JPH06308128A - Method for immunoassay of potavirus and measuring reagent - Google Patents

Abstract

PURPOSE:To make reliable inspections without a nonspecific reaction by using an antibody originating from bird's eggs which specifically react to the surface protein of rotavirus. CONSTITUTION:The specific antibody titer of eggs produced by immunized birds, particularly, the specific antibody titer of the egg yolk is measured according to an ELISA method, so that an egg with high antibody titer is selected. An antibody to anti-rotavirus is included especially in the water-soluble protein components of the egg yolk. The antibody is separated and purified. In order to separate and purity the antibody, an aqueous solution of lambda-carrageenan is added to precipitate lipoprotein, and the water-soluble protein is obtained from the supernatant. This method is easy and economical. The protein solution is processed in a column thereby to remove impurities. The solution is dialyzed after the salting out, whereby the antibody to anti-rotavirus is obtained. The antibody specifically reacts to rotavirus, hardly bringing about a nonspecific reaction. When the antibody is frozen and dried, the antibody is changed to powders which can be preserved.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological assay method for rotavirus and a reagent for assay, which utilizes an avian egg-derived antibody as an anti-rotavirus surface protein antibody.

[0002]

Rotavirus is a major causative virus of diarrheal disease in infants in winter. To make sure diagnosis of diarrheal disease caused by rotavirus, for example,
There were a method to examine the presence or absence of virus particles in the feces of patients and a method to isolate rotavirus using an electron microscope.
However, the diagnosis by these methods has a drawback that a special device such as an electron microscope is required and it takes time to obtain a result. Furthermore, since many patients with diarrhea caused by rotavirus frequently occur in underdeveloped countries and developing countries, the method using a special device has not been practical. Another way to detect rotavirus is:
There is a method using a latex reagent using an antibody or an EIA reagent. Although these immunological measurements of rotavirus are excellent in terms of speed and quantitativeness, it is necessary to use an anti-rotavirus antibody that specifically reacts with the surface antigen of rotavirus. Antibodies from mammals such as rabbits and goats have been widely used as antibodies.

[0003]

However, the antisera of mammals such as rabbits and goats sometimes cause nonspecific reaction with components of human feces, and there is a problem that the test results are unreliable. . Also, to obtain antiserum,
The immunized mammal needs to be killed. Therefore, it has been desired to find an anti-rotavirus antibody other than rabbit and goat antisera that can be used in immunological measurement of rotavirus. The present inventor has conducted intensive studies to obtain a more preferable anti-rotavirus antibody, and has found that the use of an anti-rotavirus antibody obtained from avian egg (especially egg yolk) can solve the above-mentioned drawbacks. The present invention is based on these findings.

[0004]

Therefore, the present invention relates to an immunological assay method for rotavirus, which comprises using an antibody derived from an avian egg that specifically reacts with a surface protein of rotavirus. Furthermore, the present invention relates to an immunoassay reagent for rotavirus, which comprises an avian egg-derived antibody that specifically reacts with a surface protein of rotavirus.

The present invention uses an antibody derived from avian egg as an anti-rotavirus antibody instead of using a mammalian antiserum as an anti-rotavirus antibody in the conventional method in the immunological measurement of rotavirus. Unlike the conventional method only in the point of use, the conventional rotavirus measurement technique can be applied as it is in other points.

In the present invention, eggs of any birds (eg, chickens, ducks, quails, etc.) can be used. However, chickens are easy to manage in large numbers, and thus eggs are preferred. The anti-rotavirus antibody derived from an avian egg can be obtained by isolating and purifying the egg (particularly, its yolk) laid by a bird immunized with the surface antigen of rotavirus. Here, rotavirus has a structure in which two inner and outer capsid protein layers enclose a core particle containing 11 segmented double-stranded RNAs, and an electron micrograph image of the negatively stained virus is a wheel with a diameter of about 70 nm. It is a pathogenic virus that causes a diarrhea by infecting almost all mammals and birds from mice to humans and usually orally infecting infants and young animals including humans. As the rotavirus surface antigen that can be used as an immunogen, the rotavirus particles themselves, or the structural proteins forming the inner and outer capsid protein layers, and further the infectious antigen protein present on the surface layer of virus particles prepared by genetic engineering techniques, Or a fragment thereof can be mentioned.

