de labyrinthe et/ou la surface des sphères ou autres corps plus grands que les particules recherchées est préparée de façon à permettre la liaison desdites particules à ladite surface ou auxdites surfaces respectivement, lesdites particules s'accumulant, lors du passage du liquide du gaz à travers le système de passages ou de labyrinthe, à la surface dudit système, liée directement ou indirectement aux sphères ou autres corps. L'accumulation de particules spécifiques, telles que des virus, ne se produit que si les particules se sont liées à ladite surface ou auxdites surfaces respectivement. Si aucune formation d'agrégats ne se produit, les particules de virus et les sphères de latex traversent le système de passages ou de labyrinthe et ne s'accumulent donc pas.
PCT/SE1986/000507 1986-06-10 1986-11-06 Detection de particules organiques, notamment de virus, avec technique d'enrichissement Ceased WO1987007726A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE8602591A SE8602591L (sv) 1986-06-10 1986-06-10 Sett for diagnosticering av organiska partiklar genom filtrationsteknik
SE8602591-3 1986-06-10

Publications (1)

Publication Number Publication Date
WO1987007726A1 true WO1987007726A1 (fr) 1987-12-17

Family

ID=20364772

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1986/000507 Ceased WO1987007726A1 (fr) 1986-06-10 1986-11-06 Detection de particules organiques, notamment de virus, avec technique d'enrichissement

Country Status (3)

Country Link
JP (1) JPH02501327A (fr)
SE (1) SE8602591L (fr)
WO (1) WO1987007726A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2514367A1 (fr) * 1981-10-08 1983-04-15 Pasteur Institut Procede pour la detection de germes pathogenes dans des fluides par filtration sur des immunoadsorbants, et application a la determination de la potabilite d'une eau
US4407943A (en) * 1976-12-16 1983-10-04 Millipore Corporation Immobilized antibody or antigen for immunoassay
US4459361A (en) * 1982-06-04 1984-07-10 Angenics, Inc. Ligand assay with one or two particulate reagents and filter
WO1985005451A1 (fr) * 1984-05-11 1985-12-05 Hybritech Incorporated Procede et appareil pour des analyses immunologiques
WO1986002160A1 (fr) * 1984-09-26 1986-04-10 Jan Peter Andersson Procede et dispositif de piegeage et d'analyse de particules

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4407943A (en) * 1976-12-16 1983-10-04 Millipore Corporation Immobilized antibody or antigen for immunoassay
FR2514367A1 (fr) * 1981-10-08 1983-04-15 Pasteur Institut Procede pour la detection de germes pathogenes dans des fluides par filtration sur des immunoadsorbants, et application a la determination de la potabilite d'une eau
US4459361A (en) * 1982-06-04 1984-07-10 Angenics, Inc. Ligand assay with one or two particulate reagents and filter
WO1985005451A1 (fr) * 1984-05-11 1985-12-05 Hybritech Incorporated Procede et appareil pour des analyses immunologiques
WO1986002160A1 (fr) * 1984-09-26 1986-04-10 Jan Peter Andersson Procede et dispositif de piegeage et d'analyse de particules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 89 (1978), Abstract 2665g, Clin. Chem. (Winston-Salem N.C.), 1978, 24(4), 571-9. *
PATENT ABSTRACTS OF JAPAN, Vol. 8, No. 82, (p-268); & JP,A,58 225 354, published 27 December 1983. *

Also Published As

Publication number Publication date
SE8602591D0 (sv) 1986-06-10
SE8602591L (sv) 1987-12-11
JPH02501327A (ja) 1990-05-10

Similar Documents

Publication Publication Date Title
US4962023A (en) Single incubation immuno sorbent assay method using particle labels to detect test antigen specific antibodies in presence of other antibodies
Hawkes et al. A dot-immunobinding assay for monoclonal and other antibodies
CA1200761A (fr) Dispositifs et trousses pour les analyses immunologiques
CN111398603B (zh) 一种用于检测新型冠状病毒抗体的试纸条、制备方法及其应用
EP0556202B1 (fr) Analyse de detection amelioree de ligands
US7297497B2 (en) Substrates for isolating, reacting and microscopically analyzing materials
WO1984003151A1 (fr) Matrice de points enduits d'anticorps pour la determination d'antigenes
Kelen et al. Rapid detection of Australia/SH antigen and antibody by a simple and sensitive technique of immunoelectronmicroscopy
AU2781992A (en) Solid support and assembly for quantitative and qualitative characterization of antigens
WO1988008538A1 (fr) Test et dispositif de determination du profil immunologique
US6933109B2 (en) Rapid particle detection
US5637453A (en) Whole blood method and kit for the detection of antibodies against human immunodeficiency virus (HIV) in HIV-seronegative individuals
Lasfargues et al. Experimental infection of a cat kidney cell line with the mouse mammary tumor virus
RU191660U1 (ru) Иммунохроматографическая тест-полоска для проведения экспресс-метода определения четырех групп антибиотиков в молоке с исключением возможной фальсификации образца
CN111521805A (zh) 一种2019新型冠状病毒抗原检测试剂及其制备方法
EP0537827A1 (fr) Méthode, dispositif d'essai et coffret pour essai avec un ligand de liaison spécifique, utilisant un écoulement controlé à travers une membrane de filtration
Kintzios et al. Study on the mechanism of Bioelectric Recognition Assay: evidence for immobilized cell membrane interactions with viral fragments
CN1141580C (zh) 对艾滋病与丙型肝炎病毒抗体的联合快速检测方法
Sanders et al. Routine detection of human rota virus by latex agglutination: Comparison with enzyme-linked immunosorbent assay, electron microscopy and polyacrylamide gel electrophoresis
CN1453588A (zh) 胶体金层析法检测sars冠状病毒抗原的试剂
US4814269A (en) Diagnostic testing for antibodies against microorganisms
WO1987007726A1 (fr) Detection de particules organiques, notamment de virus, avec technique d'enrichissement
US4187075A (en) Method of analyzing biological, liquid specimens for antigens or antibodies
Smith et al. Adenovirus-antibody agglutination reactions
EP0333801A4 (en) Self-contained multi-immunoassay diagnostic system

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE