JPH0253737A - Production of polyfunctional specific antibody - Google Patents

Abstract

PURPOSE:To immunize a laying hen against a complex antigen of infectious diarrhea and to simultaneously form a specific antibody corresponding to each of the complex antigen in vitellus of egg laid by the hen. CONSTITUTION:A pathogenic virus of infectious diarrhea such as rotavirus, enterogenous adenovirus, nororqvirus or calcivirus and an intestinal bacterium such as pathogenic Escherichia coli, vibrio of enteritis, salmonella or Clostridium botulinum are multiplied and cultivated by a proper animal cell and a medium, respectively, inactivated by using formalin, etc., by a conventional procedure, respectively and blended to give a complex antigen of infectious diarrhea. A laying hen is immunized against the complex antigen (at interval of once in a week or two weeks and three time or more in total) to continuously form a polyfunctional specific antibody having high antibody value during egg laying period of hen. Infectious diarrhea can be simply and safely prevented by eating the hen's egg as it is or orally administering an antibody separated from the vitellus and purified.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a multifunctional specific antibody for the purpose of preventing infectious diarrheal diseases, and more particularly to one or more pathogenic viruses of infectious diarrheal diseases and A method for producing multifunctional specific antibodies, which comprises immunizing laying hens using a mixture of one or more types of pathogenic bacteria as antigens, and simultaneously producing antibodies corresponding to each in the water-soluble protein fraction of egg yolks. Regarding.

[Conventional technology]

Infectious diarrhea is a relatively common disease encountered on a daily basis.
It occurs when a pathogenic virus or bacteria infects the gastrointestinal tract. However, diarrhea caused by gastrointestinal infection often becomes a serious problem for infants, young livestock whose immune system is not fully developed, and the elderly whose immune system is weakened. Currently, 50 million infants and children die annually from infectious diarrhea, mainly in developing countries.
Controlling the disease is an urgent issue and has become a global project of the World Health Organization (WHO) and other organizations.

Animals, including humans, have foreign substances (bacteria, bacteria, etc.) that have invaded their bodies.
It produces specific antibodies against antigens (viruses, foreign proteins, etc.) and inactivates or detoxifies foreign antigens. Most specific antibodies are produced in autologous body fluids, especially blood. In contrast, infectious diarrhea occurs when pathogenic viruses and bacteria that have orally invaded wall cells in the gastrointestinal tract, which are outside our bodies, become attached and infected. Therefore, it is said that it is difficult for the body's specific antibodies to be used effectively against infectious diarrhea. Especially in infants, the elderly, and young livestock with low autoimmunity, the production of specific antibodies is insufficient, so it is difficult to prevent infectious diarrhea with the specific antibodies produced by themselves. be.

However, recently, it has been pointed out that oral administration of specific antibodies produced in xenobiotic animals is effective in preventing this type of infectious diarrhea [CurtisR, Bartz et al.: Th
e Journalof'Inf'ectiou
s Diseasesvol, 142,439
(1980), R.

bert H, Yolken et al.: Pediatric
s vol, 81,291 (198B).

In order to prevent infection of infectious diarrhea by orally administering specific antibodies against pathogenic viruses and pathogenic bacteria, it is first possible to produce the specific antibodies at any time and in a planned manner. Must.

Typically, specific antibodies are isolated from the blood of an immunized mammal (rabbit, goat, horse, etc.) that has been killed. Therefore, mass production of the antibody necessarily requires sacrificing a large number of animals. Mammals used for immunization are not only expensive;
The blood may contain viruses, so it is not necessarily hygienically clean, requires complicated handling and care, and the yield of antibodies is not necessarily good.

On the other hand, chickens also respond to invading foreign antigens by producing specific antibodies in their own blood. Moreover, as mentioned above, in order to protect the sub-chicken organisms from attack by foreign antigens, the specific antibodies are transferred from the parent chicken blood and exist in the egg yolk. This fact was first discovered by Batterson et al. in 1962 [Patterson, R. et al.: J. Im.
munol, 89,272. (1962) Ko.

Since then, it has been reported that specific antibodies against various antigens can be obtained in the yolk of chicken eggs. However, all previous research reports have aimed to use tun antigens to produce antibodies (monofunctional antibodies) that specifically bind to the antigens in chicken egg yolks, and use them, for example, as clinical test reagents. It's just that.

