JPS6242933A - Human type monoclonal antibody against varicella zoster virus and production thereof - Google Patents

Abstract

PURPOSE:To obtain the titled antibody useful for diagnosing and remedying infectious diseases caused by Varicella Zostr Virus, by preparing a hybridoma to produce a human type monoclonal antibody to react specifically with Varicella Zoster Virus and cultivating it. CONSTITUTION:A human lymphocyte sensitized with Varicella Zoster Virus (VZV for short) or protein or glycoprotein derived from it, in the presence or absence of mitogene in vitro, is fused with a tumor cell (e.g., mouse myeloma cell) to prepare a hybridoma to produce a human type monoclonal antibody to react specifically VZV, the hybridoma and/or a cell strain derived from it is cultivated and the aimed monoclonal antibody is collected from the culture mixture. The monoclonal antibody has an antibody to recognize glycoproteins having 108K molecular weight, 105K molecular weight and 62K molecular weight and an antibody to recognize glycoproteins having 90K molecular weight and 55K molecular weight and both the antibodies are usable for diagnozing and remedying virus infectious diseases caused by VZV.

Description

DETAILED DESCRIPTION OF THE INVENTION (f) Field of Industrial Application The present invention is directed to the use of Varicella virus (varicella)
laZoster Virus +hereinafter referred to as VZV)
The present invention relates to a human monoclonal antibody (hereinafter referred to as MOA) directed against the human monoclonal antibody (hereinafter referred to as MOA) and a method for producing the same. The purpose is to
Useful for diagnosis and treatment of viral infections caused by ZV,
The purpose of the present invention is to provide a human-type MOA specific to vzv.

(b) Prior art and its problems A mouse-type MOA for vzv has already been produced (T, 0kuno et al.).

Vlrology, volume 129, page 357, 1
983).

But 1. Since mouse-type MOA is antigenic to humans, antibodies are generated when administered into the human body, causing side effects such as anaphylaxis. Therefore, humanized MOA would be particularly desirable for therapeutic purposes.

Human-type MOA includes MOA for cancer cells (
J. Schlom et al., Proc. Natl., Ac.
ad.

Sci, USA, vol. 77, p. 6841, 1980)
.

MOA for tetanus toxin, (N, N, Te
ng and others + Proc, Natl, Acad, Sc
1. USA+80 volumes.

7308 pages, 1983), MOA for measles
(0, A, 0roce et al., Nature+ 28
8 volumes, 488 pages, 1980). However, compared to mouse-type MOA, human-type MOA
Only a very limited amount is known so far,
The human MOA for VZV is also unknown. There are two reasons why human-type MOA is more difficult to produce than mouse-type MOA. The first point is that the efficiency of forming hybridomas that produce MOA is very low when using human lymphocytes, and the second point is that it is difficult to immunize humans in many cases, so it is difficult to effectively stimulate them. (was done)
It is difficult to obtain an IJ ball. In particular, the latter problem is caused by human-type MOA that is specific to infectious diseases or cancer.
This is important in the production of

(c) Means for Solving the Problems The present inventors have discovered that there is currently no way to immunize humans with VZV by sensitizing them in vitro. Considering that, as a result of Kei and σ research, v
Human lymphocytes (antibody-producing cells) sensitized with zv,
The abdomen of mouse myeloma cells and human lymphocyte cell lines* I! ! Either create hybridomas fused with the Epstein-Barr virus (Eps).
By transforming the cells with tein-Barr VIrus+ (hereinafter referred to as BBV), a cell line producing a human type MO human specific to vzv was obtained. First, the present inventors were able to culture such a Ml cell strain and obtain a human-type MOA that specifically reacts with VZV from the culture.

iii) The present invention is a human-type MOA that specifically reacts with VZV. Such a humanoid M0 person is - in vl
tro in the presence or absence of mito-diene,
vzv or a protein or glycoprotein derived from the virus (
By fusing human lymphocytes and tumor cells, vz
A method that produces a human-type MOA that specifically reacts with vVC.
It can be obtained by creating a hybridoma, culturing the hybridoma and/or a cell line derived therefrom, and collecting MOA from the culture.

