JPH01202295A - Anti-hd monoclonal antibody and its production - Google Patents
Anti-hd monoclonal antibody and its productionInfo
- Publication number
- JPH01202295A JPH01202295A JP63025507A JP2550788A JPH01202295A JP H01202295 A JPH01202295 A JP H01202295A JP 63025507 A JP63025507 A JP 63025507A JP 2550788 A JP2550788 A JP 2550788A JP H01202295 A JPH01202295 A JP H01202295A
- Authority
- JP
- Japan
- Prior art keywords
- human
- antigen
- lymphocytes
- monoclonal antibody
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は抗ＨＤモノクロナール抗体及びその製法に関す
る。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an anti-HD monoclonal antibody and a method for producing the same.
〔従来技術及びそれが解決しようとする課題〕ＨＤＨＤ
抗原ａｎｇａｎｕｔｚｉｕ−Ｄｅｉｃｈｅｒ抗原の略記
であり、ヒトとニワトリの正常組織には存在しないが、
癌化すると腫瘍組織の細胞膜上に見出され、また癌患者
の血清中に出現することが知られている。化学的には末
端にＮ−グライコリルノイラミン酸があり、いわゆる２
型の糖脂質であることがわかっている。またこの抗原は
ヒトとニワトリ以外の動物、例えばウマ、ブタ等の組織
や血清中には存在することも分かっている。[Prior art and the problems it attempts to solve] HDHD
Antigen Anganutziu-Deicher antigen, which is not present in normal tissues of humans and chickens,
It is known that it is found on the cell membrane of tumor tissue when it becomes cancerous, and also appears in the serum of cancer patients. Chemically, there is N-glycolylneuraminic acid at the end, so-called 2
It is known that it is a type of glycolipid. It is also known that this antigen exists in the tissues and serum of animals other than humans and chickens, such as horses and pigs.
以上文献１）〜１１）が参照される。References 1) to 11) are referred to above.
１） Ｈａｎｇａｎｕｔｚｉｕ、Ｍ、＋１９２４．Ｈ
６ｍａｇｇｌｕｔｉｎｉｎｅｓＭｔ４ｒｏｇ６ｎｅｔｉ
ｑｕｅｓ ａｐｒｅｓ １ｎｊｅｃｔｉｏｎ ｄｅｓｅ
ｒｕｍ ｄｅ Ｃｈｅｖａｌ、５ｏｃｉｅｔｊ ｒｏｕ
ｍａｉｎｅ ｄｅＢｉｏｌｏｇｉｅ、９１：１４５７゜
２） Ｄｅｉｃｈｅｒ、Ｈ，，１９２６，口ｂｅｒ
ｄｉｅ Ｅｒｚｅｕｇｕｎｇｈｅｔｅｒｏｓｐｅ
ｚｉｆｉｓｈｅｒ Ｈ；ｉｍａｇｇｌｕｔｉｎｉｎｅ
ｄｕｒｃｈＩｎｊｅｋｔｉｏｎ ａｒｔｉｒｅｍｄｅｎ
Ｓｅｒｕｍｓ、Ｚｅｉｔｓｃｈ、Ｈｙｇ。1) Hanganutziu, M., +1924. H
6magglutininesMt4log6neti
ques apres 1 njection dese
rum de Cheval, 5ocietj rou
maine deBiology, 91:1457゜2) Deicher, H., 1926, mouth ber.
die Erzeugungheterospe
zifisher H; imagglutinine
durchInjection artiremden
Serums, Zeitsch, Hyg.
