CH633825A5 - Preparation of sub-units of the inner capsid of the rotavirus - Google Patents

Description

The invention relates to preparations of the rotavirus which are suitable as diagnostic agents in immunological test methods.

The rotavirus particles were first discovered in the stables of free-range calves by A.L. Ferneluis et al., Archives for the Entire Virus Research, volume 37, 1972, pages 114 to 130, and were called rotaviruses under the electron microscope because of their wheel-like appearance. It was later found that particles of identical morphology were found in the stools of other species, such as pigs and humans, and were constantly associated with gastroenteritis. In fact, the rotavirus can be considered a major cause of childhood gastroenteritis.

On the surface, the particles resemble the reovirus in that they have an inner and an outer capsid, the diameter of which is 55 nm and 70 nm, respectively (TH Flewett et al., J. Clin. Path. Volume 27, 1974, pages 603 to 614) . However, the particles are morphologically different from the reovirus because the subunits in the outer capsid have a soft or smooth outer surface.

After the discovery of the rotavirus group, it was found that there were serological relationships between the viruses depending on the different types (A.Z. Kapikian et al., Science, Volume 185, 1974, pages 1049 to 1053). In fact, A.Z. Kapikian et. al., Lancet, vol. 1, 1975, pages 1056 to 1061, demonstrated that it was possible to use a virus isolated from the stool of free-range calves as a diagnostic antigen in the study of human rotavirus infection. Lately, it has been shown that serological crossbreeding between members of the rotavirus group is largely mediated by the antigens of the inner capsid (G.N. 20 Woode et. Al. In Infection and Immunity in press).

It has now been found that preparations from stools infected with rotavirus can contain considerable amounts of the inner capsid in the form of virus subunits and that the relative proportion of such subunits can be increased by appropriate treatment.

Accordingly, the invention relates to a preparation of subunits of the inner capsid of the rotavirus, which is characterized in that it is at least approximately free of complete virus particles and their fragments.

The appearance of the subunit is generally circular in the electron microscope and has a diameter of less than 2 nm.

35 The invention further relates to a method for producing and concentrating the subunits of the inner capsid of the rotavirus, which is characterized in that a sample of faecal material of a mammal infected by the rotavirus, the Sus-40 pension at 30 to 40 ° C. incubated for at least 20 minutes, the subunits removed from the suspension by centrifugation and / or filtration and concentrated.

In order to obtain a preparation of subunits of the rotavirus, one can obtain the faecal material from a mammal infected with a rotavirus, which in turn can be detected by immunoelectron microscopy using the serum from recovering mammals, the mammals either Calves, pigs or mice or children, so the faecal material is preferably suspended in a suitable buffer solution at a pH of 5 to 9, so that a suspension of about 5 to 30 wt .-%, preferably about 20 wt .-%, results. The suspension is then placed in a sealed container and incubated at a temperature of 30 to 45 ° C, preferably about 37 ° C, for at least 20 minutes, but usually for 30 minutes. The suspension is then clarified by centrifuging at an appropriate speed, so that preferably the whole virus particles and 60 fragments sediment, but the subunits cannot sediment. The supernatant liquid can then be filtered off and the resulting liquid, which is almost clear, is generally reduced to at least the 10th part, preferably the 25th part, preferably 65 by the concentration method by molecular filtration according to "Minicon" or "Amicon" ( Amicon Corp.).

The concentrate of the subunits produced in this way can be further processed by «density gradient centrifugation»

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can be cleaned using, for example, a sucrose density gradient. Furthermore, it is possible to increase the proportion of subunits in the concentrate by treating the initial faecal suspension with enzymes, such as trypsin or papain, which break down the complete virus particles into their subunit components. For example, a 0.5% trypsin solution can be used at 37 ° C for 1 hour.

The presence of virus subunits in the concentrate can e.g. by direct negative staining electron microscopy or by immunoelectron microscopy, which is preferred. When examined using immuno-electron microscopy, the preparations consist almost entirely of circular, frayed subunits, the size of which is less than 2 nm, and which are complexly bound to antibodies. Furthermore, one or two complete virus particles approximately 70 nm in diameter or large fragments thereof can also be visible.

The present subunit concentrate can be used for various immunological diagnostic tests, such as, for example, for immunoelectron microscopy, for the immunodiffusion test, for the haemagglutination test, for the “complement fixation test” and for the radioimmunoassay.

Immunoelectron microscopy can be used to diagnose the cause of gastroenteritis in a patient or to determine whether an individual has suffered recently or not or has experienced a subclinical infection. To perform such a test, a subunit concentrate is prepared as described above and mixed with a serum sample from the patient, the mixture allowed to react at room temperature and then centrifuged. The supernatant liquid is poured off and the remaining sediment plate is stained negatively with phosphotungstic acid and examined under the electron microscope for the presence of subunits which are complexly bound to antibodies.

