JPS62175426A - Antibody and spraying agent containing said substance as active component - Google Patents

Abstract

PURPOSE:To provide an antibody which is an immunoglobulin prepared from an egg laid by a hen immunized with a respiratory disease, having prophylactic effect against pathogen of respiratory disease and useful as an agent for spraying into nasal cavity or throat. CONSTITUTION:The objective antibody is an immunoglobulin prepared from an egg laid by a hen immunized with a pathogen of respiratory disease (e.g. influenza virus) and having prophylactic effect against the pathogen of said respiratory disease. The antibody is especially suitable for spraying and applicable to the respiratory tracts such as nasal cavity and throat. There is no particular restriction in the form of the spraying agent, however, an inhalation aerosol to be sprayed in the form of floating fine particles is preferable. A sufficient effect can be achieved by applying the agent at an amount to neutralize the pathogen adsorbed to the respiratory tracts. Usually, about 1-10mg of the antibody is applied before going out of door.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to antibodies and sprays, and more particularly to antibodies against pathogens of respiratory diseases and sprays containing the same as an active ingredient.

(Prior art) Microorganisms that cause respiratory tract diseases such as respiratory tract infections, viruses, bacteria, mycoplasma, and rickettsia are known, and dust particles such as pollen and dust mites are also known to be causative agents of allergic diseases. well known. For example, influenza viruses are classified into A% B and C types, but the A type virus in particular has a unique phenomenon in which subtypes with different surface antigens suddenly appear, and this happens at a cycle of approximately /θ years. It is the cause of influenza pandemics, and even within the same subtype, small mutations occur every year, resulting in repeated changes. ``Currently, the only method used to combat such influenza epidemics is inoculation with an inactivated i-vaccine prepared from a virus grown in fertilized chicken eggs.

(Problems to be Solved by the Invention) Although the above method produces antibodies within the human body, the effectiveness of this preventive injection is not satisfactory.

For example, in addition to the fact that the type of influenza virus that causes epidemics constantly mutates, once a vaccination is given, the body's immune system remembers it, and the next year, a different type of influenza vaccine is given. It is said that this causes the same antibodies as before to be produced (antigen original sin theory). Furthermore, it has been pointed out that since the proliferation of influenza viruses is limited to mucosal epithelial cells on the surface of the respiratory tract, antibodies in the blood produced by inactivated vaccines have a problem in terms of efficiency.

Further, effective preventive drugs using antibodies have not been put into practical use for the various respiratory diseases caused by the various pathogenic systems mentioned above.

(Means for Solving the Problems) The present inventors have developed antibodies against pathogens of respiratory diseases,
We conducted various studies to find antibodies suitable for so-called spray administration, and arrived at the present invention.

That is, the gist of the present invention is an immunoglobulin prepared from eggs produced by hens that have been immunized against pathogens of respiratory diseases, and an antibody that has the ability to protect against the pathogens of respiratory diseases, and an antibody that is effective against the pathogens. It is present in the spray agent as an ingredient.

Hereinafter, a detailed description of the invention will be given.

First, an example of the method for producing antibodies according to the present invention will be explained.

That is, the hens are first immunized with a respiratory disease pathogen. This pathogen causes respiratory disease through infection, and includes viruses, bacteria, mycoplasma, and rickettsiae.

Viruses include influenza virus A% B and C, parainfluenza virus/, 2, and 3.
type, R. virus, adenovirus, line virus,
Examples include coronavirus, EB virus, herpes simplex virus, measles virus, varicella virus, echovirus, and coxalaki virus.

t+t, as a bacterium, Staphylococcus aureus
), Gram-positive cocci such as Clepsilla epp (K
Examples include aerobic Durham-negative bacilli such as Lebsiella spp).

Furthermore, Mycoplasma and Rickettsia include Mycoplasma pneumoniae (M7coplasmapnθ
umoniaθ), Coxiella prunetheii (Cox
iella 'burnetii).

When immunizing hens, the above-mentioned pathogens are usually purified and used. Purification can be carried out by conventional methods, such as centrifugation, adsorption, extraction with organic solvents, electrophoresis, chromatography, etc., but differential centrifugation is the most common.