Ｉｎｆｅｋｔｉｏｎｋｒ、、１０６：５６１゜３）
５ｔｕａｒｔ＋Ｃ，Ａ、＋＾、Ｍ、Ｇｒｉｆｉｎ、に、
Ｍ、Ｗｈｅｅｌｅｒ ａｎｄＳ、Ｂａｔｔｙ、１９３６
．＾ ｔｈｅｒｍｏｓｔａｂｌｅ ａｎｔｉｇｅｎ
１ｎＢＥＦ−ｃｅｌｌｓ、Ｐｒｏｃ、Ｓｏｃ、Ｅｘｐ、
Ｂｉｏｌ、３４：２１２４） Ｄａｖｉｄｓｏｈｎ、
１．、ａｎｄ Ｐ、Ｈ，Ｗａｌｋｅｒ、１９３５．Ｔｈ
ｅｎａｔｕｒｅ ｏｆ ｈｅｔｅｒｏｐｈｉｌｅ ａ
ｎｔｉｂｏｄｉｅｓ １ｎｉｎｆｅｃｔｉｏｕｓ
ｍｏｎｏｎｕｃｌｅｏｓｉｓ、Ａｍｅｒ、Ｊ、Ｃｌ１ｎ
。Infectionkr, 106:561゜3)
5tuart+C, A, +^, M, Griffin,
M. Wheeler and S. Batty, 1936
．． ^ thermostable antigen
1nBEF-cells, Proc, Soc, Exp,
Biol, 34:2124) Davidson,
1. , and P. H. Walker, 1935. Th
nature of heterophile a
antibodies 1infectious
mononucleosis, Amer, J, Cl1n
.
Ｐａｔｈ、、５：４５５
５） Ｋａｓｕｋａｗａ、Ｒ，、ＫＪａｎｏ、Ｍ、Ｌ
、Ｂ１ｏｏｎ＋ ａｎｄ Ｆ。Path, 5:455 5) Kasukawa, R., KJano, M.L.
, B1oon+ and F.
Ｍｉｌｇｒｏｍ、１９７６、Ｈｅｔｅｒｏｐｈｉｌｅ
ａｎｔｉｂｏｄｉｅｓ ｉｎｐａｔｈｏｌｏｇｉｃ
ｈｕｍａｎ ５ｅｒａ ｒｅｓｅｍｂｌｉｎｇａｎｔｉ
ｂｏｄｉｅｓ ｓｔｉｍｕｌａｔｅｄ ｂｙ ｆｏ
ｒｅｉｇｎ ５ｐｅｃｉｅｓｓｅｒａ、ｃｌｉｎ、ｅｘ
ｐ、Ｉｍｍｕｎｏｌ、２５：１２２６） Ｙａｍａｋ
ａｗａ、Ｔ、、ａｎｄ Ｓ、５ｕｚｕｋｉ、１９５１．
Ｔｈｅ ｃｈｅｍｉｓｔｒｙｏｆ ｔｈｅ １ｉｐ
ｉｄｓ ｏｆ ｐｏｓｔｈｅｍｏｌｙｔｉｃ ｒｅｓ
ｉｄｕｅｏｒ ｓｔｒｏａｍａ ｏｆ ｅｒｙｔｈｒｏ
ｃｙｔｅｓ、１．Ｃｏｎｃｅｒｎｉｎｇｔｈｅ ｅｔｈ
ｅｒｉｎｓｏｌｕｂｌｅ １ｉｐｉｄｓ ｏｆ Ｉ
ｙｏｐｈｉｌｉｚｅｄｈｏｒｓｅ ｂｌｏｏｄ ｓｔｒ
ｏｍａ、Ｊ、Ｂｉｏｃｈｅｍ、、３８：１９９７）旧ｇ
ａｓｈｉ、Ｈ，＋Ｍ、Ｎａ１ｋｉ＋Ｓ、Ｍａｔｓｕｏ、
ａｎｄ Ｋ、０ｋｏｕｃｈｉ。Milgrom, 1976, Heterophile
antibodies inpathologic
human 5era resemblinganti
bodies stimulated by fo
Reign 5peciessera, clin, ex
p. Immunol, 25:1226) Yamak
awa, T., and S. 5uzuki, 1951.
The chemistry of the 1ip
ids of postmolytic res
idueor stroke of erythro
cytes, 1. Concerning the eth
erinsoluble 1ipids of I
yophilizedhorse blood str
oma, J. Biochem, 38:1997) old g
ashi, H, +M, Na1ki+S, Matsuo,
and K, 0kouchi.
