JPS60248626A - Preventive and remedy for infectious disease or pseudomonas aeruginosa - Google Patents

Abstract

PURPOSE:A preventive and remedy showing improved effect on infectious diseases of Pseudomonas aeruginosa, containing a monoclonal antibody having specific affinity for an antigen classified by serotype of Pseudomonas aeruginosa and directly aggregating activity on molds of Pseudomonas aeruginosa. CONSTITUTION:A preventive and remedy of infectious diseases of Pseudomonas aeruginosa containing at least one monoclonal antibody (derived from mouse or man), being reacted with only a strain belonging to one group among molds classified by serotype of Pseudomonas aeruginosa of A-M groups by serological classification, not being reacted with strains belonging to the other groups (antigen specificity classified by serum), having directly aggregating activity on molds of Pseudomonas aeruginosa, having a globulin class belonging to IgG. The monoclonal antibody on Pseudomonas aeruginosa is prepared by cell fusion method or transformation method of B cell with EB virus.

Description

DETAILED DESCRIPTION OF THE INVENTION [■] Background of the Invention The present invention relates to a monoclonal antibody that is effective in the preventive treatment of Pseudomonas aeruginosa hyposmosis.

Pseudomonas aeruginosa (hereinafter sometimes referred to as Pseudomonas aeruginosa) exists widely in nature, and is found at high rates in water, sewage, etc., and also in the oral cavity and intestine of humans and animals. This bacterium exerts pathogenicity not in general attenuated patients due to the bactericidal activity of the bacterium itself, but rather in patients with reduced attenuation resistance, such as cancer patients, people on immunosuppressive therapy, transplant patients, burn patients, and others. Newborns etc.

Currently, Pseudomonas aeruginosa is considered to be the most difficult disease to treat. In other words, not only is Pseudomonas aeruginosa resistant to almost all antibiotics that have been commonly used, but it is also easily induced to become resistant to antibiotics that have been developed in recent years. This is because there are many cases. In addition, in view of the limitations of antibiotic therapy, we are developing anti-infection protection agents, or vaccines, using bacterial body components, with the aim of increasing the host's ability to treat Pseudomonas aeruginosa. Pseudomonas aeruginosa multivalent vaccine prepared by purifying and mixing surface cloth antigens, OEP whose main component is protein as a common chemical antigen of Pseudomonas aeruginosa, or protease toxoid, elastase toxoid, or exoprotease prepared from baking soda in OEP. There are three or four types of mixed vaccines that contain toxoids.

However, it is no exaggeration to say that none of these vaccines has sufficient protection against infection or is sufficiently safe, and that a better, more practical vaccine is awaited. Furthermore, immunoglobulin therapy using passive immunization using human serum immunoglobulin preparations prepared from the serum of healthy individuals has been studied, but many of the actual preparations have low antibody titers against Pseudomonas aeruginosa (and their prevention and treatment). There are many people who question its effectiveness.

[■] Summary of the Invention 1 Summary The present invention provides that the globulin class that has specific affinity for Pseudomonas aeruginosa serotype antigens and has direct agglutination activity against Pseudomonas aeruginosa cells is IgG. The aim is to create monoclonal antibodies consisting of the following and use them as preventive and therapeutic agents, either singly or in combination, to achieve this purpose.

The target monoclonal antibody against Pseudomonas aeruginosa is produced by cell fusion of mouse myeloma cells (tumor cell line) with lung cells of mice immunized with Pseudomonas aeruginosa serotypes to produce hybridomas. In the case of human monoclonal antibodies, antibody-producing cells are created by transforming human-derived B cells with EB virus, and the antibodies produced by these cells react specifically to antigens of different serotypes. It is produced by selecting an antibody that has the agglutination activity and whose globulin class is IgG class.

2. Effects The monoclonal antibodies of the present invention, either alone or in combination, can achieve excellent preventive and therapeutic effects against Pseudomonas aeruginosa hypoinfection.

What should be noted in the present invention is that among the monoclonal antibodies that specifically react with the serotype antigens of Pseudomonas aeruginosa, the antibodies whose glabulin class is IgG and have direct agglutination activity on Pseudomonas cells are , which exhibits prophylactic and therapeutic effects against Pseudomonas aeruginosa that reacts with the antibody at extremely low concentrations and doses.