[0007] Immunization of birds can be carried out by subcutaneously, intracutaneously, intraperitoneally or intravenously administering the purified rotavirus surface antigen, optionally together with an appropriate adjuvant. The specific antibody titer of the egg (particularly, its yolk) laid by the immunized bird is measured by, for example, the ELISA method, and the egg with high antibody titer (particularly, its yolk) is selected. The anti-rotavirus antibody is contained especially in the water-soluble protein component of egg yolk. Therefore, the anti-rotavirus antibody can be obtained by using any known method for separating and purifying a water-soluble protein component from egg yolk. For example, a method of extracting egg yolk with chloroform to obtain the remaining liquid component (T. Kramer et al., Immunology;
19, 157, 1970); polyethylene glycol is added to the yolk liquid to precipitate the yolk lipoprotein, and water-soluble protein is obtained from the supernatant; or, carrageenan or xanthan gum is added to the yolk liquid to precipitate the yolk lipoprotein. , Method for obtaining water-soluble protein from supernatant (H. Hatta et al., Agric. Biol. Che.
m. 55, 2141, 1991); and a method of adding a water-soluble cellulose derivative or the like to egg yolk liquid to precipitate egg yolk lipoprotein and obtaining a water-soluble protein from the supernatant (JP-A-2-188533). it can. In particular, a method of adding a λ-carrageenan aqueous solution to precipitate a lipoprotein and obtaining a water-soluble protein from the supernatant is preferable because the operation is easy and economical.

[0008] Next, the water-soluble protein-containing solution is subjected to a column treatment (for example, DEAE-Sephacel column) to remove impurities, and then a salting-out operation is performed, and the salted-out product is dialyzed to remove anti-rota Viral antibodies can be obtained. This anti-rotavirus antibody specifically reacts with rotavirus, and almost no nonspecific reaction occurs. The IgG-type anti-rotavirus antibody thus obtained can be lyophilized and stored in powder form.

According to the present invention, the above-mentioned anti-rotavirus antibody can be used to measure, ie, detect and / or quantify the rotavirus in feces. The test sample of the present invention is not particularly limited as long as it may contain rotavirus, and is, for example, feces.

According to the present invention, for example, an anti-rotavirus antibody immobilized on a carrier and a rotavirus (antigen) in a test sample cause a specific antigen-antibody reaction, and the amount of rotavirus in the test sample is increased. Agglomeration occurs in proportion to. By measuring the degree of this aggregation, the amount of rotavirus in the test sample can be determined. Therefore, the present invention can be directly applied to the known latex agglomeration method and the like. Examples of the measuring method include visual measurement on a slide plate and an optical method (such as photometry by near infrared rays or ultraviolet-visible partial photometric method). In the present invention, the anti-rotavirus antibody can be used by adsorbing it on a commonly used carrier. The type and particle shape of the carrier are not particularly limited,
For example, synthetic polymer beads such as polystyrene or acrylic having a particle shape of 0.05 to 1.0 μm can be used.

The measurement according to the present invention can be carried out individually by hand, but it is also possible to use an automatic analyzer as in the conventional method, and it can be carried out by various ordinary equipments conventionally used. You can That is, when a test sample and an anti-rotavirus antibody immobilized on a carrier are mixed and aggregation occurs in proportion to the amount of rotavirus present in the test sample, the formation of aggregates is visually measured or transmitted. It is only necessary to spectroscopically measure the change in the light amount. In the latter case, the range of 340 nm to 900 nm can be used as the photometric wavelength.