A suitable gel for immunodiffusion is produced as a layer on a plate, into which wells of equal size are cut with the same distance from one another. The subunit concentrate is introduced into some of the wells and a test serum from the patient into the others. The gel is then left to stand at room temperature for a few hours and then checked for the reaction. If specific antibodies against the rotavirus are present in the test serum, a precipitin line forms in the gel, specifically at the point at which the serum diffusing into the medium meets the subunit concentrate which also diffuses into the medium. Using an appropriately placed reference antigen and antisera, the immunodiffusion test can be successfully used to read patient serum samples for the presence of an antibody or faecal extracts for the presence of virus antigen. The subunit concentrate is particularly suitable for the gel diffusion test, since it has been found that the subunits have a much greater mobility in the gel than complete viruses. This is evidenced by the lack of any other Precipitin line which shows the complex of the whole virus and the antibodies.

The subunits migrate quickly through the gel so that the results can be read more quickly and with greater accuracy.

Using the introduced complement fixation technique as described by Bradstreet & Taylor in Public Health Laboratory Services Bulletin, Vol. 21, 1962, page 96, the subunit concentrate can be used to diagnose rotavirus infections.

To perform a hemagglutination test, it may be possible to artificially attach the subunits to red blood cells and then insert such red blood cells into a hemagglutination test system, as described, for example, by Sequeira, P.J.L., and Eldriges, A.E. in Br. J. Vener. Dis., Volume 49, 1973, page 3, or even incorporate them into a gel system to which anti-serum and complement can be added.

An advantage of the subunit concentrate is that it reacts equally well with both homologous and heterologous antisera and is therefore a valuable general diagnostic tool, since the serological relationships between the members of the rotavirus group are mediated by the antigens of the internal component . In addition, the subunit concentrate can be easily prepared from stool samples by simple laboratory techniques and then tested using a reference serum known to contain antibodies to the rotavirus. In this way, all of the tests described above can be used to test stool samples for the rotavirus using the rotavirus antiserum.

Another object of the invention is the use of the preparation according to the invention of subunits of the inner capsid of rotavirus for the production of a rotavirus antiserum which contains antibodies against the subunits of the inner capsid of rotavirus, which is characterized in that a preparation of the subunit is given to a mammal of the inner capsid of the rotavirus, and after a few days, the mammal is re-injected with a second aliquot of the subunit preparation, which after a few more days bleeds the mammal, clots the blood and separates and removes the antiserum.

The following exemplary embodiments serve to explain further advantages of the present invention.

example 1

Production of a subunit concentrate

Faecal material was obtained from infected calves from a dairy herd in which a rotavirus infection had broken out. A 20% suspension of the faecal material was prepared in phosphate buffered saline (PBS). This suspension was placed in a tightly sealed test tube and incubated in a water bath at 37 ° C for 30 minutes. During this time, the suspension was mixed thoroughly at least three times using a mechanical mixer. The mixture was then clarified by centrifugation at 3000 g for 15 minutes.

The supernatant liquid from this process stage was passed through a “Millipore” pre-filter and a 1.2 p, m filter. The resulting liquid, which was almost clear, was then placed in a "Minicon B15" (manufactured by Amicon Corp.) and concentrated on the 25th part. Following this procedure, 1 ml of faeces gave 0.2 ml of the preparation as the final concentrate.

Example 2

Diagnosis by immunoelectron microscopy

Aliquots of 0.5 ml of the subunit concentrate from Example 1 were placed in test tubes

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and 0.1 ml of bovine serum or human serum derived from individuals suffering from gastroenteritis as well as 0.1 ml of a standard saline solution in a control test tube. The test tubes were left at room temperature for 1 hour and then centrifuged at 12,000 g for 1 hour. The supernatants were poured off and the respective sediment was stained negatively in the usual way using phosphotungstic acid. When viewed under the electron microscope, aggregates of small, circular subunits that complexed with antibodies could be seen, which proved that the gastroenteritis of the patient was caused by the rotavirus.

Example 3 Diagnosis by Immunodiffusion

A patient suffering from gastroenteritis provided the blood samples from which the serum was made.

Agarose was prepared in TRIS / ethylenediaminetetraacetate buffer, the preparation consisting of 0.9 w / v% agarose, and an aliquot of 2 ml was used to cover a glass plate. A template with two rows of 3 mm wells and 3 mm spaces was used to cut wells into the gel. One row of the wells was filled with the subunit concentrate prepared in Example 1 and the other row with the serum of a patient suffering from gastroenteritis. After filling, the gel was left overnight at room temperature.