Alternatively, lymphoblastoid cells, which produce human-type MOA that specifically reacts with vzv, obtained by transforming human lymphocytes sensitized as described above with EBV, and/or It can also be obtained from cultures of cell lines derived therefrom.

In the present invention, human lymphocytes include, in addition to the 81777 cells producing antibodies, their progenitor cells, memory cells, and the like. These cells are obtained from tissues such as human spleen, lymph nodes, peripheral blood, bone marrow, tonsils, and adenoids. but,
There are very few lymphocytes specific for a given antigen, so even if you create a hybridoma or transform it with gBV, you will not be able to use hiOA specific for that antigen with yU.
The probability of obtaining viable cells is extremely small. Therefore, in the present invention, lymphocytes are in vitro treated with antigens as follows.
o Sensitize. For in vitro sensitization, for example, lymphocytes are incubated at 0.5 x 106 to 3 x 106/
Disperse the cells in a culture medium at a density of 1110 cells and place them in a 96-well plate with 0.1 to 0.2 Il17! by,' or 24
Put 2ml each of T+ and 57E into a well plate.

As an antigen, (6) zv with inactivated staining power or its virus-derived protein or sugar kumbaku is used,
Pa ball 1′! - permeate (1,2 ng/m to 1,0
00ng/l, preferably +t 1-1100n/d
Add the antigen. Lymphocyte mitogen is also effective for antigen sensitization. Examples of mitogenes include pork lyde mitogene (hereinafter referred to as PWM), protein A1 B cell growth factor (so-called BOGF)
etc. When using PWM, 1Pg/+d
to 200μg/'% preferably about 20μg/'%
d to the lymphocyte suspension. In particular, antigen and PWM
When added together, lymphocytes specific for that antigen synergistically increase. In this way, an antigen or an antigen and a mitogen are added to a lymphocyte suspension, and cultured for 3 to 20 days, preferably 4 to 8 days, at 37°C.
The culture solution may contain various serums, such as fetal bovine serum (hereinafter referred to as FO8), calf serum, bear serum, etc. (for example, RPM11640 or Dulbecco-MEM).
is used.

The sensitized human lymphocytes thus obtained are fused with mouse or human lymphoid cell lines or transformed with BBV. Cell fusion partners include mouse minima cell line, P3X63Ag8, P3-N8.
1/1-Ag4-1, P3X63Ag8ul, 8P2/
θAg14, P3X63λgB, 6.5.3, MPOI
! -45,6, TOI, 7 or 5P-1 are used. Myeloma RPM is a human lymphoid cell line.
I8226 strain, lymphoblastoid cell u729-6 strain and ARH
77 strains, and even B lymphoma) leak. In addition, heteromyeloma derived from mouse myeloma and human lymphoid cells (N, N + Teng et al.
c* Nati, Acad, 8cl.

USA, Vol. 80, p. 7308, 1983) can also be used.

Sensitized lymphocytes and mouse or human lymphoid cell lines are fused according to known methods as follows.For example, sensitized lymphocytes and lymphoid cell lines are fused at 10:
Mix at a ratio of 1 to 1:100, preferably 1:1 to 1:20, and add a suitable cell fusion solution, such as 35 polyethylene glycol (molecular weight about 1.000 to 6.000) and 7.551r dimethyl.几P containing sulfoxide
Add M11640 and heat for 1 to several minutes at room temperature to 37°C.
Gradually dilute with ii'08 added RPM1164θ,
After washing, cell density was reduced to 0.0 with hypoxanthine-aminopterin-thymine (hereinafter referred to as HAT) selection culture medium.
Adjust to 5 to 5 x 105 pieces/d. This is 5
．． Dispense 2 ml into a 93-well plate, and culture in humid air containing 5002 at 35-38° C. for 2-4 weeks.

In the HAT medium, only hybridomas survive, and unfused 8-azaguanine-resistant lymphoid cell lines and lymphocytes die.

After culturing, the antibody titer in the culture solution is screened to select hybridomas producing the desired MOA. Radioimmunoassay (RIA) is a screening technology.
), enzyme antibody method (BLIS), fluorescent antibody method, etc.