The assay reagent of the present invention contains the above-mentioned avian egg-derived anti-rotavirus antibody in place of the antiserum of goat, rabbit, etc. in the conventional rotavirus assay reagent. Then, there is no difference from the composition of the conventional reagent for measuring rotavirus. Therefore, the measuring reagent of the present invention can contain a commonly used sensitizer, buffer and / or preservative in addition to the anti-rotavirus antibody. Examples of the sensitizer include polyethylene glycol or a surfactant (for example, a nonionic surfactant such as TWEEN-20).
Examples of the buffer solution include phosphate buffer solution, Tris buffer solution, borate buffer solution and the like. Examples of the preservative include sodium azide and the like.

[0013]

[Function] In the conventional rotavirus measurement, as an anti-rotavirus antibody, artificially immunized mice, rabbits,
Mammalian antisera such as goats, guinea pigs or horses were used. These mammals are developmentally closely related to humans and have various immunological commonalities. Therefore, these serum proteins have common antigens with each other, and also have commonities with the complement system, coagulation system, rheumatoid factor and the like. In addition, these mammals and humans have many similarities in infectious microorganisms and normal flora. Therefore, these mammals contain, in addition to the immunogen (rotavirus), an antibody having the same specificity as a human antibody as a normal antibody, and as a result, an antigen-antibody reaction occurs due to the involvement of an antigen other than the target antigen (rotavirus). However, a non-specific reaction appears when viewed from the target antigen.

On the other hand, birds are phylogenetically located between fish and mammals and are more distant from humans than mammals, but their immune mechanisms are sufficiently advanced. Therefore, by using an avian antibody that is taxonomically distant from humans, the occurrence of nonspecific reaction is prevented or reduced, only the desired antigen-antibody reaction appears, and more accurate measurement and diagnosis can be performed. Further, in birds, since the antibody can be separated and purified from the egg, the antibody can be obtained without killing the individual.

[0015]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1: Preparation of anti-rotavirus antibody (1) Antigen Culture of rotavirus as an antigen was performed using monkey kidney-derived cells (MA104 strain) as host cells. That is, MA104 cells were placed in roller bottles containing 10% fetal calf serum containing minimum essential medium (MEM).
The cells were cultivated by rotation until a monolayer was prepared, and the medium was replaced with a MEM medium containing no fetal bovine serum to infect human rotavirus Wa strain serotype I. After that, trypsin added M
The culture was continued for about 2 to 4 days until cell degeneration was observed in the EM medium. The rotavirus culture solution was centrifuged (10,000
The cell-derived residue was removed by rotation (30 minutes), and the resulting supernatant (rotavirus culture supernatant) was further centrifuged (40,
000 rotations, 2 hours) to precipitate virus particles,
A suspension of the precipitate in 20 mM phosphate buffer was used as a purified rotavirus antigen in the following experiments.

(2) Immunization The purified rotavirus antigen was diluted with physiological phosphate buffer to adjust its infectious titer to 10 ⁷ FCFU (fluorescent antibody positive focus unit) / ml, and 1 ml of the diluted solution was used. After mixing with 1 ml of complete Freund's adjuvant, it was injected into the muscles of laying hens for immunization. Immunization was performed once every two weeks for a total of four times.

(3) Preparation of anti-rotavirus antibody After adding an equal amount of deionized water to the yolk of the egg laid by the immunized chicken and homogenizing with a homomixer, an aqueous solution of λ-carrageenan (8 mg) equivalent to the yolk (1 mg) / Ml) was added and mixed. The resulting lipoprotein aggregates were removed by centrifugation, and the resulting centrifugation supernatant was adjusted to a final concentration of 20 mM with phosphate 2
By dissolving sodium and adjusting the pH to 8.0 with 3N hydrochloric acid, and loading it on a DEAE-Sephacel column previously equilibrated with 20 mM phosphate buffer (pH 8.0), and washing the column with the equilibration buffer. After removing the non-adsorbed protein, the protein adsorbed on DEAE-Sephacel was eluted with 200 mM phosphate buffer (pH 8.0). 15% sodium sulfate in the obtained eluate
(W / v), and the resulting precipitate was collected by centrifugation and dissolved in 10 mM phosphate buffer (pH 8.0).
This salting-out operation with sodium sulfate was repeated 3 times, and finally dissolved in 10 mM phosphate buffer (pH 8.0), dialyzed against the same buffer, freeze-dried, and a specific egg-derived antibody against rotavirus. (Hereinafter, anti-Rota-IgY
Referred to as). The purity of the obtained antibody had a single peak by gel filtration.

