JPS6129463B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the diagnosis of gastroenteritis caused by rotavirus, and in particular to rotavirus preparations suitable for use as diagnostic reagents in immunological testing methods.

Rotavirus particles were determined by Juane Lewis, A.
L. It was first discovered in the toilet bowls of calves with diarrhea by Ferneluis, AL, et al.
Archiv fu¨r die Gesamte
Virusforschung, 1972, 37 , 114-130] and was called rotavirus because of its wheel-like appearance under an electron microscope. It was then established that virus particles of the same form were found in the feces of other species, such as pigs and humans, and that they were consistently associated with gastroenteritis. In fact, rotavirus is considered one of the major causative agents in infant gastroenteritis.

The particles apparently have an inner capsid and an outer capsid, each with a diameter of 55nm.
It is similar to reovirus in that it has a wavelength of 70 nm and 70 nm [Frewett, T. Flewett, TH et al., J. Clini. bread. (J.Clin.Path.) 1974, 27 , 603−
614]. However, this particle consists of subunits located outside the two capsids.
It is morphologically different from reoviruses because it has a smooth or continuous outer surface.

After the discovery of the rotavirus group, it was recognized that serological relationships existed between viruses associated with different species [Kapikian, A. Zett.
(Kapikian, AZ) etc., Science,
1974, 185 , 1049-1053]. In fact, further Kapikian, A. Zett. (Kapikian, AZ) et al. showed that virus isolated from the feces of calves with diarrhea can be used as a diagnostic antigen when studying rotavirus infection in humans (Lancet, AZ).
1975, 1 , 1056-1061). More recently, it has been recognized that serological cross-over between members of the rotavirus group is mediated to a large extent by inner capsid antigens [Ud, G., et al. N. (Wooden,
GN) etc., Infection and Immunity).

It has been discovered in accordance with the present invention that preparations of rotavirus-infected feces can contain significant amounts of the inner capsid in the form of viral subunits, and that the relative proportions of such subunits can be increased by appropriate treatment.

Thus, one feature of the present invention is to provide a rotavirus inner capsid subunit formulation that is substantially free of intact virus particles and fragments thereof.

When viewed under an electron microscope, this subunit usually has a circular shape and a diameter of less than 2 nm.

The second feature of the present invention is to suspend a fecal sample obtained from a mammal infected with rotavirus,
The rotavirus inner capsid subunits are prepared by incubating the suspension at 30 to 45°C for at least 20 minutes, removing the subunits from the suspension by centrifugation and/or filtration, and concentrating the suspension. An object of the present invention is to provide a method for producing and concentrating.

To obtain rotavirus subunit preparations, rotaviruses as demonstrated by immunoelectron microscopy using convalescent serum collected from mammals, including calves, mice, pigs, or children. Obtain feces from an infected mammal. This feces is suspended in a suitable buffer solution with a pH of 5 to 9 to form a 5 to 30% suspension, preferably approximately 20% by weight. This suspension is then placed in a closed container and kept at a temperature of between 30 and 45°C, preferably around 37°C, for at least 20 minutes, usually 30 minutes.
The suspension is then preferably clarified by centrifugation at a speed sufficient to precipitate all virus particles and fragments, but insufficient to precipitate subunits. The supernatant liquid is then filtered and the resulting almost clear liquid is at least
Concentrate 10 times, but preferably 25 times, preferably by either the Minicon or Amicon molecular filtration concentration methods.

The subunit concentrate obtained by this method may be further purified by concentration gradient centrifugation, using, for example, sucrose concentration. Furthermore, it is possible to increase the proportion of subunits in the concentrate by treating the initial fecal suspension with enzymes capable of breaking down intact virus particles into their component units, such as trypsin or papain. be. For example, a 0.5% trypsin concentration can be used for 1 hour at 37°C.

The presence of viral subunits in the concentrate
It may be demonstrated by direct negative stain electron microscopy or immunoelectron microscopy (preferred). When tested using immunoelectron microscopy, the formulation consists of almost completely circular, rounded subunits, which are less than 2 nm in size and capable of forming complexes with antibodies. Furthermore, one or two approximately 70 nm in diameter
One complete virus particle or a large fragment thereof may be visible.

The subunit concentrates of the invention can be used in several immunological diagnostic tests, such as immunoelectronoscopy, immunodiffusion, hemagglutination, complement fixation, and radioimmunopotency assays.