Instead of creating hyperdomas, we used poorly produced lymphocytes with E.
Cells that continue to produce EBV, such as B
When the 95-8 cell line is cultured, the culture supernatant contains infectious EBV. RB this culture supernatant.
As a source of V to transform lymphocytes.

When transforming, set the BBV to 0, for example.
．． 01-10, -10 = preferably 0.2-1 transform unit (T
I) 5o) Add 7077 balls to 5.

Then, incubate at 37°C for about 1 to 2 hours.
1, then remove the free virus by 16 (hearts) and remove the resulting iaBv infection by 10-
2fJ '% Li' □ SCA RP M I l 6
Distribute 40 j pieces jlk K. This 1411 cells i゛ii fluid (wholesale 1 ship 400~so, ooo pieces/n
Inoculate 0.2 ml of I) into a 6-well plate (and, for example, ;) at 7°C in a 00□ incubator. Screen as in the case of dormers.

The M OA IG live cell line thus obtained is cloned by the limiting dilution method or the soft agar method. cloned hybridoma or E
BV) Transformants are stored in a cell storage tank containing liquid nitrogen.

The cell line (cell a) of such a hybridoma or EBV l-transformant and/or its derived cellularity ranges from +1 to +1! ','c TZj method C large i+t
When cultured, it is possible to obtain 1 → human-type MO&, which has a clear nl effect, from the culture supernatant! Ru. This bar f noridoma again! 11. ! Transfer to the material: tf I-tel 1 [Useful, human type 1110 Af
You can also get a'. Kakarokata〃〈By Funera 1+
MOAlt, which is characteristic of the present invention, specifically reacts with VZV. -+: (-, especially human type M (JAV
I is the molecule jjtl Fl 8, 105Kli and 62
5 characteristics to recognize ni no 搏, t1℃J and more fj
Furthermore, the human type M OA V 2 ha, molecule 1q 9 (
Recognizes the protein anti-j to lK to55. And in the presence of VC + rg body for both ■1 and ■2 -〇, M OAIA
It has excellent lower phase activity, inactivating VZV by a factor of 50 at concentrations below 17μg/17.

Hereinafter, the present invention will be explained in detail with reference to Examples.

2) Example Example VZ Hypuri that produces a human-type MOA for ViC
Doma (1) Preparation of VZV antigen VZV (Ka
waguchi I strain) was inoculated, and when a cell degeneration appeared, the cells were removed with a rubber policeman. 1 cell
After being destroyed by ultrasonication for 1 minute,
It was centrifuged for 0 hours. On top of that, a was placed on a layer of cesium lypride and centrifuged at 23,000 rpm for 3 hours. Arm degree 1.
■8v gathered between 4 and 1.2. This virus fraction was further precipitated by centrifugation at 25,000 rpm for 1.5 hours, and the precipitate was dispersed in phosphate buffered saline for 3 seconds for 5 seconds.
Ultrasonication was performed twice.

The virus preparation thus obtained was inactivated by ultraviolet rays and Ta irradiation, and then used in the following experiments.

(2) Sensitization of lymphocytes with VZV Culture medium A (RPMI 1640+) of human tonsillar lymphocytes.
20cm fetal bovine serum + 20mM 1110PE
884-2rn Glutamine'-1, mMNa Pyruvine N +0.02 +ny/tel Serine +80μ97
2.6 x 10' pieces/l1l
It was suspended in l. In addition to this, the VZV obtained in (1) above (
Final concentration 17 ng protein/ml) and P
WM (20lt g/'') was added and cultured for 3.5 or 7 days. The lymphocytes were cultured at C5 (6) and used for the following cell fusion.

(3) Mouse miniroma cells P3X63,
P2H4 was cultured in culture medium B (ILI'MI 1
640 + 10 Chi Dead Serum + 2 mM Glutamine + 80
Cells were cultured (μg/ml gentamicin) at a concentration of 6 × 105 cells/ml when used.
Met. The 4 groups of sensitized lymphocytes (3 wells combined) and P2H4 from (2) above were separately treated with serum-free PM1.
It was fkS-purified twice with 1640. Lymphocytes of each group and 5x1
0'fb! I) 3! -TI and were combined in a test tube. 15 (discharged at 10 rpm for 5 minutes, and discarded the supernatant. The cell pellet was well dispersed by tapping the test tube.