This was discovered for the first time by the present inventors.

[■] Detailed description of the invention 1. Pseudomonas aeruginosa used In the present invention, the classification of Pseudomonas aeruginosa used was in accordance with the serological classification determined by the serotype review committee sponsored by the Pseudomonas aeruginosa research group (1975). (Table 1).

2. Production of monoclonal antibody The monoclonal antibody against Pseudomonas aeruginosa according to the present invention is produced by cell fusion method and transformation method using DB virus.

The production of monoclonal antibodies by cell fusion method is basically based on the method of Kohler and Milstein et al.
．． 285-308 (1980). The production of monoclonal antibodies by this method generally includes (1) preparation, (2) cell fusion, (3) cloning, (4) detection of monoclonal antibodies, (5) preparation of large quantities of monoclonal antibodies, and (6) purification of antibodies. It consists of processes.

Next, we will add detailed explanations for each step.

(1) Preparation A solution containing hypochycanthin, aminopterin, and thymidine (hereinafter referred to as 1 (A, T) and (U) is prepared.

It is convenient to store all of these as 100 times concentrated solutions. In particular, it is convenient to prepare a stock solution by combining hypoxanthine and thymidine (HT). Each stock solution was sterilized by filter filtration (0.2μ membrane filter →), divided into appropriate amounts, and stored frozen at -20°C.Next, commercially available Dulbecco, m
A complete medium (hereinafter referred to as 1 decoy) consisting of medium, sodium pyruvate, penicillin-streftomycin, L-glutamine, 2-mercaptoethanol, and fetal bovine serum is prepared. This can be stored at 4°C for 6 weeks. Darbetsuko M above
It is also possible to use commercially available RPMI-1640 medium instead of EM medium.

Then DMBMKHAT or HT stock solution was added to H
Prepare AT medium or HT medium. and separately NaCl, KCI, Nap, )JP in redistilled water.
A GKN salt solution containing O4,2H20, NaH2PO4, I-(201 Gluco-\, and phenol red) is prepared.

For cell fusion, the HVJ method and polyethylene glycol (PE) are used.
G) There are two methods, but the latter method is more useful in terms of how to handle increases. There are various types of PEG depending on their average molecular weight, but usually PEG-1000, P
It is appropriately selected from among Ea-, 4ooo, and PEG-6000. The molecular weight to be used will vary depending on the cells to be fused and the purpose, but in general, the smaller the molecular weight, the less viscous and easier to handle, but the greater the cytotoxicity. Cell fusion rate and cytotoxicity are particularly dependent on the concentration of PBG. Also, since there are lot differences between manufacturers, the PEG to be used is determined by considering the above points. The concentration of PEG used is generally between 42.5% and 50% concentration. Furthermore, dimethyl sulfoxide (DMSO) and poly-L-arginine are known to increase fusion efficiency.

However, since DMSO increases the cytotoxicity of the PEG solution, it is necessary to take measures such as shortening the fusion time and repeatedly and thoroughly washing after the fusion operation.

As feeder cells used to promote proliferation of hybridomas, mouse peritoneal macrophages or tile lymphocytes are suitable. In either case, collect cells aseptically and prepare a cell suspension. Wash three times with culture solution and centrifuge at 400×g for 10 minutes at room temperature. After washing, count the number of living cells using Trivan blue solution. A survival rate of 95% or higher is always used.

(2) Cell fusion The animal-derived antibody-producing cells used for cell fusion vary depending on the reduced partner cells, i.e. tumor cell lines, in the HAT medium used for fusion, but in general mice and rats are used. It is often used. Fusion of mouse tumor cell lines and rabbit or human antibody-producing lymphocytes is often inefficient in terms of the frequency of noiplidomas. In the present invention, mouse-derived antibody-producing lymphocytes are used for the following reasons. Mouse tumor cell lines that have lost all immunoglobulin-producing ability are useful, and these cell lines have a high fusion efficiency with mouse-derived antibody-producing lymphocytes and a high incidence of hybridomas. In particular, it is known that mouse IgG, regardless of class, binds to protein A, making it easy to purify antibodies.