Immunoelectronoscopy can be used to diagnose the cause of gastroenteritis in a patient or to determine whether an individual has recently suffered from, or currently has, a subclinical infection. . To perform such a test, a subunit concentrate is prepared as described above, mixed with a serum sample taken from a patient, allowed to interact at room temperature, and then centrifuged. The supernatant is discarded and the crystal globules are neutrally stained with phosphotungstate and then examined under an electron microscope for the presence of subunits in complex with the antibody.

In immunodiffusion, a suitable gel is produced in layers on a plate with equally sized and equally spaced holes cut therein. Some wells are filled with subunit concentrates, others with test serum taken from the patient, and the gel is then left at room temperature for a few hours before testing the reaction. If rotavirus-specific antibodies are present in the test serum, precipitated strands will form in the gel where the serum diffusing in the solvent meets the subunits being diffused. With standard antigens and antisera in place, immunodiffusion tests can be successfully used to test serum samples taken from patients for the presence of antibodies to viral antigens or fecal extracts. Subunit concentrates are particularly advantageous for gel diffusion studies. This is because the complete virus is found to be more mobile in the gel than the complete virus, as evidenced by the absence of any other precipitated strands representing the complete virus-antibody complex. There is. Therefore, the subunits migrate through the gel quickly and results can therefore be read faster and more reliably.

As described by Bradstreet and Taylor [Public Health Laboratory Services Bulletin]
Services Bulletin), 1962, 21 , 96], subunit concentrates can be used in the diagnosis of rotavirus infection using methods for establishing complement fixation.

To perform a hemagglutination test, subunits are artificially added to red blood cells, and the red blood cells are then incorporated into a hemagglutination test system [for example, Br. J. Vener.
Dis., 1973, 49 , 3 Sequeira, P. J.A.
L. (Sequeira, PJL) and Eldrizi, A. E. (Eldridge, AE)] or just incorporate them into a gel system in which antiserum and complement are added.

One advantage of subunit concentrates is that the serological relationships that occur between members of the rotavirus group are mediated by internal component antigens, making this concentrate a useful general diagnostic tool. It can react equally well with type and variant antisera. Furthermore, subunit concentrates are very easily prepared from stool samples by simple laboratory techniques and tested using standard sera known to contain antibodies to rotavirus.
All of the tests described above can thus be used to test stool samples for rotavirus by using rotavirus antisera.

Accordingly, the present invention also relates to formulations of rotavirus antisera containing antibodies against rotavirus inner capsid subunits.

The invention further relates to a method of producing rotavirus antisera containing antibodies against rotavirus inner capsid subunits.
This method involves injecting a mammal with a rotavirus inner capsid subunit formulation, a few days later injecting the mammal with another portion of the subunit formulation according to the invention, and a few more days later injecting the mammal with a rotavirus inner capsid subunit formulation. This involves exsanguinating the blood, allowing the blood to clot, and separating and removing the antiserum.

The advantages of the invention will become more apparent from the following description of the embodiments of the invention, which are in no way intended to limit the invention.

Example 1 Production of subunit concentrate Feces are obtained from infected calves from a dairy herd where explosive rotavirus infection has occurred. Make a 20% suspension of fecal matter into buffered saline (PBS)
(PH approx. 7.2). This suspension is placed in a tightly capped tube and kept at 37°C on a water bath for 30 minutes. During this time, the suspension is thoroughly mixed at least three times using a mechanical stirrer. Mixture then 3000 for 15 minutes
Clarify by centrifugation at g.

From this step, the supernatant is passed through a Millipore prefilter fitted with a 1.2 μm filter. The almost clear liquid obtained is then placed in a Minicon B15 (Amicon) and concentrated 25 times. Using this method, 0.2 ml of the preparation is obtained as a final concentrate from 1 ml of feces.

Example 2 Diagnosis by immunoelectron microscopy A portion (0.5 ml) of the subunit concentrate produced in Example 1 was placed into a tube, and bovine or human serum (0.1 ml) obtained from an individual suffering from gastroenteritis was added. to each tube and standard saline solution (0.1 ml) to the control tube. Leave the tube at room temperature for 1 hour, then
Centrifuge at 12000g for 1 hour. The supernatant is discarded and the crystal globules are neutrally stained with phosphotungstate in the usual manner. When examined under an electron microscope, small circular subunit aggregates in complex with antibodies were seen, proving that the patient's gastroenteritis was caused by rotavirus.

