JP2008169142A - Production method of antibody against food poisoning bacteria - Google Patents

Abstract

A method for producing a large amount and homogeneous antibodies against food poisoning bacteria using birds having higher immunity than mammals, a diagnostic agent for food poisoning bacteria using the antibody, and further useful for the prevention and treatment of food poisoning, Provision of an edible composition that can also be applied to chicken allergy patients.
A method for producing an antibody against food poisoning bacteria, in which food poisoning bacteria are ingested into ostriches and IgY antibodies are extracted from the eggs. A diagnostic kit for food poisoning bacteria using the antibody. Furthermore, food poisoning bacteria specific antibody content using at least a part of ostrich eggs.
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Description

  The present invention relates to a method for producing an antibody against food poisoning bacteria. More specifically, the present invention relates to a method for producing antibodies against food poisoning bacteria from eggs using birds, particularly ostriches.

  Birds are known to have much higher immunity than mammals. This is said to be because birds have undergone a completely different evolutionary process from mammals. In addition, the antibodies possessed by avian females are excreted out of the body as eggs, so they are also used for the production of antibodies.

  Chickens are animals that have been studied and used in practice for the production of such antibodies. For example, an antigen is inoculated into a chicken and an antibody specific for the antigen is formed in the chicken. A method for obtaining a large amount of a material containing a specific antibody from the whole egg, egg yolk or egg white of the egg laid by the chicken after the antibody is formed is disclosed (see Patent Document 1).

However, it was considered that there was a problem with mass productivity in chicken eggs, and an antibody production method with improved mass productivity was proposed. Patent Document 2 discloses a technique in which E. coli, Helicobacter pylori, and Salmonella are antigenized and a young chick is inoculated with three kinds of antigens simultaneously so that an anti-pathogenic bacterial antibody coexists in one egg. Yes. That is, a chicken immunized with a plurality of antigens is made to produce a plurality of antibodies, and a plurality of antibodies are obtained from one chicken egg.
Japanese Patent Application No. 62-215534 JP-T-2004-517115

  A chicken has a small egg, and it is necessary to use a large number of chickens if it produces antibodies in large quantities. Different chickens have different immunity and different antibodies are produced. Therefore, even if the same antigen is used, if the immunized individuals are different, they are not exactly the same. Therefore, the obtained antibody has a problem that lot differences due to individual differences occur. When using antibodies, it is desirable that they are as uniform as possible with no difference between lots.

  In addition, since there are relatively many people who are allergic to chicken eggs, there is a problem that when taking a specific antibody-containing substance obtained from chicken eggs orally and aiming at preventive or therapeutic effects, attention must be paid to allergic reactions. is there.

  The present invention has been conceived for solving such problems. In the present invention, an ostrich (Struthio camelus) is used as a bird, and an antibody is obtained from the egg. Further, a specific antibody-containing product is obtained from the egg.

  Ostrich eggs are approximately 20-30 times that of chicken eggs, and the amount of antibody that can be obtained from the eggs is equivalent. Therefore, a large amount of antibody can be obtained from one ostrich, which means that the difference between lots can be reduced.

  In addition, ostrich eggs are less allergic than chicken eggs. Therefore, when an antibody-containing product obtained from an egg is orally ingested to achieve a preventive or therapeutic effect, an effect of easily avoiding the risk of an allergic reaction is obtained.

  When an ostrich is immunized with an antigen, specific antibodies are produced in the blood, of which IgY moves to the yolk and is excreted simultaneously with egg laying. If IgY is extracted from this egg yolk, a large amount of a specific and highly sensitive antibody can be obtained, which can be used as a diagnostic / testing agent or therapeutic agent. It is possible to purify 2 to 4 g of IgY from one egg yolk and obtain 400 g of antibody per year from one ostrich. In other words, it is possible to produce a diagnostic kit with a small lot difference of about 40 million or more in one bird. If a large number of ostriches are used, an enormous amount of antibody can be produced, which is useful for industrial use.

  Here, an antibody against Salmonella Enteritidis (hereinafter referred to as SE), which is famous as a food poisoning fungus, was produced in ostrich egg yolk.

  A commercially available SE antigen vaccine (trade name: Layer Mun SE) (Daiichi Pharmaceutical Co., Ltd.) (1 ml) was immunized biweekly (intramuscular injection) to laying ostriches, and yolks were collected at 4 weeks from the first immunization. Purification of egg yolk is performed as follows.

  First, 5 times the amount of TBS (20 mM Tris-HCl, 0.15 M NaCl, 0.5% NaN3) and the same amount of 10% dextran sulfate / TBS are added to the yolk and stirred for 20 minutes.

  Next, 1MCaCl2 / TBS is added in the same amount as egg yolk, stirred and allowed to stand for 12 hours. Thereafter, the supernatant is recovered by centrifugation at 15000 rpm for 20 minutes.

  Further, ammonium sulfate is added so that the final concentration is 40%, and the mixture is allowed to stand at 4 ° C. for 12 hours. After standing, the mixture is centrifuged at 15000 rpm for 20 minutes to collect the precipitate. Finally, it is resuspended in the same amount of TBS as egg yolk and dialyzed with TBS. This process enabled the recovery of 90% or higher purity IgY. 2 to 4 g of IgY could be purified from one egg yolk.

  FIG. 1 is a photograph of the results of examining the presence of antibodies using an agar gel precipitation reaction on the yolk extract of SE non-inoculated (1) and inoculated ostrich (4 weeks after immunization) (4). . The center of the photograph (A) is the result of the influenza virus HA antigen used as the antigen. When the antigen is present, a white sedimentation line appears.

  In FIG. 1, 1 is a phosphate buffer (PBS), 2 is an egg yolk of an SE non-inoculated ostrich, 3 to 6 are SE-inoculated ostriches (4 weeks after immunization) and 4 yolk extracts are added. In PBS and non-inoculated ostrich, egg yolk does not show a white sedimentation line and it can be concluded that no antibody is present. On the other hand, in the case of 4 inoculated ostriches, white sedimentation appeared. Therefore, the presence of the antibody could be confirmed in all ostrich eggs inoculated with SE.

  Since about 100 eggs are obtained from one ostrich female during the spawning season from April to September, a maximum of about 400 g of antibody (IgY) can be obtained. Since antibodies obtained from the same individual are the same antibody even if the egg changes, it is possible to produce a large amount of antibodies with no difference between lots. Further, the antibody obtained here can be purified and applied as a therapeutic agent for Salmonella-infected patients (food poisoning).

  In addition, even if egg yolk is used as a food containing a specific antibody without purifying the antibody, it can be applied for prevention and treatment of Salmonella infection. In this case, it may be used as it is, but it may be used after powder drying. In particular, ostrich eggs are extremely low in egg allergy, so it can be taken even in chicken allergy patients.

  Furthermore, if egg yolk powder is contained in the laying hen's feed, it is possible to prevent the Salmonella infection of the chicken and to produce a salmonella-free chicken egg.

Photograph of the result of examining the presence of antibody using agar gel precipitation reaction

Claims (4)

Inoculating avian females with food poisoning bacteria;
A method for producing an antibody against food poisoning bacteria, comprising the step of extracting IgY from the yolk of eggs laid by an individual inoculated with said food poisoning bacteria. The method for producing antibodies against food poisoning bacteria according to claim 1, wherein the birds are ostriches. A diagnostic kit using the antibody obtained according to claim 1. A specific antibody-containing product comprising at least a part of eggs laid by an individual who inoculates an ostrich female with a food-poisoning bacterium and ingested the food-poisoning bacterium.