JP2013163662A - Antibody-including pollen allergy protective material - Google Patents

Abstract

Allergy is caused by binding of IgE of mast cells to an antigen allergen and secreting histamine and the like. Therefore, it is considered that allergy can be prevented by preventing the binding between IgE and allergen.
The antibody-containing pollen allergy protective material of the present invention is an antibody-containing pollen allergy protective material comprising an antibody obtained from an egg laid by an ostrich immunized with a pollen allergen. The antibody-containing pollen allergy protective material of the present invention also includes an antibody obtained from an egg laid by an ostrich naturally affected with hay fever.
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Description

  The present invention relates to an antibody that protects against pollen allergy obtained from ostriches.

  In recent years, hay fever is an allergic disease that affects people all over the world including Japan. It causes various symptoms such as tears, runny nose, sneezing, cough, fever, headache, tediousness, etc., and the economic damage due to the decrease in work efficiency is enormous. The cause of hay fever is mainly pollen of cedar and cypress, and a specific protein among them is considered as an allergen. The allergens of cedar pollen are Cryj1 and Cryj2, and the allergens of cypress pollen are Chao1 and Chao2. In fact, it has been demonstrated that antibodies to one or more of these allergens are produced in the blood of hay fever patients.

  Conventionally, as a method for suppressing allergy to pollen, an inhibitor that suppresses the production of cedar pollen-specific IgE antibody has been known (Patent Document 1). In this patent document, an inhibitor containing bacteria isolated from human intestines or fermented foods is disclosed. And it is said that it is suitable that a bacterium is Enterococcus faecalis FERM BP-10284 (AIST / patent microorganism deposit center deposit number).

JP 2007-091694 A

  Allergies are caused by mast cell IgE binding to an antigen allergen and secreting histamine and the like. Therefore, it is considered that allergy can be prevented by preventing the binding between IgE and allergen.

  The present invention has been completed by obtaining an antibody from ostrich that prevents pollen allergens from binding to IgE by binding to pollen allergens outside the body. More specifically, the antibody-containing pollen allergy protective material of the present invention is an antibody obtained from an egg laid by an ostrich immunized with a pollen allergen. The antibody-containing pollen allergy protective material of the present invention also includes an antibody obtained from an egg laid by an ostrich naturally affected with hay fever.

  Since the allergic defense material of the present invention specifically adsorbs to pollen allergens outside the body, the pollen allergen can be prevented from binding to IgE in the body. Therefore, there is an effect that allergic symptoms can be suppressed.

It is a photograph which shows the result of a Western blot. It is a photograph which shows the findings after 1 hour of the person who applied PBS (control), pollen allergen, pollen allergen + antibody of the present invention.

  In the present invention, antibodies were extracted from eggs laid by ostrich suffering from hay fever, and a large amount of antibodies against the above-mentioned pollen allergen was successfully recovered and completed. The life of an ostrich is surprisingly long at 60 years, and because of its open breeding (outdoors), there is a high probability of being sensitized by pollen. Therefore, it is considered that the possibility of hay fever inevitably increases.

  Since ostriches are birds, antibodies produced in the body are transferred to eggs. That is, if an ostrich naturally affected with hay fever is used, a large amount of antibodies against pollen allergy can be recovered from the egg. 400 grams of antibody per year (800 rabbits) can be obtained from one ostrich.

  If the antibody is used, it becomes possible to neutralize pollen allergens outside the body (to lose antigenicity), and to suppress allergenicity to humans. These antibodies can be used as hay fever prevention masks, air conditioner filters, cosmetics, eye drops, foods (candy and gum), mouth washes, and nasal drops. It can also be applied to inactivate pollen allergens in spaces, walls and clothes as sprays and sprays.

  That is, the point of the present invention is that an antibody is obtained from an egg of a hay fever ostrich, the antibody suppresses human pollen allergy, and even if an ostrich is immunized with only Cryj1 of cedar pollen, Cryj2, Chao1, Chao2 The production of antibodies that cross.

  When blood of ostriches raised outdoors was collected, antibody production to Cryj1, Cryj2, Chao1, Chao2 was observed in 4 out of 15 animals (ELISA). When the egg yolk antibody was purified from the egg yolk of eggs laid from these ostriches, it reacted strongly with Cryj1, Cryj2, Chao1, Chao2 (ELISA, Western Blotting). In other words, antibodies against pollen allergens have migrated to the egg yolks of ostriches affected by hay fever and can be collected in large quantities.

  When ostrich egg yolk antibody is added to the filter paper impregnated with the allergen and applied to the forearm of a pollen allergic patient (human) (patch test), when filter paper of only allergen (Cryj1, Cryj2, Chao1, Chao2) is applied However, the filter paper impregnated with allergen and antibody did not show any allergic reaction. In other words, it was proved that human pollen allergy is suppressed by the ostrich antibody.

