WO1987007726A1

WO1987007726A1 - Detection of organic particles, particularly viruses, with enrichment technique

Info

Publication number: WO1987007726A1
Application number: PCT/SE1986/000507
Authority: WO
Inventors: Jan Peter Andersson; Rolf Nybom
Original assignee: Jan Peter Andersson; Rolf Nybom
Priority date: 1986-06-10
Filing date: 1986-11-06
Publication date: 1987-12-17
Also published as: JPH02501327A; SE8602591D0; SE8602591L

Abstract

Method of diagnosing organic particles, particularly virus, bacteria, fungus, and pollen, by concentration technique, wherein liquid or gas to be examined for determining the presence of said particles, if any, in body liquid is passed through a passage or labyrinth system and accumulated particles, if any, then are detected. A passage or labyrinth system is used for the concentration, having a permeability allowing the passage of said particles. The surface of the passage or labyrinth system and/or the surface of spheres or other bodies larger than the particles sought for, is prepared so as to bind said particles to said surface or surfaces, respectively, said particles at the passage of the liquid or gas through the passage or labyrinth system accumulating on the surface of such system directly, or indirectly bound to the spheres or other bodies. The accumulation of specific particles, e.g. virus, takes place only if the particles have bound to said surface or surfaces, respectively. If no formation of aggregates takes place, the virus particles and the latex spheres will pass through the passage or labyrinth system and thus will not be accumulated.

Description

DETECTION OF ORGANIC PARTICLES,PARTICULARLY VIRUSES, ITH

ENRICHMENT TECNIQUE

The invention relates to a method of diagnosing

5 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,

10 then are detected.

It is a pronounced want to be able to determine rapidly and simply by using conventional apparatus if a specific virus is present in a body liquid, e.g. serum, spinal fluid, saliva, or urine, to be able to put in as

15 rapidly as possible the medical care required due to the presence of said virus. The conventional diagnosing methods now available do not satisfy this want.

A conventional method for diagnosing virus is serological diagnostics. When virus attacks the

20 organism, 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

25 by taking samples from the patient. However, this cannot take place until one or two weeks after the attack, because not until then there is a sufficient amount of antibodies, and serological fluid will not turn positive until there is a surplus of antibodies. This period in

30 many cases is too long to make possible that an effective therapeutic treatment is put in.

The serological diagnostics is an indirect test, because the generated antibodies are determined and identified. There is also a direct test which involves

35 inoculation of the sample on a tissue culture. Such so-called 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.

There s also immunofluorescence wherein a tissue sample is taken from the patient, e.g. from the mucous membrane in the nose, and the body cells of this sample are incubated with fluorescent antibodies directed towards the virus sought for. If this virus is present and thus is bound to the antibodies there is obtained a specific staining of the body cells infected by the virus. This conventional diagnosing method is rapid - it can be performed in three to five hours - but it is relatively cumbersome and insensitive.

This is true also for the fourth existing method for diagnosing virus, hybridizing, which involves staining of cells from a tissue sample from the patient with anticomplementary viral DNA or RNA marked by radioactivity or fluorescence. Alternatively, viral DNA or RNA can be demonstrated by electrofores in so-called northern or southern blotting technique against a known DNA or RNA probe.

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.

As mentioned above, the invention is based on conventional concentration technique which has not, however, previously been applied to the diagnosing of virus. On the other hand, 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. In that case, 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.

In order to make possible that virus is collected on a filter by applying the filtration technique it is required that 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. Thus, in case of 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. At viremia, 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. On the basis of this knowledge the method of the invention has obtained the characteristics appearing from claim 1.

In order to explain the invention in more detail embodiments thereof will be described below by means of examples with reference to the accompanying drawings in which

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, and FIG. 6 is a diagrammatic picture illustrating the application of antibodies on a filter surface.

EXAMPLE 1.

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

5 antibodies 11 in the constant region thereof (Fc), while the variable, regions of the antibodies 12 are free to bind to the relevant antigen thereof. For e.g. CMV (cytomegalovirus) the antibodies 12 can consist of monoclonic mouse antibodies directed towards the cover

10 of the virus particles.

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.

15 Initially, 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

20 liquid then being diluted in balanced saline solution, so-called PBS (phosphate buffered saline solution) having a pH of 7.4, serum being diluted in the relationship 1:100, spinal fluid 1:20, saliva 1:5, and urine 1:2.

₂₅ After incubation 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. In that case the filter preferably comprises a hydrophilic polycarbonate filter

₃₀ having exact apertures obtained by protone technique and having a size ranging from 0.8yum to 10yum. 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. 2 and 3, 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. In FIG. 4, 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.

Alternatively, 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) . However, 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. In this case, 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. 5 where virus designated 13 is bound to the radioactively marked antibodies 14 which can comprise Iι₂₅* ^A^^tβr washing three times, the remaining radioactivity on the filter surface is measured by so-called RIA (radioimmunoassay) in a ga mameter.

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. This is diagrammatically shown in 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. Existing virus, if any, will stick directly to the filter surface by specific binding to the virus antibody and cannot be removed by washing not even by four succeeding washings. If virus has not been bound to the surface, it will be removed by washing. 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. Finally, 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.

If the filter consists of a translucent material, the detection can take place by emitting light therethrough.

Claims

10CLAIMS

1. Method of diagnosing organic particles, particularly virus, by concentration technique, wherein liquid or gas to be examined for determining the

5 presence of said particles therein, if any, is passed through a passage or labyrinth system and accumulated particles, if any, then are detected, c h a r a c t e r i z e d in that a passage or labyrinth system is used for the concentration, having a

10 permeability allowing the passage of said particles, and that the surface of the passage or labyrinth system and/or the surface of spheres or other bodies larger than the particles sought for, is prepared so as to bind said particles to said surface or surfaces,

15 respectively, said particles at the passage of the liquid or gas through the passage or labyrinth system accumulating on the surface of such system directly, or indirectly bound to the spheres or other bodies.

2. Method as in claim 1,

20 c h a r a c t e r i z e d in that the surface of the passage or labyrinth system and/or the surface of the spheres is prepared to bind covalently to said surface or surfaces, respectively, antibodies of a specific kind directed towards the particles sought for.

25 3. Method as in claim 2, c h a r a c t e r i z e d in that said antibodies directed towards the particles sought for, are bound to said surface or surfaces, respectively, by means of an intermediate antibody.

30 4. Method as in claim 2 or 3, c h a r a c t e r i z e d in that reactive groups are bound to said surface or surfaces, respectively, to bind covalently the antibodies directed towards the particles sought for, or to the intermediate antibodies,

35 respectively. 5. Method as in claim 4, c h a r a c t e r i z e d in that the antibodies or the intermediate antibodies, respectively, are bound to the reactive groups by carbodiimide binding or by another covalent binding.

6. Method as in any of claims 1 to 5, c h a r a c t e r i z e d in that the concentration is effected on a passage or labyrinth system of a translucent material to allow emitted light to pass therethrough for detection.