Example 2: Anti-Rota-IgY sensitized latex
Preparation (1) Anti-Rota-IgY solution 0.1 M Tris-HCl buffer (pH) was added to freeze-dried anti-Rota-IgY powder to a concentration of 10.0 mg / ml.
8.0) (hereinafter referred to as 0.1 M Tris buffer) to give an anti-rota-IgY-containing solution. In addition, anti-rota-I
The concentration (mg / ml) of gY was determined by dividing the absorbance at 280 nm of the contained liquid by 1.35. (2) Antibody-sensitized 10% latex suspension [IMMUTEX CA-P
S: Nippon Synthetic Rubber Co., Ltd.] was added with a 4-fold amount of 0.1 M Tris buffer to obtain a 2% latex suspension. An equal amount of anti-rota-IgY-containing solution was added to this, and after stirring at room temperature for 5 hours, the supernatant was removed by centrifugation (18,000 rpm, 15 minutes). A 0.5% bovine serum albumin (hereinafter referred to as BSA) solution was added to the precipitated latex so as to be a 2% suspension, the mixture was stirred at room temperature for 30 minutes, and then the supernatant was removed by centrifugation. Distilled water was added to the precipitate, the mixture was stirred at room temperature for another 30 minutes, and the supernatant was removed by centrifugation.
A 50 mM borate buffer solution (pH 8.5) containing 0.05% BSA (hereinafter referred to as borate buffer solution) was added to a concentration of 5% and mixed uniformly. The obtained mixed solution was used in the following experiment as a reagent for detecting rotavirus by the latex agglutination method.

Example 3: Preparation of sensitized latex for control Using an egg-derived antibody (hereinafter referred to as IgY) prepared in the same manner as in Example 1 from eggs of non-immunized chickens,
The latex was sensitized under the conditions described in Example 2 and used as the control IgY-sensitized latex.

Example 4: Anti-Rota-IgY sensitized latex
Culture supernatants rotavirus prepared in reactive Example 1 (1) of the culture supernatant of Evaluation (1) Rotavirus,
Latex reagent 3 prepared in Example 2 was diluted with physiological phosphate buffer 4- to 64-fold, and 35 μl of each diluted solution.
5 μl was added and mixed. A sample in which latex agglutination occurred after rocking for 3 minutes was determined to be rotavirus-positive.
The results are shown in Table 1. In Table 1, + indicates positive and-indicates negative.

[0021]

[Table 1]

In the rotavirus culture supernatant, latex agglutination was observed even when diluted up to 16-fold. On the other hand, no aggregation was observed in the results of the same experiment using the control IgY-sensitized latex prepared in Example 3. (2) Reactivity with specimen confirmed presence of rotavirus by electron microscope After diarrhea patient stool specimen with pathogen virus identified by electron microscope was suspended in physiological phosphate buffer to a concentration of 10% After centrifugation (3,000 rotations, 10 minutes), the precipitate was removed to prepare a sample extract. 35 μl of this extract
Anti-Rota-IgY sensitized latex prepared in Example 2 and control IgY sensitized latex prepared in Example 3 3 each
After adding 5 μl and mixing and shaking for 3 minutes, the presence or absence of latex aggregation was determined. Judgment was made such that a sample that aggregated only with the anti-rota-IgY-sensitized latex was positive, and a sample that did not aggregate with either latex was negative. Table 2 showing the results
As is clear from the above, all the samples in which the presence of rotavirus was confirmed by electron microscopy were positive. On the other hand, one of the causative viruses for diarrhea, Small Ro
und Structured Virus (hereinafter referred to as S
RV) and a sample in which the presence of adenovirus was confirmed by an electron microscope were both negative without causing aggregation.