１９７７、Ａｎｔｉｇｅｎ ｏｆ ”Ｓｅｒｕｍ ５ｉ
ｃｋｎｅｓｓ”ｔｙｐｅ ｏｆＨｅｔｅｒｏｐｈｉｌｅ
ａｎｔｉｂｏｄｉｅｓ ｉｎ ｈｕｍａｎ ５ｅｒ
ａ；１ｄｅｎｔｉｆｉｃａｔｉｏｎ ａｓ ｇａｎｇｌ
ｉｏｓｉｄｅｓ ｗｉｔｈ Ｎ−ｇｌｙｃｏｌｙｌｎｅ
ｕｒａｎ＋１ｎｉｃ ａｃｉｄ、Ｂｉｏｃｈｅｍ、Ｂｉ
ｏｐｈｙｓ。1977, Antigen of “Serum 5i”
ckness”type ofHeterophile
antibodies in human 5er
a;1dentification as gangl
iosides with N-glycolyne
uran+1nic acid, Biochem, Bi
ophys.
Ｒｅｓ、Ｃｏａ＋ｎ＋ｕｎ、、７９：３８８゜８）
Ｍｅｒｒｉｃｋ、Ｊ、Ｍ、、Ｒ，５ｃｈｉｆｆｅｒｌｅ
、に、Ｚａｄａｒｌｉｋ、Ｋ。Res, Coa+n+un, 79:388゜8)
Merririck, J. M., R., 5chifferle.
, in Zadarlik, K.
Ｋａｎｏ ａｎｄ Ｆ、Ｍｉ１ｇｒｏｍ＋１９７７．１
ｓｏｌａｔｉｏｎ ａｎｄｐａｒｔｉａｌ ｃｈａｒａ
ｃｔｅｒｉｚａｔｉｏｎ ｏｆ ｔｈｅ ｈｅｔｅｒ
ｏｐｈｉｌｅａｎｔｉｇｅｎ ｏｆ １ｎｆｅｃｔｉ
ｏｕｓ ｍｏｎｏｎｕｃｌｅｏｓｉｓ ｆｒｏｍｂ
ｏｖｉｎｅ ｅｒｙｔｈｒｏｃｙｔｅｓ、Ｊ、Ｓｕｐｒ
ａｍｏｌｅｃｕｌａｒＳｔｒｕｃｔｕｒｅ、６：２５７
９） Ｈｉｇａｓｈｉ＋Ｈ，、Ｙ、Ｎ１５ｈｉ、Ｙ、
Ｆｕｋｕｉ、に、Ｉｋｕｔａ、Ｓ、Ｕｅｄａ。Kano and F, Mi1grom+1977.1
solation and partial chara
cterization of the heter
ophileantigen of 1nfecti
ous mononucleosis fromb
ovine erythrocytes, J. Supr.
amolecularStructure, 6:257 9) Higashi+H,, Y, N15hi, Y,
Fukui, N., Ikuta, S., Ueda.
Ｓ、Ｋａｔｏ、 Ｍ、Ｆｕｊ ｉ ｔａ、 Ｙ、Ｎａｋ
ａｎｏ、 Ｔ、Ｔａｎｉｇｕｃｈｉ 、 Ｓ。S, Kato, M, Fujita, Y, Nak.
ano, T., Taniguchi, S.
５ａｋａｉ、Ｍ、５ａｔｏ ａｎｄ Ｍ、Ｎａ１ｋｉ、
１９８４．Ｔｕｍｏｒ−ａｓｓｏｃｉａｔｅｄ ｅｘｐ
ｒｅｓｓｉｏｎ ｏｆ ｇｌｙｃｏｓｐｈｉｎｇｏ−１
ｉｐｉｄ Ｈａｎｇａｎｕｔｚｉｕ−Ｄｅｉｃｈｅｒ
ａｎｔｉｇｅｎ ｉｎｈｕｇｇａｎ ｃａｎｃｅｒｓ
、Ｊｐｎ、Ｊ、ｃａｎｃｅｒ Ｒｅｓ、（Ｇａｎｎ）
。5akai, M, 5ato and M, Na1ki,
1984. Tumor-associated exp
reaction of glycosphingo-1
ipid Hanganutziu-Deicher
antigen inhuggan cancers
, Jpn, J, cancer Res, (Gann)
.