Example 3 Diagnosis by immunodiffusion A blood sample is taken from a patient suffering from gastroenteritis and serum is made from it.

Prepare agar consisting of 0.9% w/v agar in TRIS/EDTA buffer and use a portion (2 ml) to cover the slide.

Holes are made in the gel using a template with two rows of 3 mm holes spaced 3 mm apart. The first row of holes is filled with the subunit concentrate prepared in Example 1, and the other row of holes is filled with serum from a patient suffering from gastroenteritis. Once filled, the gel is left at room temperature overnight.

Examination of the gel the next day reveals that a line of precipitin has formed in the gel between the well containing the subunit concentrate and the well containing the patient's serum; this may be due to the patient's condition. This indicates that the gastroenteritis in patients is caused by rotavirus.

Example 4 Diagnosis by complement fixation test Human serum to be tested for the presence of antibodies against rotavirus was first diluted 1:2 with veronal buffered saline (VBS) and kept on a water bath maintained at 56°C for 30 minutes. Heat inactivate. These sera were then passed through a 0.025ml "U" bottom microtitration plate.
Titrate to a 1:2 to 1:256 dilution with 1:256 volume of VBS. Each serum is titrated in two parallel columns. To the first dilution series, add a 0.025 ml volume of rotavirus subunit preparation diluted 1:8 in VBS. The second dilution series is used as an antiserum control, with 0.025 ml VBS added to the serum instead of antigen. Antigen controls were also prepared without the addition of test serum.
Includes antigen added to 0.025 volume of VBS.

Complement kept at -70°C is diluted 1:50 in VBS and a volume of 0.025 ml is added to all wells. Complement control is added in the usual manner. The plate is then covered and left overnight at 4°C.

The next day, antiserum against sheep red blood cells, i.e., hemolyzed serum kept at -20°C, was added to VBS at 1:1.
Dilute from 100 to 1:800 and incubate at 37°C for 30 minutes with an equal volume of 4% sheep red blood cells to form a hemolytic system. At the same time, the plates are moved to 37°C so all components of the system are at the same temperature. Add a 0.025 ml volume of hemolysis system to each well and shake the plate gently. Incubate the plate for an additional 1/2 hour at 37°C, shaking at 15 minute intervals. It is then returned to 4°C for 2-3 hours before being read.

Lysis of red blood cells indicates the presence of antibodies against rotavirus in the serum sample tested.

Example 5 Preparation of antiserum against rotavirus inner capsid subunit The rotavirus inner capsid subunit preparation (2 ml) was mixed with an equal volume of Freund's complete supplement and divided into two aliquots of 2 ml each. One aliquot is injected subcutaneously into two guinea pigs. After 21 days, a second aliquot is administered to the guinea pigs in the same manner. Thirty-four days after the first dose, the guinea pigs are bled and the blood is allowed to clot. The fluid collected after coagulation becomes an antiserum against rotavirus inner capsid subunits.

Claims (1)

[Scope of Claims] 1. A rotavirus inner capsid subunit preparation consisting of a rotavirus inner capsid subunit having a circular shape and a diameter smaller than 2 nm and substantially free of intact virus particles and fragments thereof. . 2. A method for producing a rotavirus inner capsid subunit preparation consisting of a rotavirus inner capsid subunit having a circular shape and a diameter smaller than 2 nm, which does not actually contain intact virus particles or fragments thereof, and hand,
Suspend a fecal sample from a rotavirus-infected mammal and hold this suspension for at least 20 minutes.
A manufacturing method comprising collecting and concentrating subunits from a suspension by centrifugation and/or filtration while maintaining the temperature at 30 to 45°C. 3. The method according to claim 2, wherein feces are suspended in a buffer solution at pH 5 to 9. 4. The method according to claim 2 or 3, wherein the suspension is kept at a temperature of approximately 37°C. 5. Removal of subunits from suspension by centrifugation at a speed sufficient to precipitate all virus particles and fragments, but insufficient to precipitate the subunits. The method described in any one of the above. 6. A method according to any one of claims 2 to 5, wherein the initial fecal suspension is incubated with proteolytic enzymes. 7. The method according to claim 6, wherein the protease is trypsin or papain.