[Problem to be solved by the invention]

Various infectious diarrheal diseases are caused by multiple factors including pathogenic viruses and pathogenic bacteria. Therefore, in order to prevent infectious diarrhea by oral passive immunization using specific antibodies, a mixture of specific antibodies that neutralize the infectivity of various pathogenic viruses and pathogenic bacteria, that is, a multifunctional It is practical to prepare and utilize specific antibodies. However, at present, no attempt has been made to actively prepare and apply multifunctional specific antibodies for the purpose of preventing such infectious diarrhea, at least as far as the field concerned is concerned.

In the case of chickens, it is generally known that increasing the number of virus strains used as antigens makes it difficult to increase the antibody titer against each strain. This is an obstacle in producing multifunctional specific antibodies using chicken eggs for the purpose of preventing infectious diarrhea.

[Means to solve the problem]

The inventor's basic research has shown that 1) unlike mammals, egg-laying hens can withstand immunization injections of many types and large amounts of antigens, and 2) antibodies produced in body fluids are concentrated and transferred to the yolks of chicken eggs fairly rapidly. 3) It was confirmed and clarified that these functions are maintained at the same level for a long period of more than half a year by repeated administration of antigen. In other words, unlike viviparous mammals, oviparous birds have the characteristic of imparting a significant amount of antibodies to their eggs in order to protect the next generation of oviparous life, and the present invention was successful based on this principle. can.

The present inventor aims to industrially produce multifunctional specific antibodies with high antibody titers that specifically bind to viruses and bacteria that cause infectious diarrhea and neutralize their respective infectivity. After conducting extensive research for this purpose, we discovered the following facts and completed the present invention.

1) Immunize laying hens with antigens that cause infectious diarrhea,
The specific antibody obtained from the chicken egg yolk showed approximately the same antibody titer as the specific antibody obtained from the blood of a similarly immunized rabbit.

2) Shock death due to booster immunization was common in rabbits, but not at all in egg-laying hens.

3) The amount of specific antibody obtained from the whole blood of one rabbit was approximately the same as that obtained from the yolks of about 10 chicken eggs.

4) Even if laying hens are immunized with antigens that cause infectious diarrhea, there is no effect on their egg production rate, and the average amount of immunization per chicken is
She laid 254 eggs a year.

5) If the booster immunization was repeated at certain regular intervals, the antibody titer of the specific antibody in the egg yolk of the chicken was maintained at a high level throughout the egg-laying period of the chicken.

6) If egg-laying hens are immunized with a diarrhea complex antigen containing one or more infectious diarrhea viruses and pathogenic enterobacteria isolated using blood agar medium using hemolytic rings as an indicator, only infectious diarrhea can be detected. Specific antibodies with higher titers were obtained in the egg yolks for each virus than those immunized with the disease virus alone. At the same time, specific antibodies against the pathogenic intestinal bacteria used were also obtained in the egg yolk.

The present inventor completed the present invention as a result of detailed research into the biological characteristics of antibody production in laying hens as described above. That is, the present invention immunizes laying hens with an infectious diarrhea complex antigen consisting of one or more pathogenic viruses of infectious diarrhea and one or more pathogenic enterobacteria; The present invention relates to a method for producing multifunctional specific antibodies characterized by simultaneously producing specific antibodies corresponding to each of the complex antigens in egg yolk.

The pathogenic viruses that cause infectious diarrhea referred to in the present invention are viruses that cause diarrhea in humans and domestic animals, such as rotavirus, enteric adenovirus, norouvirus, calicivirus, astrovirus, coronavirus, and predavirus. On the other hand, pathogenic enterobacteria refers to bacteria that are isolated from the diarrhea side of patients with diarrhea using hemolytic rings on blood agar plates as an indicator, and most of them are usually pathogenic bacteria that cause diarrhea in humans and livestock. Escherichia coli, Vibrio parahaemolyticus, Clostridium perfringens,
These include Campylobacter, Staphylococcus, Salmonella, Clostridium botulinum, and Bacillus cereus.

