JPH02501327A - Detection of organic particles, especially viruses, using improved techniques - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Re-゛ni Tol　1　ni　Rus゛ The present invention relates to a method for diagnosing organic particles such as viruses, bacteria, fungi and pollen. is tested to determine the presence, if any, of said particles therein. The liquid or gas to be If so, the accumulated particles are detected.

To ensure that any medical treatment required due to the presence of the specific virus is administered as soon as possible. In order to can be quickly and easily determined using conventional equipment. There is a clear need for something that can be done. Conventional diagnostic methods currently available cannot achieve this desired goal. does not satisfy.

The traditional method for diagnosing viruses is serological diagnostics. virus is alive When attacking an object, antibodies against the virus are present in the patient's blood or other body fluids. generated. The method relies on the presence of antibodies produced in body fluids after a virus attacks. antibodies are measured and such antibodies are detected by taking samples from patients. It is based on processes that are identified as However, this It does not occur until 1 or 2 weeks later. Because there are enough antibodies No, and the serological fluid will not turn positive until there is excess antibody. It's your body. This period often does not allow for effective therapeutic treatment. Too long to be possible.

Serological diagnosis is an indirect method since the antibodies produced are measured and identified. It's a test. Direct tests also exist that involve inoculating samples into tissue cultures. Ru. Such so-called virus tissue isolation is even more troublesome and time-consuming. a.m. for 3 to 5 days, but usually 6 to 8 weeks, to obtain test results. Requires culture.

Also, a tissue sample is taken from the patient, for example from the nasal mucosa, and this The sample's somatic cells are infected with fluorescent antibodies directed against the virus being sought. Curated immunofluorescence also exists. This virus exists and antibodies When combined, specific staining of somatic cells infected by the virus is obtained. Ru. Although this traditional diagnostic method is rapid (it can be done in 3-5 hours), the However, it is relatively complex and insensitive. Antibiotics marked by radioactivity or fluorescence For diagnosis of viruses that hybridize with complementing viral DNA or RNA Also for the fourth method (which involves staining cells from tissue samples from patients) Also, this is correct. On the other hand, viral DNA or RNA is the so-called known DNA Electrophores in the Northern method or Sachin method for A or RNA probes -(electrofores).

The object of the present invention is to directly identify and also quantify viruses or other viruses. An object of the present invention is to provide a method for rapidly diagnosing microorganisms.

However, as mentioned above, the present invention has not been applied to virus diagnosis so far. Not based on traditional enrichment techniques. On the other hand, this technique is described in U.S. Pat. No. 4,449, it is applicable as far as the determination of the presence of bacteria is concerned. I've been In this case, the concentration is unique for every filter. on the acting filter disc; its porosity is such that the particles, in this case, The bacteria being searched for do not pass through the filter disc and the surface of that disc collected above. The bacteria collected on that filter disc are then transferred to a suitable Staining with dyes or incubation with fluorescent antibodies is applied.

Viruses can be collected on filters by applying filtration techniques. In order to be effective, the filter must be very dense, i.e. It is required that the holes be as small as an elbow. Because viruses, for example, This is because they are considerably smaller than bacteria. A filter with pores of about 50 nm is Related, but viruses that also pass through filters with such small pores For example, there are hepatitis viruses with a size of 20 to 4 Qr+m. This kind of file The router will quickly become clogged. That is, this is only a very small amount of liquid may be allowed to pass through the filter before clogging occurs. means. Therefore, a circular shape with a diameter of 8 cranes and a hole of the order of 50 nm For a filter of only 20% of liquid with a protein concentration of 0.5/- Only ~30u can be passed from them before filter clogging occurs.

With viremia, the virus is present in the serum at a concentration of 10'' to 107 particles/mZ. exist, and this is due to very few viral particles (0,5 μ/- protein). O ~ 3xlO”/30x serum) is diluted to a serum concentration on the filter surface. Available. They are widely dispersed and further coated by a protein layer They will then be tested by using currently known methods. It cannot be used for detection.