Add 0.5ml of polyethylene glycol solution (RPM
I 1640 5.75 t: Mal+polyethylene glycol 1000 3.5 In/+dimethyl sulfoxide 0.75 rAt) (approximately ml of Pl-G solution) was added to allow the cells to slowly migrate to lγ. After 1 minute (1,5 ml R,P M' I 1640 was added, after another minute 1 ml IL P tvl I and after another 2 minutes 4 Tnl of HAT culture (R, I'MI)
640 + 20 multifetal bovine serum + 80 μg/lI + /gentamacin + 95 μM hypoxanthine + 0.4 μM aminopterin + 1.6 μM thymidine), and after another 2 minutes, 4 mt of HAT culture medium was added. Finally, 1 #7 (t) of 25 d cells were closed with 5 ml of HAT medium. These were plated on one culture plate (96 wells) and incubated at 37°C for 5 ml of cells.
2-containing air. - Half of the culture solution was replaced with fresh HT culture solution (HA'T without humans) every week, and Hyzuridoma was obtained.

(4) Measurement of human anti-VZV antibodies The IgM or IgG anti-VZV MOA of the culture supernatant of the hybridomas thus formed was measured by enzyme antibody method. That is, in order to measure anti-VZV antibodies, VZV (Kawaguchi strain) 2 μg protein/gold was immobilized in each 96-well 7' rate of Falcon Microtest ■. This plate contains 60μ of Noka Ipridoma culture soil! was added and left at room temperature for 1 hour. and 1%
Hank's saline solution containing BS (bovine serum albumin)
Goat anti-human II
G antibody or anti-1gM antibody-alkaline phosphatase (2000-fold dilution) at 60μ! In addition, 1 o'clock I! at room temperature! 1 reaction. After further washing three times with HBSS-BSA, P-2) p-phenyl phosphate was IM:
) Ethanolamine+1 rn M M g O! 2
100μ of a solution of 0.6mg/d S in a pH 9.8 solution! added. 40 after 30 to 60 minutes
Absorbance at 5 mμ was measured.

The results are shown in Table 1.

Table 1 Example 2 Lymphocytes were isolated from B viscera surgically removed from a patient with autoimmune thrombocytopenia by the Ficoll-Bac method. These lymphocytes were treated with V Z V (17 ng/ml) and P WM (
20 μg/mt) and sensitized for 6 days. The thus sensitized lymphocytes were subjected to cell fusion in the same manner as in Example 1 (3). Regarding the hybridoma culture supernatant thus formed, total human IgG and IgMS V
IgG1 and IgM reacting with ZV-infected lie 1 cells,
More non-infectious Hel? IgG & I that react with t83 cells
gM was measured by enzyme antibody method. The results are shown in Table 2 as the number of people per 96-well plate.

From Table 2, it can be seen that selective IgG type M0 production occurs in vzv.

Furthermore, it can be seen that the number of hyzuridomas producing such yzv-selective MO individuals is significantly increased by VZV+PWM sensitization. Such hybridomas appear even with PWM alone (6155), but compared to sensitization with vzy alone (9/27), non-selective hybridomas are present.

191 Example 3 Cloning of anti-vzv antibody-producing hybridomas and specificity of anti-vZVMOA.

(1) Cloning Cells were taken out from the anti-VZV activity-positive wells, the number of cells was counted, and the cells were plated in a 96-well culture plate at 1 cell/well or 0 cells/well. 3 to 4 weeks 11. Since the cells proliferated sufficiently after Il, anti-VZV was added to the culture supernatant.
Screening was performed to see if there was antibody activity. In this way, the derived cells were selected from the one that was seeded at 1 cell/well as much as possible. This operation was repeated 2 to 4 times to ensure that all clones had anti-VZVA activity.

Three 71 hybridomas were established by mouse x human cell fusion. Those 1 hybridoma (6 to 25 μm)
It produced an MOA of 106 g/day. These were named vl, v2, and v6.