After culturing these pathogenic viruses and pathogenic enterobacteria of infectious diarrhea in appropriate animal cells and culture media, and inactivating each using formalin etc. by a conventional method,
The antigens are mixed to form the infectious diarrhea complex antigen of the present invention.

Immunization of laying hens using the complex antigen is carried out by a conventional method. That is, any method such as intramuscular injection, subcutaneous injection, or intravenous injection may be used, but it is preferable to use a combination of these methods to produce the multifunctional specific antibody of the present invention.

The amount of immunization can be up to 50 μg of protein per bird or one time in the case of viruses, and up to 1X10'' cells in the case of pathogenic intestinal bacteria, but it is difficult to immunize without affecting the egg production rate. However, it may be difficult to obtain a high antibody titer if the amount of protein is less than 10μg for viruses or less than 1X10' for bacteria, so the immunization dose should be set to 1/2 of each amount. It is desirable to do more than that.

Immunization is performed a total of three or more times at intervals of once every week or every two weeks. The antibody titer in chicken egg yolk usually increases from the second immunization and reaches its maximum value about one week after the third immunization. This highest value persists for about three months thereafter. If the antibody titer decreases after that, if the patient is immunized once again, the antibody titer will recover to almost its previous maximum value about one week later, and this maximum value will continue for about three months. That is, by using chickens, it is possible to produce multifunctional specific antibodies in the yolks of chicken eggs that have a continuous high antibody titer during the egg-laying period of the chickens.

Multifunctional specific antibodies produced using the infectious diarrhea complex antigen of the present invention can be obtained by immunizing chickens using only the pathogenic virus of infectious diarrhea without using pathogenic intestinal bacteria. The antibody titer is higher than that of the obtained antibody. This is a noteworthy fact, and is thought to be because the pathogenic intestinal bacteria used at the same time have some kind of enhancing effect on the production of viral antibodies.

In actual application of the multifunctional specific antibody produced by the present invention that is effective for preventing infectious diarrhea, chicken eggs containing the multifunctional specific antibody may be eaten as they are, or whole egg powder or egg yolk powder may be eaten. It can be used as a raw material, or if necessary, the multifunctional specific antibody can be purified from chicken egg yolk and used for oral administration. The multifunctional specific antibody can be prepared from egg yolk using a natural polysaccharide such as lambda carrageenan as an egg yolk lipoprotein precipitant, and the egg yolk antibody is purified from the centrifuged supernatant (both egg yolk water-soluble proteins). (Patent application number 62-194083) is M
Although R alone is industrially applicable, there are no particular limitations on the manufacturing method.

The present invention will be explained in more detail with reference to Examples below.

〔Example〕

Example 1. Comparison of immunotolerance to crude infectious diarrhea complex antigen in viviparous animals (rabbits) and oviparous animals (laying hens) and the amount of total antibodies obtained. After adding an equal volume of % formalin saline and leaving it overnight, the supernatant was centrifuged (1,000 rpm, 5 minutes) and filtered through a membrane with a pore size of 5 μm to obtain an infectious diarrhea complex antigen solution. The complex antigen is 1m! Human
The sample contained 5.2 x 10' FCFU (fluorescent antibody positive focus units) of 1 Tavirus Wa strain and 2.3 x 10' pathogenic enterobacteriaceae isolated on the basis of hemolytic rings on a blood agar medium. The complex antigen solution was intramuscularly injected into 10 rabbits and 10 egg-laying hens each at 2mff1 once per bird to serve as immunization groups. Also, 0.5 for each two birds.
In the same manner, 2 mρ of % formalin saline was injected intramuscularly to prepare a solution.

Immunization was performed every two weeks for a total of 4 times. For rabbits, blood was collected every two weeks, and whole blood was collected two weeks after the final immunization, and the serum was separated according to a conventional method. The mixed serum was mixed in equal amounts with the neutralizing antibody titer ILJ regular number. did. On the other hand, for laying hens, all the egg yolks of eggs laid every two weeks were collected and homogenized, a portion of which was diluted 10 times with a lambda-carrageenan aqueous solution (1 mg/mρ), and then centrifuged 'a' (3, 00 rpm for 10 minutes), and the supernatant was used as a solution equivalent to a 10-fold dilution of egg yolk. Using the obtained rabbit mixed serum and a solution equivalent to a 10-fold dilution of egg yolk, the neutralizing antibody titer against human rotavirus Wa strain was measured by the following method.