Among mouse strains, BALB/C mice are most preferred, and other mouse strains may be used depending on the antigen. 8-12 in females to collect antibody-producing lymphocytes.
Week-old mice are best. In addition, antibody-producing lymphocytes of mice to be subjected to cell fusion to obtain monoclonal antibody-producing hybridomas of the present invention are A = M in Table 1.
By culturing Pseudomonas aeruginosa strains belonging to each group, and immunizing mice by subcutaneous or intraperitoneal administration using detoxified washed bacterial cells obtained by formalin treatment or a lipopolysaccharide fraction obtained from the bacterial cells as an antigen. You can get it. At this time, the adjuvant used for immunization is
Freund's adjuvant, alum, Bordetella pertussis, etc. can be used. Depending on the type of antigen, the adjuvant, administration route, frequency, and amount of antigen used will differ.
When Pseudomonas aeruginosa cells are used as the antigen, 1-100 μg of the antigen is prepared in water-in-oil form with an equal amount of 70 India incomplete adjuvant and administered intraperitoneally 2 to 5 times. Harvest mouse stock and use it for cell fusion. In addition, as a pretreatment for cell fusion, stimulating the immunized mouse's stored lymphocytes with antigen in vitro or transferring them together with the antigen into a mouse host that has been irradiated with X-rays can greatly increase the number of target antibody-producing cells. As a result, hybridomas can be obtained efficiently.

In the present invention, the HAT medium-reduced tumor cell line used for cell fusion is p3-NSI/1=Ag4. t(
Abbreviation N5-1) or P 3-X 63-A g8,
Mouse cell lines commonly used for cell fusion such as ul (abbreviation P3'ul) are suitable. Cell fusion occurs when tumor cells are proliferating best (104-505/l)
go to

Next, actual cell fusion is performed as follows.

The plaque from an immunized animal is removed aseptically and a lymphocyte fluid containing antibody-producing cells is prepared. Mix approximately 10 times the amount of lymphocyte fluid with the tumor cells of the partner cells, centrifuge, and remove the culture medium by aspiration. Loosen the pellet, and add 1 m of 45% PEG pre-warmed to 37°C.
Add l.

Gently shake the test tube from time to time to ensure that the cells collide well with each other. After 5-6 minutes, about 50 ml of DMEM is added to stop the reaction. Centrifuge and aspirate the supernatant. 3
Slowly add HAT medium warmed to 7°C to loosen large cell clumps.

Then, dispense into Saewell or 96-well culture plates. Replace half of the medium with HAT medium every 2-3 days. After repeating this procedure for about two weeks, the culture supernatants of the wells in which hybridoma growth was observed are examined for the production of the target antibody using various assay methods to search for antibody-producing hybridomas.

(3) Cloning Once the hybridoma is established, perform cloning as soon as possible. As a method, 3 pieces of 7m
The limiting dilution method consists of diluting the suspension of hybridomas suspended in DMBM with DMBM-agar in equal volumes and dispensing 0.2 ml/well into a 96-well plate. The mixture was mixed one by one, spread on Betrizois, and cultured in a CO2 incubator at 37°C for about 10 days.
The soft agar method, which involves observing the resulting colonies with the naked eye or a low-magnification stereomicroscope and lifting them with a Pasteur pipette; the fibrin gel method, which uses fibrin gel, which is easy to operate and has high cloning efficiency; Apart from this method, any method can be used, such as a cell sorter method in which hybridomas are allowed to react with fluorine cent microspheres bound to an antigen, and hybridomas producing the desired antibody are selected as single clones.

The present inventors empirically determined the limit dilution ratio, which is easy to operate, at 1°C and 1°C.

(4) Detection of monoclonal antibodies Assay methods for detecting monoclonal antibodies include the following methods. One is a method using red blood cells bound to protein A, which shows excellent sensitivity and specificity among methods for detecting specific antibodies. In addition, the agglutination reaction method, which determines the antibody titer by observing the degree of agglutination, is the simplest method to determine the globulin class or subclass. This is an ophthalony method in which the supernatant can be precipitated with an antibody and then confirmed by developing it on a 5DS-polyacrylamide gel or the like. In addition, antigens or cells are immobilized on a U-shaped microtiter plate made of polyvinyl chloride, and culture supernatant is added thereto and incubated to cause an antigen-antibody reaction. After washing, radioimmunoassay or enzyme immunoassay (EIA) is used to examine radioactivity or enzymatic activity in each well.
There is also.