Immunization was carried out in several doses, 700-1000μm/
It is common to apply this treatment to the abdomen, crotch, armpits, etc. about 1/2 times. Eggs produced by the hens are obtained approximately 1 month after immunization.

The desired immunoglobulin is prepared from the eggs. For example, specific fractionation can be achieved by collecting yolks and purifying and concentrating antibodies using polyethylene glycol. The immunoglobulin obtained from this yolk is
It is G.

On the other hand, from the white meat, for example, concentration using ammonium sulfate and Sephadex G-10θ" and G-,200
The immunoglobulin obtained by fractionation using is IfA.

The antibodies thus prepared have the ability to protect against the above-mentioned pathogens and are particularly suitable for spray administration. Spray administration is applied to the respiratory airways of the nasal cavity and throat.

The spray agent suitable for the above administration is not particularly limited, but an inhalation aerosol type that is sprayed as fine suspended particles (aerosol) is suitable. The aerosol itself can be formed by a conventional method. The particle size is 3~!
The thickness is generally about Qμm, but it is preferably about 70 to 30μm from the viewpoint of deposition efficiency in the respiratory airways.

Antibodies are generally dissolved using distilled water, physiological saline, or the like. In addition, various stabilizers, as necessary,
For example, gelatin etc. can also be added.

The dose may be any amount that neutralizes pathogens adsorbed to the respiratory tract, and it is usually sufficient to administer the antibody before going out so that the amount of the antibody is about 70 μm.

(Example) Hereinafter, the present invention will be further explained in detail with reference to Examples.

Example/ (Influenza virus) (purification) Influenza virus WSN (H/N/) and A/
Udorn/ 307/7.2 (HJ N, 2) to 1
0 day old chickens) 200 IJ fertilized eggs each were inoculated,
After culturing at 4t°C for 34t, the infected chorioallantoic fluid was collected. Differential centrifugation 7 times (7,QθOrpm 20 minutes, 7.00 Orp
m76 hours) and sucrose concentration gradient centrifugation twice (30~jQ%
The virus in the chorioallantoic fluid was purified by 36,0θθrpm/hour), and the pH containing 0,00/M KDTA was
It was stored suspended in 71% 0.07M Toss buffer.

(Immunity) / W8N (H/N/) 200,0θomhU/
ml 9”9/enemyJ A/U(lorn/J(77
/ 7J (H3Nu) ioo, oooHm/xl
xrrs3/y13 control purified virus/,! - with 7.5% complete Freund's adjuvant as Wlo/5t using a male G needle.
The above antigen was injected subcutaneously into the abdominal cavity and lower axilla of three month-old white leghorn hens three times (day 0, day 2, and day 27).

Eggs obtained up to 2Z days after the /th injection were divided into groups as shown in Table/, and the yolks were pooled for each group. With each group / θθ Take the yolk of punishment / θ
0 punishment of 0.0/M IJ acid buffer (θ,/M
7t of polyethylene glycol (pwat, ooo
) and stirred for 75 minutes.
Next,? , centrifuged for 75 min at 0θOrpm, above.

! The supernatant was filtered through gauze, polyethylene glycol (PFiG40θθ) was added, and the mixture was further pressed for 75 minutes. , centrifuged at θ00 rpm for 75 minutes.

The obtained precipitate was diluted with 70me phosphate buffer (0.7M H
act, pH 7,j) and dissolved in it/,% t
Add polyethylene glycol (pvatoθ0)/! The mixture was pressed for a minute and then centrifuged at J', 000 rpm for 75 minutes. Discard the supernatant and continue with tr, o o.

Centrifuge for 75 minutes at rpm and add 10. θ of l
, 0/j M phosphate buffer (pHF, 0) was added (obtaining the desired antibody).

table /.

0 4 /% /r, 2J λ7747
6 Ta? //J /21 /7%day IA/IBIOI IFI
GIHI Engineering 11(6)” (9) α1(
6) αOI A 1. BIOI
D I Ift +IAIBI OII [
il I P' + 0
1 1 α4 (61 (43 *) The value in parentheses indicates the number of eggs.

<Hemagglutination inhibition (H-technique) test> H-technique test was performed on the antibody obtained in Example.