The invention referred to herein applies concentration techniques to accumulated viruses. In order to It is necessary to use a tract or labyrinth system and to ensure that the virus particles can be passed through and nevertheless stopped on said system. Based on the knowledge that it is necessary to have virus particles that are

Based on this knowledge, the method of the invention obtains the features of claim 1.

In order to explain the invention in more detail, its aspects are illustrated by way of example with reference to the accompanying figures. Therefore, it will be described below. here, FIG. 1 is a diagram illustrating the application of antibodies to latex spheres, Figure 2 shows the binding of virus particles present to specifically directed antibodies. This is a micro-photograph illustrating how those spheres form aggregates when Figure 3 is a microphotograph illustrating the expansion of aggregate formation; Figure 4 is a microphone illustrating an isolated latex sphere with no virus attached. It is a photo, Figure 5 is a diagram illustrating the application of radioactively labeled antibodies to latex beads. ri, and FIG. 6 is a diagram illustrating the application of antibodies onto the filter surface.

Example 1゜ 4. Otnn size latex balls 10 (Batch 17155) (Po ly-science, from LISA), inoculation method induced by gamma rays. carboxylated by a method (this is described in European Patent Application No. 8585008). -7), the carboxyl group is added to the latex as shown in Figure 1. Bonded onto the surface of the sphere. Rabbit anti-mouse antibody 11 is then applied to the surface of the sphere. covalently bonded by carbodiimide to the carboxyl group and then probed A specific type of mouse monoclonal antibody 12 directed against the Ru. The antibody 12 is bound to the antibody 11 in its constant region (Fc), but the anti-antibody The variable region of body 12 is free to bind its appropriate antigen. For example, CMV (site For megaloviruses), antibody 12 is directed against the covering of virus particles. It can consist of a monoclonal mouse antibody that has been tested.

Antibody-binding spheres are incubated in the appropriate number in the liquid to be tested. (105 latex balls/test fluid). This fluid can be, for example, serum, spinal cord It can include fluid, saliva or urine. However, first of all, the liquid The table was heated for 10 minutes at approximately 150 rpm to allow cells or cell debris to settle out. It is centrifuged in a centrifuge. Aspirate the superior synovial fluid from them, and above latex spheres for 1 hour at 37°C, then the liquid was diluted in a dissolved salt solution, so-called PBS (phosphate buffered solution) with a pH of 7.4. , dilute serum in a 1:100 ratio, saliva in a 1:5 ratio, and urine in a 1:2 ratio. Dilute with

After incubation, the test liquid as a whole is 8404B21-4 and has a filter of the embodiment described. Filter by using a sample holder. In this case, that filter is Preferably with a precise pore obtained by proton technique and with a diameter of 0.8 to 1 It comprises a hydrophilic polycarbonate filter of size in the zero range. That flap The pore size of the filter depends on the detection system used for final determination of the virus. selected. With such a filter, large amounts of liquid or gas can pass through the filter. can be filtered without clogging. Trademark NUCLE with appropriate filter registered Can be used as OPORE. Latex binding virus, i.e. CMV. The spheres will clump together. That is, they have some latex balls and nodules. Complex formation of combined virus particles forms clumps on the surface of the filter (Figures 2 and 3), however, latex spheres with no virus attached are will lie isolated on the Luther surface (Figure 4). Then uncombined The virus will pass through the filter. Because the virus particles This is because it is considerably smaller than the Luther hole.

If the filter pores are larger than the latex bulb, and the latex bulb , even if they do not bind viruses and do not form complexes, It will pass through Luther.