Among various assay methods, one may be selected that suits the purpose. Basically, what is important is that it be short, sensitive, and inexpensive. The present inventors conducted the following EIA
law is used.

Specifically, Pseudomonas aeruginosa cells were used as antigens as membrane filter-Kl quantitative dots, and after reacting with the culture supernatant of a hybridoma, a rabbit anti-mouse immunoglobulin antibody labeled with peroxidase (DAKO) was reacted. The donor immuno binding assay method (DIBA method, A
analytic biochem, 119, 14
2, 1982).

(5) Preparation of large amounts of monoclonal antibodies Methods for preparing large amounts of monoclonal antibodies include expansion culture methods, liposomes; large-scale culture methods using microcapsules, and antibodies that appear in the serum by transplanting hybridomas into nude mice. In addition, the preparation method consists of increasing the amount of ammonium sulfate, DEAE-cellulose, protein A- from the ascites of the mice by transplanting the hybridoma intraperitoneally into BALB/C mice pre-administered with pristine.
There are preparation methods such as purification using a column such as Sepharose, and any of these methods can be applied to the present invention. In particular, in the present invention, the No. 5 hybridoma is transplanted intraperitoneally into BALB/C mice pretreated with an immunosuppressant (pristine).
From ascites that accumulates due to the proliferation of ibridoma
We have adopted a method for large-scale preparation of monoclonal antibodies from Akira.

(6) Purification of antibodies - Non-specific purification The monoclonal antibodies of the present invention can be purified from ascites etc. by a combination of known methods such as gel filtration, ion-exchange cellulose chromatography, and salting out with ammonium sulfate. I can do it.

■Antigens were bound to Sepharose 4B (Pharmacia) or Affigel-10 (Bio-Rad) activated with specific purified cyanogen bromide, and the immunoglobulin fraction or ascites was poured into a column to insolubilize it, and antibodies were bound to it. Affinity chromatography, which purifies the adsorbed antibody by eluating it with a urea solution or the like, can also be used if necessary. Furthermore, when the monoclonal antibody of the present invention is IgG, it is recommended to use an affinity column on which Flotin A is immobilized because purification is easy.

3. Production of human monoclonal antibodies Human monoclonal antibodies against Pseudomonas aeruginosa according to the present invention are produced by transforming human B cells with BB virus.

In general, human monoclonal antibody production methods include ■Human-to-human hybridoma method ■Mouse-human hybridoma method ■Transnoemation method using EB virus, etc.

Method (2) has so far been an incomplete technique due to the lack of excellent human myeloma cells, poor proliferation of the desired hybridoma, and missing genes. Method (2) involves the problem that, since the method is mouse-human, even if the gene is inserted, the gene deletion phenomenon occurs immediately, making it difficult to establish a stable strain.

Therefore, the present inventors produced the human-derived monoclonal antibody of the present invention using the EB virus transformation method, which is said to be capable of producing relatively stable antibody-producing cell lines.

h) For B cell materials, peripheral blood of healthy individuals with production of anti-Pseudomonas aeruginosa antibodies in the serum and removed lymph nodes (e.g. tonsils) of patients are used. - Mononuclear cell fraction containing B cells was concentrated and used by Conley gravity centrifugation method.

The basic technique for transforming human B cells using EB virus is a known method (for example, Nature
267.52', 1977, Record of the 4th Annual Meeting of the Japanese Society of Immunology, p. 399, 1974), B cells were infected using the culture supernatant of a cell line that continuously produces EB virus as a virus source, and mutated into a proliferative type. DIBA from the cell population
(in this case, using Kappel's peroxidase-labeled goat anti-human immunoglobulin antibody as the second antibody), the target antibody-producing cell population was screened, and then a rosette formation method using known antigen-sensitized sheep red blood cells and soft agar. A monoclonal monoclonal antibody-producing clonal cell line of the present invention was obtained by a colony formation method.