  Next, the result of artificially immunizing an ostrich with an allergen (Cryj1) is shown. For the first immunization, 30 μg of Cryj1 was mixed with Freund's complete adjuvant and inoculated into the lumbar muscle of a female ostrich. For booster immunization, 2 weeks after the first immunization, 30 μg of Cryj1 was mixed with Freund's incomplete adjuvant and inoculated into the lumbar muscle of the female ostrich. The antibody was purified from ostrich eggs laid after 2 weeks of booster immunization.

The antibody purification method is shown below. Purification of antibody (IgY) from egg yolk was performed as follows. First, 5 times the amount of TBS (20 mM Tris-HCl, 0.15 M NaCl, 0.5% NaN ₃ ) and the same amount of 10% dextran sulfate / TBS are added to the egg yolk and stirred for 20 minutes. Then, 1MCaCl ₂ / 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. Next, ammonium sulfate is added to a final concentration of 40%, and the mixture is allowed to stand at 4 ° C. for 12 hours. Then, it centrifuges at 15000 rpm for 20 minutes, and collects 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.

<Measurement by ELISA method>
The antigen reactivity of the obtained antibody was measured by the following ELISA. 100 μl of 2 μg / mL of Cryj1, Cryj2, Chao1, and Chao2 was placed in each well of a 96-well microplate for ELISA, and allowed to stand at room temperature for 2 hours. Then, after washing 3 times with PBS, 100 μl of a commercially available blocking solution (Block Ace: Dainippon Sumitomo Pharma Co., Ltd.) was placed in each well and allowed to stand for 2 hours. Then, after washing 3 times with PBS, 50 μl of serial dilutions of ostrich IgY antibody before and after immunization (100 mg, 200-fold and 2-fold serial dilutions using 2 mg / mL as a stock solution) are added to each well at room temperature. Left for 1 hour.

  Then, after washing 3 times with PBS, 100 μl of peroxidase-labeled anti-ostrich IgY rabbit polyclonal antibody (self-made) was placed in each well and left for 45 minutes. After washing 3 times with PBS, color was developed for 30 minutes with a commercially available color kit for peroxidase (Sumitomo Bakelite), and the absorbance (450 nm) was measured with an ELISA plate reader. The obtained result was shown by the highest dilution factor which becomes 2 times or more the light absorbency value of IgY (egg yolk antibody) of normal ostrich (ostrich not suffering from pollinosis). The results are shown in Table 1.

  The female ostrich bred outdoors has antibodies against allergens (Cryj1, Cryj2, Chao1, Chao2) in blood (serum). Furthermore, these antibodies have also migrated into the yolk. Serum and egg yolk antibodies from ostrich immunized with Cryj1 alone can also cross Cryj2, Chao1, and Chao2 (ie, immunizing only one allergen yields an antibody that crosses cedar and cypress allergens).

<Western blot method>
20 μg each of Cryj1, Cryj2, Chao1, and Chao2 was electrophoresed by 8% SDS-PAGE, transferred to a PVDF membrane, and then an antibody (1000-fold diluted) obtained from the egg yolk of hay fever ostrich (No. 1) was used for 1 hour at room temperature. Reacted. After washing with PBS containing a surfactant, HRP-labeled anti-ostrich IgY rabbit antibody was reacted at room temperature for 1 hour. After washing with PBS, it was visualized with an X-ray film using an ECL kit. The results are shown in FIG. It can be seen that the antibody recovered from the egg yolk of hay fever ostrich reacts strongly with cedar pollen and cypress pollen.

<Effect of ostrich antibody on pollen allergy (patch test)>
A soy allergen (Cryj1: 20 μg and Cryj2: 20 μg) soaked in a circular filter paper having a diameter of 7 mm was applied to the forearm skin of 26 volunteers with pollen allergy. The above-mentioned allergen-containing filter paper added with an ostrich antibody (20 μg of egg yolk antibody obtained from the egg yolk of hay fever-affected ostrich (No. 1)) was applied to another part of the forearm skin of the same person. In addition, as a control, a filter paper soaked with PBS alone was applied to another part of the person's forearm skin. The degree of redness of the coated part of the filter paper after 24 hours was observed. Table 2 shows the results.

  When only cedar pollen antigen was impregnated, 24 out of 26 ostrich antibodies were added, and 24 out of 26 subjects were confirmed to be red, but when ostrich antibody was added, 3 The name was only reddish.

  FIG. 2 shows a photograph after 1 hour when the above is applied to another part of the forearm skin of the same person. Allergic reaction (redness) to cedar pollen was suppressed by adding ostrich antibody. After the bandage, all three remain.

  If the antibody is used, it becomes possible to neutralize pollen allergens outside the body (to lose antigenicity), and to suppress allergenicity to humans. These antibodies can be used as hay fever prevention masks, air conditioner filters, cosmetics, eye drops, foods (candy and gum), mouth washes, and nasal drops. It can also be applied to inactivate pollen allergens in spaces, walls and clothes as sprays and sprays.

Claims (2)

  Antibody-containing pollen allergy protective material consisting of antibodies obtained from eggs laid by ostrich immunized with pollen allergen.   An antibody-containing pollen allergy protective material consisting of antibodies obtained from eggs laid by ostriches naturally afflicted with hay fever.