Detection of virus and latex sphere aggregates on filters can be done in different ways. It can happen. One embodiment is according to Swedish Patent Application No. 8404821-4. Deposition and vacuum evaporation of the sample onto the filter surface in the sample holder; Gold or platinum ions with a thickness of 0.5-5 nm by so-called buffeting The filter surface is then subjected to scanning electron microscopy. Analyze with a microscope. Next, the electrical Using a filter with a 0.8-pore size fixed in a conductive holder. use The drawings seen have the appearance illustrated in FIGS. 2-4. No. 2 and 3, the latex spheres are coated with specific antibodies from body fluids, It has virus particles bound to its surface and the virus and latex A complex of many aggregates of spheres is formed. The bond between the latex spheres is particularly It can be clearly seen in the figure.

In contrast, in Figure 4, the latex spheres coated with specific antibodies , devoid of viruses. Their surfaces are completely smooth, and their Latex spheres exist isolated on the filter surface.

On the other hand, it binds viruses and aggregates that cannot pass through the filter. The latex spheres that form together with the virus are oxidized by peroxidase. This can be determined by marking the spheres and, if the substrate is supplied, its The filter may become yellow due to latex spheres deposited on its filter surface. It turns yellow-green because of the obvious fact. The color response can be determined by spectroscopic analysis or by ELI. A system similar to that used for ZM enzyme-linked immunosorbent assays The stem can be measured by eye. However, this reaction method If the virus does not form an aggregate by binding to that particular virus, then larger than the diameter of the latex sphere so that it passes through the filter. Requires a large filter pore diameter. In this case, with a pore size of the order of 10- filter is used.

A more improved type of detection method is also used to detect viruses coated and filtered. The latex spheres deposited on the surface of the This can be achieved by coating the latex sphere with a specific antibody, and this This is done by supplying said second antibody to the filter where it is collected.

If a virus is present in the latex gem, it will be marked by its radioactivity. The target antibodies bind to virus particles and are then removed from the filter surface by washing. However, if there are no viruses on the latex beads, the The second radioactive antibody will be washed away from the filter. This is shown in Figure 5. The virus shown and named here as 13 can contain 1125. The antibody 14 is bound to a radioactively marked antibody 14. After washing three times, filter The remaining radioactivity on the surface is measured by so-called RIA (radioimmunoassay). Measure with gamma sense.

Yet another detection method is to use a second antibody 14 directed against a specific virus. Marked by oxidase, this antibody binds, and is not removed by washing. If not, after addition of the substrate a yellow-green reaction is obtained and the same method as above is used. It consists of something that can be measured.

Example 2゜ Alternatives to the binding of rabbit anti-mouse antibodies to latex spheres in the manner described in Example 1. In particular, the binding of these antibodies is similar to European Patent Application No. 8585008-7. Directly onto polycarbonate filter discs that would be treated in the same way. and the filter is coated with antibodies covalently attached to the filter surface. Carboxylation by T-ray induced inoculation method, which allows binding of Fc moieties. silized. This is illustrated in Figure 6, where 15 indicates the filter holder. and 16 shows the filter disk, after cleaning the filter surface 4 times. , filter the test liquid through the filter, then apply it to the surface of the virus. Add specific mouse monoclonal antibodies directed against it. If there is , the viruses present are filtered by specific binding to their viral antibodies. It adheres directly to surfaces and cannot be removed even by four consecutive washes. stomach. If the virus is not bound to the surface, it is removed by washing There will be. The pore size on the filter surface is determined by the diameter of the virus particle. is selected to be about 10 times larger than . Detection is the same as described in Example 1. (scanning electron microscopy, radioimmunoassay or ELIZ) by A). For filtration, Swedish Patent Application No. 8404821-4 A filter holder of the type described in is used.

The filter allows the presence of different viruses to be determined in one filtration to make different sizes or different shapes on the surface of different levels Different types of latex spheres and placed at different levels for virus deposition can be done. The filter also has many wells spaced apart from each other. and the bottom of which has different samples in it. A filter is supplied to receive the Finally, the filter is porous It can be formed by a unique latex sphere supported by a sole.

The filters each have one more for concentration of virus and latex spheres. can be replaced by a passageway or maze system.