The monoclonal antibody of the present invention can be prepared in large quantities from the antibody-producing clonal cell line thus obtained by collecting the culture fluid by mass culturing using spinner flasks, roller bottles, etc., and then using it as described in the method for purifying mouse monoclonal antibodies. It is possible to purify in a similar manner.

As mentioned above, the monoclonal antibody of the present invention has specific affinity for the serotype antigen of Pseudomonas aeruginosa derived from mice and humans, and has direct agglutination activity against Pseudomonas aeruginosa cells. The globulin class it has is IgG, and these properties can be defined by the method shown below. In other words, the specific affinity for the serotype antigen of Pseudomonas aeruginosa means that it reacts only with strains belonging to one of the serotypes of Pseudomonas aeruginosa in groups A and -M in Table 1. , meaning that it does not react with strains belonging to other groups (for example, it reacts only with bacteria of group A and does not react with other groups B-M).

In addition, the direct agglutination activity of the monoclonal antibody of the present invention against Pseudomonas aeruginosa cells refers to the direct agglutination activity of Pseudomonas aeruginosa cells, as shown in Example IK. Refers to antibody activity that directly aggregates.

Furthermore, when the globulin class of the monoclonal antibody of the present invention is derived from a mouse, the purified antibody has a heptaphryl 83
Gel using 00 (Pharmacianehi)? Perpatterned and peroxidase-labeled goat anti-mouse IgG antibody (
Kanohel Co., Ltd.) DIBA method, and in the case of human origin, the gel filtration pattern of the purified antibody using heptaphacryl S-300 and the peroxidase-labeled goat anti-human IgG antibody (
Kappel Co., Ltd.) I) IBA method, respectively, means those identified as IgG.

As exemplified in the test examples, the monoclonal antibody of the Thor invention exhibits excellent preventive and therapeutic effects against Pseudomonas aeruginosa infection. is preferably used in the form of an injection. EXAMPLES The present invention will be explained in more detail by illustrating Examples and Test Examples below.

Example 1 Serotype: Preparation of mouse monoclonal antibody against Pseudomonas aeruginosa and its properties 1) Strains used Pseudomonas aeruginosa type B in Table 1 is DLOO2 strain E type ID1130
Strain, type G ATCC10145 strain, type ID101.0
Each strain was used.

2) Cultivation of bacteria Each type of P. aeruginosa was cultured overnight at 37°C on an ordinary agar medium, the grown cells were suspended in physiological saline, and 0.5 ml of it was added to the synthetic medium of Weight et al. (J, Biochern, , 8, 3.

7], 1.1.978) was inoculated into a Sakaro flask containing 1501η1, and cultured with shaking at 37°C for 16 hours. After culturing, formalin was added to the Sakaro flask to a concentration of 0.3%, thoroughly mixed, and left at room temperature for 1 hour. The culture solution was then centrifuged (12,000×g for 30 minutes) to collect bacterial cells, and washing with physiological saline and distilled water and centrifugation were repeated to obtain formalin-treated bacterial cells.

3) Immunization Immunization 1 was carried out by making an emulsion of 0.3% formalin-treated bacterial cells of each type of P. aeruginosa in incomplete Freund's adjuvant and intraperitoneally administering it to BAL B/C mice 5 times every other week. ~Ta.

4) Cell fusion 4 days after the final immunization, 5 x 108 mouse tile cells and 5 x 1.0' N5-L mouse myeloma cells ([f
The hybridomas (obtained by cell fusion with fi in the presence of 50% polyethylene glycol) were dispensed into a 96-well flat-bottomed plate, and ]-IAT (containing hypoxanthine, aminopterin, and thymidine) was added.1. 5% in Dulbecco's MEM medium supplemented with 10% tallow serum.
Cultured at 37°C in the presence of σ. The presence or absence of anti-Pseudomonas aeruginosa monoclonal antibodies in the culture supernatant of wells in which hybridoma growth was observed was determined using the enzyme immunoassay method (andlytical assay).
Biochemis-try 119, 142-14
．． 7, 1982. It was measured using the IBA method below. The DIBA method is a 96-well U-bottom multiplayer 1.
A membrane filter (3.1 mm square) on which 0.4 μg of 0.3% formalin-treated 0.3% formalin-treated P. pyogenes per 1 dot was immobilized as an antigen and a membrane filter (3.1 mm square) and the above culture supernatant 10
0 μm at room temperature for 30 minutes, then reacted with peroxidase-labeled Tsuzagi anti-mouse and immunoglobulin antibodies (manufactured by DAKO) for 30 minutes, developed color using chlornaphthol as a substrate, and visually observed on a membrane filter on which the antigen was immobilized. Those in which color development was observed were judged to be positive for antibody production. Hybridomas in which monoclonal antibody production was observed in the culture supernatant were further cloned using limiting dilution method.
Monoclonal hybridomas were grown in flasks and treated with the immunosuppressant pristane (Aldrich).
Treatment BAL] A monoclonal antibody was extracted from the mouse ascites fluid obtained by intraperitoneal transplantation of 3/C mice using protein A-Sepharose (Pharmacia), an affinity column (for IgG), and heptaphryl 5300 (for IgG). Purified.