(Measurement of neutralizing antibody titer) A dilution series was prepared for each of the prepared rabbit mixed serum and a solution equivalent to a 10-fold dilution of egg yolk using physiological saline. That 50μ! 100FC of human rotavirus Wa strain
After mixing with 50 μl of virus solution containing FU (fluorescent antibody positive focus! 11th place) and incubating at 37°C for 1 hour, MAIO4 cells (11 kidney cells) IXlo”
Cells/wells were infected. After culturing for 37°CCl2O hours, each FCFU was measured, and the virus 100F
The maximum dilution factor sufficient to inhibit CFU infectivity by 50% was determined and used as the neutralizing antibody titer in serum and egg yolk.

In addition, the amount of antibodies in the mixed rabbit serum was measured using anti-rabbit antibody goat serum, and the amount of antibodies in the egg yolks of chicken eggs was measured by quantitative immunodiffusion method using Wazabe antibody rabbit serum in a conventional manner.

(Results) Comparison of immune tolerance of Una V' and egg-laying hens against the infectious diarrheal complex antigen Of the 10 rabbits used for immunization, one rabbit received shock after the second immunization, and three rabbits received shock after the third immunization. Died. On the other hand, no shock death was observed in the laying hens used for immunization during the immunization period of this experiment.

Compared to viviparous animals (rabbits), oviparous animals (laying hens) are thought to have acquired superior resistance to artificial booster immunization by rapidly transferring antibodies in their blood into the egg yolk.
1III.

Change in neutralizing antibody titer against human rotavirus Wa strain Table 1 shows the change in neutralizing antibody titer in rabbit mixed serum and chicken egg yolk.

The neutralizing antibody titer in chicken egg yolk was found to be equivalent to that in rabbit serum. Regarding laying hens,
As a result of reimmunization when the neutralizing antibody titer decreased (approximately every three months), it was shown that the neutralizing antibody titer in chicken egg yolks recovered after one week.

Table 1. Changes in neutralizing antibody titers in rabbit serum and egg yolk against human rotavirus Wa strain (1 mark indicates week of immunization) Comparison of total antibody amount 15.6 mg of antibody was contained per mj2 of rabbit serum. This experiment In this case, an average of 56 m per rabbit was collected for whole blood collection.
l of serum were obtained (n=6). Therefore, the average amount of antibody obtained from one rabbit is 873.6 mg. on the other hand,
Each chicken egg yolk contained an average of 102 mg of antibody.

In this experiment, the average number of eggs laid by immunized hens was 254 eggs/year (n=10
), and that of control chickens averaged 245 pieces/year (n=2
)Met. Therefore, the average amount of antibody obtained from one immunized chicken was 25.908 g, which corresponded to the amount of antibody obtained from 297 rabbits.

As mentioned above, the neutralizing antibody titer against human rotavirus Wa strain was not significantly different between rabbit serum and chicken egg yolk, and the excellent immune tolerance of laying hens to infectious diarrhea complex antigens was also demonstrated. Since the amount of antibodies equivalent to approximately 30 rabbits can be obtained, it is better to use egg-laying hens and their egg yolks to prepare multifunctional specific antibodies against pathogenic viruses and pathogenic bacteria that cause infectious diarrhea. It has been shown to be superior to using blood.

Example 2. Effects of single and complex antigen administration as immunization conditions on the antibody titer of diarrhea pathogenic virus antibodies produced in chicken egg yolk.Human rotavirus Wa strain cultured in MA104 cells (monkey kidney cells) and 293 cells (embryonic kidney cells).

Enteric adenovirus 41 cultured with transformed cells)
20μg of each strain with virus protein and 2X of pathogenic E. coli cells isolated from stool with watery diarrhea in children
Each laying hen was immunized by intramuscularly injecting 1 m2 of an infectious diarrhea complex antigen solution containing 10' antigens. After the first immunization, 2
The same amount of the complex antigen was boosted every week for a total of 3 immunizations. After separating and homogenizing egg yolks from eggs produced by immunized chickens for 3 months after the final immunization, this egg yolk liquid was diluted 10 times with a lamb goo raginan aqueous solution (1 mg/ml), and then centrifuged (3,00 rpm). , for 10 minutes), and the supernatant was added to the egg yolk.
Antibody titer was measured as a solution equivalent to 0-fold dilution.