If the filter is made of translucent material, the detection emits light through them This can be done by

February 9, 1986 Mr. Kunio Kogawa, Commissioner of the Patent Office 1 Display of patent application PCT/SE86100507 2 Name of the invention Method for detecting organic particles, especially viruses, by improved techniques 73 Patent Applicant Address: Shefpal, Nis-13300 Saltsjöbaden, Sweden Stegen 15 Submission date of amendment October 16, 1987 (reception mortar) i, S゛- 1. A method for diagnosing organic particles, especially viruses, by concentration techniques (here , the liquid to be tested to determine the presence, if any, of said particles therein. or gas is passed through a passageway or labyrinth system and accumulates, if any. particles are detected) and has a permeability that allows the passage of said particles. A passageway or labyrinth system surface is used for said concentration and/or The surface of a ball or other object that is larger than the particle to be viewed is are prepared to bind the particles to multiple surfaces and pass through a passageway or labyrinth system. With the passage of a liquid or gas, the particles are directly onto the surface of such system. accumulates or is indirectly bonded to a ball or other object and to an ionized metal layer. Passages with balls or other objects perhaps located on the surface after more coating or the surface of the maze system is exposed to a scanning voltage for detection of the presence of said particles, if any. A method characterized in that it is examined by means of a submicroscope.

2. The surface of said passageway or maze system and/or the surface of said ball or other object. each surface with a specific type of antibody directed against the particle to be discovered. or prepared for covalent bonding to multiple surfaces. The method described in box 1.

3. The particles directed towards the particles to be probed are each activated by an intermediate antibody. claim 1, wherein the surface or surfaces are bonded to said surface or surfaces. the method of.

4. Reactive groups covalently bind antibodies directed against each probed particle. conjugated to said surface or surfaces or to an intermediate antibody, respectively, in order to The method according to claim 2 or 3, characterized in that:

5. The antibody or intermediate antibody, respectively, may have a carbodiimide bond or other covalent bond. Claim 4, characterized in that it is bonded to the reactive group by a bond. the method of.

6. The concentration allows emission of light through the translucent material for detection. , characterized in that the material is provided on a passageway or labyrinth system. The method according to any one of items 1 to 5.

National FGI11 inspection lost Quantity I--Hibiki-----^IM+"-'"口PCT/5E81'100507

Claims (6)

[Claims] 1. A method for diagnosing organic particles, especially viruses, by concentration techniques (here , the liquid to be tested to determine the presence, if any, of said particles therein. or gas is passed through a passageway or labyrinth system and accumulates, if any. particles are detected) and has a permeability that allows the passage of said particles. A passageway or maze system is used for said concentration, and said passageway or maze system is The surface of the system and/or the surface of a sphere or other object larger than the particle being searched , each prepared to bind said particles to said surface or surfaces, and a channel or The passage of a liquid or gas through a labyrinth system causes the particles to directly accumulated on the system or indirectly coupled to said sphere or other object. How to characterize it. 2. The surface of the passageway or maze system and/or the surface of the sphere is covered by the particles to be explored. Each surface or surfaces shares a specific type of antibody directed against the child. A method according to claim 1, characterized in that it is prepared for binding. 3. The particles directed against the particles to be probed are each activated by an intermediate antibody. claim 1, wherein the surface or surfaces are bonded to said surface or surfaces. the method of. 4. The reactive groups covalently bind antibodies directed against each probed particle. conjugated to said surface or surfaces or to an intermediate antibody, respectively, in order to The method according to claim 2 or 3, characterized in that: 5. antibodies of said antibodies or intermediates, carbodiimide bonds or other covalent bonds, respectively. Claim 4, wherein the reactive group is bonded to the reactive group by bonding. Method. 6. The concentration allows the emission of light through the translucent material for detection. , characterized in that the material is provided on a passageway or labyrinth system. The method according to any one of items 1 to 5.