Among the various anti-Pseudomonas aeruginosa monoclonal antibody-producing hybridomas obtained by We found 18 hybridoma strains.

Of these, 6, 4, 5, and 3 hybridomas reacted specifically to type E, type B, type G, and type ■, respectively. Among these hybridomas, most of the strains were of the globulin class ■ρ, and only two strains produced IgM. The results are shown in Table 2.3.4.5.

The direct agglutination activity of monoclonal antibodies against Pseudomonas aeruginosa cells was measured by the following method. A suspension of dead bacterial cells of each serotype of Pseudomonas aeruginosa treated with 0.3% formalin or heat treated at 120°C was placed in a 96-well U-bottom multiplate (Falcon) (suspended in PBS,
540=0.4) was dispensed into 50 μm portions. Next, a monoclonal antibody diluted with PBS (-1) was dispensed into 50 μm portions, and the plate surface was covered with a sealer (
Cover with Linpro Plate Sealer) and incubate at room temperature for 10 minutes.
The mixture was shaken for a minute and left to stand at 37°C for 1 hour. Further, the plate was allowed to stand at 4° C. to io° C. for 15 hours or more, and then the plate was returned to room temperature, and the agglutination reaction image of the bacterial cells was observed and judged through the back of the plate.

Test Example I Protective activity against Pseudomonas aeruginosa infection of mouse monoclonal antibodies that specifically react with each type of P. aeruginosa according to serological classification. The ability to prevent infection against bacteria was investigated. 501 g of a monoclonal antibody that specifically reacts with each bacterial type was administered to each group of 5 BALB/C female mice at the age of 10 weeks.

500ng, 5μg, 50z1g, 500
gg and 5mg were each administered intraperitoneally, and 2 hours later, a strain specific to each bacterial type (ITDtt3o(
E type), IIDLOO2 (B type), ATCC]014
5 (type G), IIDtoto (type I)) was challenged intraperitoneally with 1×106. In the control group, only physiological saline was administered instead of the monoclonal antibody. The survival rate of mice in each administration group was determined on day 7 after Pseudomonas aeruginosa infection. The results are in Table 2
Surprisingly, among the various monoclonal antibodies, only antibodies with a globulin class of IgG and strong agglutination activity were found to have an extremely strong effect on preventing infection with Pseudomonas aeruginosa.

Example 2 Creation of human monoclonal antibody against serotype Pseudomonas aeruginosa and its properties 1) E B virus B95 which produces and releases EB virus (hereinafter referred to as EBV)
-8 cells were cultured in a medium containing RPMI-1640 and 20% FC8, and the culture supernatant on day 7, near the stationary phase, was separated and used as a virus source.

2) Isolation of lymphocytes Heparinized peripheral blood collected in advance from a healthy person with high anti-Pseudomonas aeruginosa antibodies was mixed with physiological saline or PBS.
(After diluting 2 times with -1, it was slowly layered on 1/4 to 1/3 amount of Lymphopresop to avoid disturbing the interface. After layering, it was centrifuged at 1550 rpm for 30 minutes at room temperature. After centrifugation, Take out the white cloudy cell layer of the interface layer with a Pasteur pipette, and soak the cells in PBS (→ at 140 rpm,
Centrifugal washing was performed for 10 minutes (first time), then at 100 rpm for 10 minutes (second and third times). Cells are RPMI 1
5×101064y were resuspended in 640 solution.