As a comparative test, egg-laying hens were immunized under the same conditions as described above with 20 μg of each virus as a protein amount, and 1 mj2 of each solution contained simultaneously and separately. A solution equivalent to a 10-fold dilution of the egg yolk was prepared from the egg yolk.

As a control, 1 mff1 of physiological saline was administered per laying hen under the same conditions, and a solution equivalent to a 10-fold dilution of egg yolk was prepared in the same manner.

(Measurement of neutralizing antibody titer against virus) The method for measuring the neutralizing antibody titer shown in Example 1 was followed. However, for enteric adenovirus 41, 293 cells were used as infected cells.

(Measurement of agglutinating antibody titer against pathogenic Escherichia coli) The bacterial suspension used for immunization was diluted with physiological saline to prepare a suspension with an absorbance (turbidity) of 1.0 at 600 nm. 50 μ of a solution equivalent to a 10-fold dilution of egg yolk diluted to various ratios with physiological saline! After mixing and leaving overnight, the presence or absence of bacterial aggregation on the bottom of the microwell was examined. The agglutinating antibody titer was expressed as the maximum dilution ratio of egg yolk that agglutinated bacteria.

Table 2. Effects of immediate and complex antigen administration as immunization conditions on the antibody titer of diarrheal pathogenic virus antibodies produced in chicken egg yolk (results) As shown in Table 2, human rotavirus Wa strain, enteric adenovirus 41 and pathogenic Antibodies from chicken eggs obtained by immunization with a mixture of E. coli as antigens have higher neutralizing antibody titers against each virus than antibodies from chicken eggs obtained by immunization with each virus alone or individually. was found to be quite large. Of course, since the antigen used to produce the same effect contained pathogenic Escherichia coli, a sufficient agglutination antibody titer against the same bacterium was observed. Therefore, this antibody is considered to be effective as a specific antibody for infectious diarrhea, not only for preventing viral diarrhea but also for preventing diarrhea caused by pathogenic bacteria.

〔Effect of the invention〕

According to the present invention, when producing anti-viral antibodies in chicken eggs using laying hens, by immunizing them with a mixture of pathogenic intestinal bacteria of completely different origins as antigens, the neutralizing antibody titer against the virus is clearly increased. It became possible.

This fact is considered to be an effective method that can naturally be applied to the production of specific antibodies against various antigens in addition to the use of chicken eggs.

Furthermore, according to the present invention, when immunization is performed by mixing or physically immobilizing not only various pathogenic viruses that cause infectious diarrhea but also various pathogenic bacteria, a mixture of specific antibodies that neutralizes their infectivity can be obtained. It has been revealed that multifunctional specific antibodies can be produced in the yolk of chicken eggs. In addition, by using chicken eggs, it is considered possible to produce specific antibodies against viruses and pathogenic bacteria that cause diarrheal diseases for the purpose of preventing this type of infectious diarrheal disease on an industrial scale.

Chicken eggs are a food that humans have eaten since prehistoric times.
In particular, it is known that its protein nutritional value is superior to that of milk. Particularly in Japan, it has become common to eat raw eggs, and in this case, the antibodies in the egg yolk are eaten as food.

The present invention produces multifunctional specific antibodies that are effective in preventing infectious diarrhea in chicken eggs, and administers them orally, either as egg yolks or after separation and purification as necessary. It has the advantage of making it possible to prevent diarrheal diseases.

This invention is part of the research results conducted with the aim of preventing infectious diarrhea through oral passive immunization, but it contributes to the proof of infectious diarrhea, which is currently a widespread problem worldwide. This is thought to provide a means by which this can be done.

Claims (1)

[Claims] A laying hen is immunized with an infectious diarrhea complex antigen consisting of one or more pathogenic viruses of infectious diarrhea and one or more pathogenic intestinal bacteria, and the complex is added to the yolk of eggs produced by the immunized hens. A method for producing a multifunctional specific antibody, characterized by simultaneously producing specific antibodies corresponding to each antigen.