(2) Specificity of MOA The Zab class and L-type of V1, V2, and V6 were determined. Sheep anti-human IgG, anti-human) 1gG2, anti-human 1
g Octa-P- using G3 anti-human 1gG4
The subclass was determined based on the construction method. As a result, v11v
Both 2 and v6 reacted only with the anti-Human IgG1 antibody.

In addition, as a result of BLISA using alkaline phosphatase-goat IgG anti-human or anti-human IgG, three MOA
Both were found to have large mirrors.

In order to examine the immunospecificity of Vl, v2, and v6, we used BLI8A with various viruses or their host cells as antigens.
I did this.

The results are shown in Table 3.

Although vl, v2, and v6 reacted with all six strains of VZV tested, they did not react with their host cell HBL at all. In addition, vl reacted weakly to H8V types 1 and 2, but
v2 and v6 did not react. In general, neither MOA reacted to OMV or EBV.

Table 3 Example 4 Determination of antigen molecules recognized by anti-VZV-MOA Antigen molecules were determined by immunoprecipitation and nylium dodecyl sulfate-polyacrylamide gel electrophoresis (8DS-PAGB).

Human brother and sister cells < HE L ) on the second day of culture were suspended in a monolayer culture (64 m) with trypsin. V of Ml
ZV infected cells were inoculated. When cell degeneration (OPE) of 80q6 or more appears after 48 hours, the medium was diluted with 1
00μci)! J thium glucosamine lactate ((1
,6-3H(N)) glucosamine;20
ci/mmol; NEN Re5search Pr
oducts.

Boston, M person) 1/10 glucose midfielder
iM-2%FO8 or 5d of 50/IIcI ("8
)-methionine (400ci/mmol; N
BN Research Products) was replaced with 1/10 methionine MEM-2% FO8. The cells were further cultured at 37°C for 18 hours, and when opE had spread throughout the monolayer cells, the cells were collected. The collected cells were washed and treated with 10 mM Ti1a H01 (pH 7,
4), 0.15M NaOf, 1mM phenylmethylsulfonyl fluoride (81gna Ohmlcal
s 00. ST Laues, Mo), 1mM
Ethylene dianine tetra vinegar H11% Trlton X
-100, o, 1% sodium dodecyl sulfate, 1
It was dispersed and solubilized by sonication in tindium deoxycholic acid (RIPA buffer). This was superimposed on a 60% sugar solution, and insoluble materials were removed by ultracentrifugation at 100,000'g for i hours, and the supernatant was used as an antigen.
0 (3n)-glucosamine-labeled antigen fraction (200 μ) stored frozen at 0°C.

(400,000 cpm) or (358) methionine-labeled antigen fraction (200 p11.200,000 cpm)
and 71 Izuridoma supernatant 150/14 were mixed and allowed to react at room temperature for 30 minutes and then at 4°C for 11 minutes. The next morning, pre-swelled Poutine A-Sephap-su 0L.
4B beads (containing 3 mgr in 100 μl source solution: Phar+nacla Flne Ohe
mlcals-Inc, e Tokyo) was added and heated at 4°C.
The mixture was allowed to react for 3 hours. Beads containing immune complexes were pelleted by centrifugation and washed 5 times with 2 d RI PA buffer and then 2 times.
d with 10 mM Tr IH0+ (pH 6,8).
Washed twice. The washed immune complex is 110114
of 5D8-sample buffer was added and eluted at 100°C.

8Df9-PAGE electrophoresis of solubilized samples was carried out in Tr at room temperature.
ys Hot-Glycine-S D S Mild 517 liquid system (
It was carried out according to the standard method of PH8,3). After soaking Slab Guru in IM sodium salicylate aqueous solution,
Dry on paper. The dried glue was exposed to KODAKX-OMAT S film at -70°C for 7 to 21 days.

The results are shown in Figure 1. MOA vl is molecule jl108
It was recognized that v2 recognized glycoproteins with molecular weights of 90 and 55, while v2 recognized glycoproteins with molecular weights of 90 and 55.

In FIG. 1, vl (human row) and v2 (row B) were precipitated with (35s)-methionine labeled viral antigen. vi (row O) and V2 (row D) were precipitated with [3H]-glucosamine labeled antigen.