That is, 0.2% of each antibody shown in Table λ using physiological saline! A series of one-fold dilutions of @l was prepared.

Next, influenza virus (VEI) was added to this diluted antibody.
N or Udorn) 0. ．． 2j- was added, mixed well, and allowed to stand at 32° C. for 3θ minutes to allow the antigen and antibody to react.

Next, θ,j% chicken red blood cell float-1BQθ,jm/ was added to the tube, mixed well, and left at room temperature for 7 hours, after which the image of the bottom of the tube was read. The reciprocal of the highest dilution of the antibody that completely suppresses hemagglutination is defined as the H antibody titer. The results are shown in the table below.

That is, it can be seen that antibodies against WSN and TTdorn viruses specifically react with each virus.

<Neutralization test> A neutralization test was conducted on the antibody obtained in Example. That is, a series of 10-fold serial dilutions of each antibody shown in Table 2 was prepared using physiological saline. Then this diluted antibody 0
．． /! Influenza virus (WSN strain or Udorn eP) solution o,ir rttl (,2,
000Pl! Add U/ff/), mix well,
The plate was left standing at ℃ for 7 hours to allow the virus and antibody to react. Then, that 0. MDOK (Maclin-Darby) with Itut stamped on the hole plate made by Coaster Co., Ltd.
caning Kidney) monolayer culture cells were inoculated into the wells, and the virus was adsorbed to the cells by dipping/time molding. j medium-2,
5'me was overlaid and cultured for 3 days at J%oo2@degree and J%'C. Viruses that multiply in infected cells are released and spread infection to neighboring cells, but as influenza virus-infected cells degenerate and die, a small group of degenerated cells, called black, is formed around the initially infected cells.

This black can be observed with the naked eye. /PFU (pla
que forming unit), but
This corresponds to 7 infectious virus particles.

Count the number of blacks on the plate cultured for 3 days, and calculate the number of blacks (100 blacks) when no antibody is added.
The reciprocal of the highest antibody dilution at which the antibody was diluted to less than 10% was defined as the neutralizing antibody titer. The results are shown in the table. That is, it can be seen that antibodies against VEIN and Udorn viruses have specific neutralizing ability against each virus.

Table - Hf neutralizing antibody WS'lJ Udorn WSN
tTdorn(/IIEI!ri
(/stj)ws*N /A 44t -
B/, 0.2% -λooθ Kukoo.

C,20ge-Fj/ko0O-G! r/,, 200 + Hj/ Ko00 - Tech j/@2θO- Udorn, 2A -' - Virus B -2!6 0 -! /2 D-Co560 R-10,2%0 <200 20000 Control 3A -<,!
00 (−〇OB −− C−− E −− F −− a −− (in vivo test) The antibody obtained in Example / was diluted (/θO) or ///θ diluted (/θO) with distilled water. /θ-1), 0, /*l was sprayed into the nasal cavities of No. mustard to examine the effectiveness of the virus, and the number of No. mustard positive for the virus is shown in Table 3.

In other words, it can be seen that antibodies against Udorn virus specifically protect against Udorn virus infection. Antibodies; return; /'Muster's number Lungs Nasal cavity (VEI
N) j 10 j 0 0 hiro
10 σ, /R1! Oj 0 04 PB8
(Effects of the Invention) The antibody according to the present invention is effective in spray administration, which is suitable for efficiently preventing respiratory tract infections caused by pathogens of respiratory diseases.

Applicant Shimizu - Masato Fumiyo
Patent Attorney Hase Yo - Others/Names

Claims (5)

[Claims] (1) An immunoglobulin prepared from eggs produced by hens that have been immunized against a pathogen of a respiratory disease, and an antibody that has the ability to protect against the pathogen of a respiratory disease. (2) The antibody according to claim 1, wherein the pathogen is a virus, bacteria, mycoplasma, or rickettsia. (3) The antibody according to claim 1 or 2, wherein the virus is an influenza virus. (4) The antibody according to claim 1, wherein the immunoglobulin is IgA or IgA. (5) Immunoglobulin prepared from eggs produced by hens that have been immunized against pathogens of respiratory diseases, and containing antibodies that have the ability to protect against the pathogens of respiratory diseases as an active ingredient for use in the nasal cavity or throat. spray agent.