3) Removal of TIJ lymphocytes (concentration of 8977 cells) 0.5% A
ET treated 5RBCI volume, mononuclear cell suspension (5X 10
6 cells/rnl) 1 volume and 5 RBC-absorbed FC80.2 volumes were mixed in a centrifuge tube (pointed bottom), left at room temperature for 5 minutes, and then centrifuged at LOOO rpm for 5 minutes at 4°C. After centrifugation,
It was left standing in water for 1 hour. After the reaction, gently pipette the cell pellet with a Pasteur pipette, make a suspension, place it on Lymphoprep, and incubate at 1550 rpm for 30 minutes at 4°C.
It was centrifuged at

Cells that did not form rosettes on the interface after centrifugation (897
7 cells) were taken out and centrifuged and washed 3 times in PBS (-1) in the same manner as in 2 above.Cultures were cultured at 1 x 107 cells/rnl (when there are many) to 2.5 x 10 cells/ffII (when there are few). Floated in the liquid.

4) Establishment of cell line by BBV infection 8977 sphere suspension (I x 107 - 2.5 x 106f [W/ml) per well of a culture plate (24 wells)
) 0.1 ml of EBV solution was added to 1 ml of O, and incubated at 37° C. for 60 minutes in a carbon dioxide gas incubator.

After incubation, 8 ml of culture solution was added to bring the total volume to 1 ml, and static culture was performed at 37° C. in a CO2 gas incubator. After EBV infection, 4 mouths 5 were added. Thereafter, the culture was continued by replacing the culture medium every 4 to 5 days, and after 6 weeks, a culture plate with holes in which vigorous cell proliferation was observed was used. Antibody production ability was measured using a labeled goat anti-human immunoglobulin antibody. The cell population in which antibody production was observed was cloned by rosette formation method and soft agar colony formation method. The cloned cell line, which showed strong proliferative ability through cloning, was expanded and cultured using the Starling System K (manufactured by Intermethod), and the culture supernatant was purified using an affinity column using each type of progenitor bacteria as an antigen. The results are 6th and 7th
, 8 and 9.

The present inventors have isolated 14 hybridoma strains that produce human monoclonal antibodies that specifically react with each serological type of Pseudomonas aeruginosa. Among these, there are 4 and 5 hybridomas that specifically react to type E, B, G, and ■, respectively.
There were 3 stocks, 2 stocks, and 3 stocks. Incidentally, the agglutination activity of the human monoclonal antibody was also measured by the method shown in Example 1.

Test Example 2 Protective activity against Pseudomonas aeruginosa infection of human monoclonal antibodies that specifically react with each serum type of Pseudomonas aeruginosa Human monoclonal antibodies that specifically react with each type obtained in Examples 5, 6, 7, and 8, respectively We investigated the protective effect of antibodies against Pseudomonas aeruginosa infection.

The results of the study according to the method of Test Example 1 are shown in Tables 6 to 9. In the case of human monoclonal antibodies, as with mice, among various monoclonal antibodies, only antibodies of the immunoglobulin class IgG and having strong agglutination activity were found to have extremely strong protective effects against Pseudomonas aeruginosa infection.

Example 3 Solution The monoclonal antibody exhibiting anti-Pseudomonas aeruginosa activity obtained in Example 1 was dissolved in a 3M/10 amino acetic acid solution to give a globulin concentration of Lmg/ml K.

The material obtained in step 1 was filtered into a membrane filter. 20 (manufactured by Nuclepore) and aseptically dispensed into vials in 1 ml portions to obtain a liquid preparation of the substance of the present invention.

Example 4 Freeze-dried preparation The monoclonal antibody obtained in Example 1 that was effective against Pseudomonas aeruginosa infection was dissolved in physiological saline to which amine acetic acid was added to a final concentration of 3ΔOM to give Lmg/Fn I. Furthermore, mannitol was added to a concentration of 50 mg/1ml, and the membrane filter was filled with Lucribore No. 20. sterile filter using Nuclepore),
A lyophilized preparation of the substance of the present invention was obtained by aseptically dispensing the amount into vials and freeze-drying.