When the gel was examined the next day, it was found that a line of precipitin, indicating gastroenteritis, had formed in the gel between the wells containing the subunit concentrate and the patient serum who suffered the patient who caused rotavirus.

Example 4

Diagnosis using the complement fixation test

Human sera to be tested for the presence of antibodies to the rotavirus were first diluted 1: 2 in a veronally buffered saline (VBS) and heat inactivated in a water bath kept at 56 ° C for 30 minutes. These sera were then titrated over a "U" -shaped microtiter plate from 1: 2 to 1: 256 dilutions in the VBS,

using amounts of 0.025 ml. Each serum was titrated in two parallel rows. To the first series of dilutions, 0.025 ml of the rotavirus subunit preparation was added at a 1: 8 dilution in VBS 5. The second series of dilutions were used as an antiserum control and 0.025 ml of VBS was added to the sera instead of the antigen. Antigen control was also included, adding the antigen to volumes of 0.025 ml VBS without adding test serum io.

The complement, stored at -70 ° C, was diluted 1:50 in VBS and 0.025 ml added to all wells. Complement control was added in the usual way. The plate was then covered and left at 4 ° C overnight.

The following day, antiserum to sheep red blood cells, i.e. hemolytic serum stored at -20 ° C, diluted 1: 100 to 1: 800 in VBS and incubated with an equal volume of a 4% sheep red blood cell suspension at 37 ° C for 30 minutes to form a hemolysin system. At the same time, the plate was heated to 37 ° C so that all components of the system were at the same temperature. Units of 0.025 ml 25 of the hemolysin system were added to each well and the plate was gently shaken. The plate was further incubated at 37 ° C for half an hour and shaken at 15 minute intervals. It was then re-stored at 4 ° C for 2 to 3 hours before reading.

30 If red blood cells had been lysed, this indicated the presence of antibodies to the rotavirus in the test serum sample.

Example 5

35 Production of antiserum to the inner

Capsid subunit of rotavirus

2 ml of a preparation of subunits of the inner capsid of the rotavirus was mixed with an equal volume of 40 Freund's “complete adjuvant” and divided into 2 aliquots of 2 ml each. An aliquot was applied subcutaneously to two guinea pigs. 21 days later, the second aliquot was applied to the guinea pigs in the same way. 35 days after the first 45 administration to the guinea pigs, the guinea pigs were bled and the blood clotted. The fluid obtained after clumping showed complete antiserum to the subunits of the inner capsid of the rotavirus.

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Claims (12)

633 825 2nd PATENT CLAIMS 1. Preparation of subunits of the inner capsid of the rotavirus, characterized in that it is at least approximately free of complete virus particles and their fragments. 2. Preparation according to claim 1, characterized in that the subunits are circular in shape. 3. Preparation according to claim 1 or 2, characterized in that the diameter of the subunits is less than 2 nm. 4. A method for producing a preparation of subunits of the inner capsid of rotavirus according to claim 1, characterized in that a sample of faecal material is suspended from a mammal infected with a rotavirus, the suspension is incubated at 30 to 45 ° C for at least 20 minutes and the subunits are removed from the suspension by centrifugation and / or filtration and concentrated. 5. The method according to claim 4 for the preparation of a preparation according to claim 2 or 3. 6. The method according to claim 4 or 5, characterized in that the faecal material is suspended in a buffer solution at a pH of 5 to 9. 7. The method according to any one of claims 4 to 5, characterized in that the suspension is incubated at about 37 ° C. 8. The method according to any one of claims 4 to 7, characterized in that the subunits are removed from the suspension in such a way that the latter is centrifuged at a speed which is sufficient to sediment all the virus particles and their fragments, but is not sufficient to to sediment the subunits. 9. The method according to any one of claims 4 to 8, characterized in that the initial faecal suspension is incubated with a proteolytic enzyme. 10. The method according to claim 9, characterized in that trypsin or papain is used as the proteolytic enzyme. 11. Use of the preparation according to claim 1 for the production of a rotavirus antiserum which contains antibodies against the preparation of subunits of the inner capsid of rotavirus, characterized in that a first aliquot of the preparation of subunits of the inner capsid of rotavirus is injected into a mammal, after a few days a second aliquot of the same preparation is injected, after a few more days the mammal bleeds, the mammalian blood clots and the antiserum is removed from the blood cake. 12. Rotavirus antiserum which contains antibodies against the preparation of subunits of the inner capsid of the rotavirus, produced according to claim 11.