The arrow on the right indicates the mobility of the standard protein. 1, K has a taste of kilodaltons.

゛Still, ■6 is all vl etc. (same ton A body movement pattern shown)
-It is.

Example 5 Anti-vzv-me (OA virus-neutralizing anti-VZV
- The supernatant of MOA-producing cells was stained, MOA was precipitated with 50% saturated ammonium sulfate, and passed through phosphate-grade saline. The presence of MOA in these preparations was determined by the immuno-dimensional acid expansion method using anti-Human IgG antibodies. Diluted 3 times serially and diluted the solution 1 (20 (IAI) and VZ
V (Mr. Kawaguchi.

4000 PFU, 100 μl) and guinea pig complement (5
100 μl diluted solution or 5 times f'08-ALB
M (10 (1/71)) was mixed and cultured at 1-137°C for 1 hour. This virus mixture was added to the previously cultured monolayer culture of HEL cells and incubated at 37°C for 1 hour. The virus was adsorbed.After that, 2.5≠Me・j′−
5% FO8-MEM containing nilulose
KMi: JL, 5 to 71jli1jCO□Cooked in culture dish. That story, infection u z 1. , cells were fixed with lumin at 1()≠71-1 and 0.15%
Stain with crystal violet 1 and count the number of plaques ii
It turned red.

So' J a dumb, M OA, V l, v2 and V 6
The virus activity was as shown in the fourth column.

It can be seen that ■1 and ■2 have excellent neutralizing activity, with the amount of MOA required to inactivate the virus by 5° being less than 1 μg/m.

Table 4 Example 6 Preparation of human x human hybridoma 1.2 × 106 cells in (2) valley L1 of Example 1 /
The sag was adjusted to match ml V. This net January'd
i'7 A! I put it in the hole of 24 holes of 62 holes each. There, anti-It (vzv.

Kanbun-3 was added to the mixture (Kawa 11 strain) at a concentration of 4.9 μg protein/ml, and PWM was added at a concentration of 20 μg/ml. 00□6 days 11 hairs 1 with inkcovator
Lymphocytes were sensitized to the antigen by culturing.

[111-(Human lymphoblastoid cells 729-6 were cultured Xi 13 (TtL'MI 1640 + 10% FO8
-4-2mM glutamine + 8 () μg/ml gentamicin). Details when using: If
The sensitized lymphocytes were pooled in 12 wells and washed twice with serum-free IFPM 11.640.

After washing the 729-6 cells twice, 2x1.07 cells of 729
-6 cells were mixed with the sensitized bulbs in a centrifuge tube. ]
, 50 (l r p m) for 5 min, and the supernatant was discarded. The cell pellet was removed by tapping the test tube.
Well dispersed. EG solution was added and the cells were gently aged for 1 day.

After 1 minute, 2 shoulders j R, I' M I 1.64 (Add 1, and then for another minute, temporarily 4 W/ n P M I 11
After 2 minutes of further iC, 8at HAT culture (jLI'M
II 640 + 20% F Ci S + 80μg/gogentamicin + 95μM hypokiri = 7tin + 0.4μN
(aminopterin + 1.6 μM thymidine), and after another 2 minutes, 15 m of HA '11' culture was removed. added.

Finally, incubate 1 (10Iit #Il) with HA, T culture medium.
+ month 14i +% free liquid. Place this on a culture plate (96
(holes) were sown in 4 sheets and doubled in size at 37°C in 5% 00□ sound aqueous air.

Hybridomas were obtained by replacing half of the culture solution with fresh IIT culture solution every -week. -C(-), antibodies were measured after 3 to 4 weeks of culture.

Growth of high noridomas was observed in 118 out of 384 wells on 4 plates. As a result of screening these cultures and supernatants for anti-ZV antibody activity, 16 wells were positive. Antibody titers against the insensitive f(QIIEL) were not measured, and the human-type Pi(OA) growing in these holes is considered to be specific for I.
It was gG.