Example 5 Liquid and lyophilized preparations of human monoclonal antibodies that specifically react with serum type E microorganisms ■) Large-scale preparation of human monoclonal antibodies ■ Large-scale cultivation of monoclonal antibodies No. 3 obtained in Example 2 The producing strain was placed in a serum-free medium (H
B tot medium (manufactured by Fujirebio) was adapted. Next, the conditioned cells were cultured in a 5e Starling system (manufactured by Intermed) and used for the purification described below.

■Mass-purified Protein A-8 epharose 4 B (7
-r (manufactured by Ruqsia) or DEAE 5ephace
About 15 m of globulin 1il11 cars using l
I got g.

2) Prepare 100μ of the above globulin fraction in the same manner as in Solution Example 3.
A liquid preparation of the substance of the present invention with a concentration of g/fnl was obtained.

3) Freeze-dried preparation A freeze-dried preparation of the substance of the present invention was obtained in the same manner as in Example 4 from the globulin fraction obtained by mass purification using the above method.

Example 3 Therapeutic effects using mouse monoclonal antibodies specific for each serum type of Pseudomonas aeruginosa In Test Example 1, the therapeutic effects of monoclonal antibodies No., 1, 4.5, ■, ■, B, and BlY, which were found to have preventive effects, were determined. conducted an experiment.

A group of 5 BALB/C female mice aged 10 weeks were intraperitoneally challenged with 1 x 106 bacteria that react with each seclonal antibody in the same manner as in Test Example 1. Two hours later, the above monoclonal antibody 1100n ~ lomg was administered intraperitoneally, and the DE50 value was reached on the 7th day. The results are shown in Table 10. Although the therapeutic effect of each monoclonal anti-1 antibody, which had a strong preventive effect in Test Example 1, decreased from seedlings to seedlings, a therapeutic effect was also observed.

Test Example 4 Therapeutic effect using human monoclonal antibodies specific to each serum type of Pseudomonas aeruginosa Monoclonal antibody N that was found to have a preventive effect in Test Example 2
Treatment experiments were conducted in exactly the same manner as in Test Example 3 for cases o, 3, ■, A, and X. The results are shown in Table 11. Although the therapeutic effect of each of the monoclonal antibodies that showed a strong preventive effect in Test Example 2 was reduced to about 1/10, therapeutic effects were also observed.

Table 2 I IgG + 10o-20Dng 2 xgM+ >1.5 5 IgG) 700 μg 4 IgG + 301] ng 5 IgG + 10000-200 ngG>10q *Peroxidase-labeled goat anti-mouse IgG antibody and the same labeled goat anti- It was measured by the DIBA method using a mouse IgM antibody (Kabbell).

Table 3 ■IgG + 300ng II IgG + 400ng ill Igo 800μg IV ■gG-+, s Misaki *Same as Table 2 Table 4 BIgG+1oo~2oong 0 1gM -1-1,0MisakiDIgG>7ooμg Table 5 y IgG + , 100-200 ng Table 6 2 Igo -) 0.7-3 IgG + 400 ng *Peroxidase was detected using a labeled goat anti-human IgG pile and the same labeled goat anti-human egM antibody (Cubbell).
Measured by IBA method and sent to T.

Table 7 I IgM + s o o pg IIIgM-2,0~ ] [Igo + 1010n-200 n'll rg, a + 600ng Table 8 Table 9 x IgG + 1oμg Y engineering gM +2.0Kg *Same as Table 6 Table 10 08 19 5 0.9 1 2, O n’2.5 B16 E, 3.0 X4.5 Y’　1.3 Table 11 ! l38 N 6. I A9. O X 10.0

Claims (1)

[Scope of Claims] 1. A monoclonal monoclonal whose globulin class is IgG, which has specific affinity for the serotype antigen of Pseudomonas aeruginosa and has direct agglutination activity against Pseudomonas aeruginosa cells. 2. A prophylactic and therapeutic agent for Pseudomonas aeruginosa hypostaining disease containing a small number of antibodies. The prophylactic and therapeutic agent for Pseudomonas aeruginosa hyposmosis according to claim 1, wherein the monoclonal antibody is of human origin.