[0023]

[Table 2] The above results indicate that the anti-rota-IgY sensitized latex can be used as a rotavirus test reagent.

Example 5: Anti-Rota-IgY Sensitized Latex
A specificity (1) Main pathogens cultured bacterial diarrhea of pathogenic E.coli pathogenic E. coli [Esch
erichia coli) 477-4 (ST); obtained from the Institute for Microbial Diseases, Osaka University] (hereinafter, E. coli
477-4) and Escherichia coli 591-2 (LT) [obtained from the Institute for Microbial Diseases, Osaka University] (hereinafter E. coli).
591-2) and 2 strains of heart infusion liquid medium (hereinafter referred to as HI broth) 30
ml was inoculated. 17 hours, 41 hours, 65 hours, and 137 hours after the start of shaking culture, the whole amount of the culture solution was collected, and immediately centrifuged to separate the cells and the supernatant. The obtained supernatant was used for the experiment after removing the bacterial cells by aseptic filtration. The cells were suspended in physiological saline, washed 3 times by centrifugation, and then suspended in physiological phosphate buffer to make the total volume 3 ml. (2) Reaction of Pathogenic Escherichia coli with Anti-Rota-IgY Sensitized Latex Under the same conditions as in Example 4, cross-reactivity between anti-rota-IgY sensitized latex and pathogenic E. coli cells or culture supernatant was examined. It was As is clear from Table 3 showing the results, the anti-rota-IgY-sensitized latex did not react with the cells of pathogenic Escherichia coli or the culture supernatant. As a control,
HI broth (negative control) and the rotavirus culture supernatant of Example 1 (1) (positive control) were used. From the results of Examples 4 and 5, it can be seen that the anti-rota-IgY antibody is a highly specific antibody that does not cross-react with the causative microorganisms of diarrhea such as SRV and pathogenic Escherichia coli.

[0025]

[Table 3]

Example 6: Correlation with EIA method To examine whether or not the anti-rota-IgY according to the present invention specifically detects the surface antigen of rotavirus in a test sample, patient specimen 97 was used. About the example, a correlation experiment was performed between the method of the present invention and the rotavirus detection method by the ELISA method in which a biotin-labeled antibody and peroxidase-labeled streptavidin were combined. The results are shown in Table 4. ELI
The 52 negative samples by the SA method were completely in agreement with the method of the present invention, but among the 45 positive samples by the ELISA method, 4 samples having a significantly small antigen amount were negative by the method of the present invention.

[0027]

[Table 4] As is clear from the results in Table 4, the method of the present invention is
It showed a high correlation with Method A. Therefore, the measuring method of the present invention can specifically detect rotavirus in a clinical test sample.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, as an anti-rotavirus antibody, an avian egg-derived antibody is used in place of a mammalian antiserum, so that it is possible to suppress the occurrence of non-specific reactions, and more accurate than conventional methods. High and accurate rotavirus measurement can be performed. Also, it is not necessary to kill the immunized animal to obtain the antibody.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroshi Kita 1-11-4 Higashikanda, Chiyoda-ku, Tokyo Within Yatron Co., Ltd. (72) Inventor, Hitoshi Hatta 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Solarization Gaku Co., Ltd. (72) Inventor Ken Tsuda 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd. (72) Inventor Yu Fujiki 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd. (72) Inventor Kim Takehisa 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Solar Engineering Co., Ltd.

Claims (2)

[Claims] 1. An avian egg-derived antibody that specifically reacts with a surface protein of rotavirus is used.
Rotavirus immunological assay. 2. A reagent for immunological measurement of rotavirus, comprising an avian egg-derived antibody that specifically reacts with a surface protein of rotavirus.