Example 7 Preparation of shape-transforming cells using i>nv (1)IuH
Preparation of human B cell-derived EBV BO2
-8 Keyakin cells were returned from suspension and culture medium 0 (RPMI
1.64 +1+I OφF OS +501. U,
/vIl streptomycin + 50 μg/ml penicillin G potassium) to 4I12
l/me teIui embedding, at 37℃ 3]] songs cultured. The cells were again cultured in new culture medium C at 151 cells/d, and cultured at 1111 for another 2 days. The final concentration I x 1.05 (j,',
l/ml for another 7 h at 37 °C in fresh culture medium C.
3. Culture supernatant containing WBV after inter-culturing. (+00rpm
The mixture was centrifuged 4 times for 30 minutes and further filtered through a Millipore filter with a pore size of 0.45 μm. The I(BV preparation I+! solution obtained by
TD5o/i! Met.

(Sensitization of lymphocytes with 21VZV and transformation of lymphocytes with B 13V Human pi visceral lymphocytes were incubated in culture medium 13 (see (3) of Example 1) at 2X
106 cells/ml were suspended. Add this cell suspension to 1.
2i/each were placed in the wells of a 24-well plate. vzy to this
(Kawaguchi-like) 4.9 ng protein/-1 was added and cultured in a C02 incubator for 4 days.

The thus sensitized lymphocytes were treated at 1 +500 Or pm.
After centrifugation for 5 minutes, the cell pellet was well dispersed by tapping the test tube. To this, the EBV prepared powder obtained in (1) above was added to 0.5 T I) so/c
Add in a volume of e I 1 and heat at 37°C with occasional stirring.
Incubated for 0 minutes.

Finally, culture solution 1) RPMI 164 o+t o%F
OS - 1-2mM glutamine + 1mM Na-pyruvic acid 0.02mg/ml serine + 80μg/ml
ml gentamicin) and add 1.2ml to a 96-well plate.
X 10' cells/hole/o, 2 mt
The cells were plated in a culture medium and cultured at 37° C. in air A containing 500 square meters. -AJ Replace the beef oil culture solution with new culture solution 1) every 1 liter to eliminate lymphoblastoid MII cells.

Lymphoblastoid cells were added to the wells of all 4 plates. As a result of determining the anti-VZV antibody titer till the transform point 4 AJ, 238 out of 384 wells were injected with the antibody. However, before cloning began, many +n+ complements had ceased to produce antibodies and continued to produce antibodies.

Twelve clones were cloned by soft agar method. Three of them are anti-VZV human MOA.
It was established as a lymphoblastoid cell line that produces .

[Brief explanation of drawings]

Figure 1 shows the antigen recognized by anti-VZV, MOA, 8DS
-PAGE electropherogram. Figure 1

Claims (1)

[Scope of Claims] 1. A human monoclonal antibody that specifically reacts with varicella zoster virus. 2. The human monoclonal antibody according to claim 1, which recognizes glycoprotein antigens with molecular weights of 108K, 105K, and 62K. 3. The human monoclonal antibody according to claim 1, which recognizes glycoprotein antigens with molecular weights of 90K and 55K. 4. Human lymphocytes sensitized with varicella zoster virus or proteins or glycoproteins derived from the virus are fused with tumor cells in vitro in the presence or absence of mitogenes to produce varicella zoster. Varicella zoster treatment, which involves creating a hybridoma that produces a human monoclonal antibody that specifically reacts with the herpes virus, culturing the hybridoma and/or a cell line derived therefrom, and collecting the monoclonal antibody from the culture. A method for producing human monoclonal antibodies that specifically react with the rash virus. 5. The method for producing a human monoclonal antibody according to claim 4, wherein the tumor cells are mouse myeloma cells. 6. The method for producing a human monoclonal antibody according to claim 4, wherein the tumor cells are human-derived lymphoid cell lines. 7. Transforming human lymphocytes sensitized with varicella zoster virus or proteins or glycoproteins derived from the virus in vitro in the presence or absence of mitogenes by Epstein-Barr virus. Lymphoblastoid cells and/or cell lines derived therefrom that produce human monoclonal antibodies that specifically react with varicella zoster virus are cultured, and the monoclonal antibodies are collected from the culture. A method for producing a human monoclonal antibody that specifically reacts with varicella zoster virus.