７５：１０２５゜
１０） Ｈｉｒａｂａｙａｓｈｉ＋Ｙ、、Ｈ，Ｈｉｇａ
ｓｈｉ、Ｓ、Ｋａｔｏ、Ｍ、Ｔａｎｉｇｕｃｈｉ。75:1025゜10) Hirabayashi+Y,,H,Higa
shi, S., Kato, M., Taniguchi.
ａｎｄ Ｍ、Ｍａｔｓｕｍｏｔｏ、１９Ｂ？、０ｃｃ
ｕｒｒｅｎｃｅ ｏｆ ｔｕｍｏｒ−ａｓｓｏｃｉａ
ｔｅｄ ｇａｎｇｌｉｏｓｉｄｅ ａｎｔｉｇｅｎｓ
ｗｉｔｈＨａｎｇａｎｕｔｚｉｕ−Ｄｅｉｃｈｅｒ
ａｎｔｉｇｅｎｉｃ ａｃｔｉｖｉｔｙｏｎ ｈｕｍ
ａｎ Ｍｅｌａｎｏｍａｓ、Ｊｐｎ、Ｊ、Ｃａｎｃｅｒ
Ｒｅｓ。and M, Matsumoto, 19B? ,0cc
urence of tomorrow-associa
ted ganglioside antigens
with Hanganutziu-Deicher
antigenic activity hum
an Melanomas, Jpn, J, Cancer
Res.
（Ｇａｎｎ）、７８：６１４゜
１１） ０ｈａｓｈｉ、ｖ、、Ｔ、５ａｓａｂｅ４．Ｎ
１５ｈｉｄａ＋Ｙ、Ｎ１５ｈｉ。(Gann), 78:614°11) 0hashi, v,, T, 5asabe4. N
15hida+Y, N15hi.
ａｎｄ Ｈ，旧ｇａｓｈｉ＋１９８３．）ｌａｎｇａｎ
ｕｔｚｉｕ−Ｄｅｉｃｈｅｒｈｅｔｅｒｏｐｈｉｌｅ
ａｎｔｉｇｅｎ ｉｎ ｈｕｍａｎ ｒｅｔｉｎ’ｏ
ｂｌａｓｔｏｍａｃｅ１１ｓ、Ａｍ、Ｊ、Ｏｐｈｔｈａ
ｍｏｌ、＋９６；３２１゜ところで、このＨＤ抗原を微
量測定したり、あるいはその性状のさらに詳細な検討を
するためにはそのモノクロナール抗体が用意される必要
がある。しかし、ここに解決されるべき課題が提起され
る。すなわち、前記されるごとく、このＨＤ抗原はヒト
とニワトリ以外の動物の血清中には通常に存在している
ために、用意されるべきモノクロナール抗体はその製造
過程の培養において無血清培地が使用され、しかもヒト
或いはニワトリ型のものでなければならない。例えば細
胞培養にあたり胎児仔牛血清が配合されている通常の培
地を使用した場合には、該血清中にすでにＨＤ抗原が存
在しているので、抗原刺激による新たなモノクロナール
抗体を得ることができない。また例えば抗原刺激に抗体
を産生せしめるにあたり常法に従いマウスを使用した場
合には、該マウス中にすでにＨＤ抗原が存在しているの
で、前記と同様に抗原刺激による新たなモノクロナール
抗体を得ることができない。従って、所定の抗体産生細
胞はまずヒト細胞が使用され、無血清培地で培養され、
次にこれをＨＤ抗原で抗原刺激することにより用意され
るものでなければならない。しかし、このような困難な
条件を満足させながら所定の抗体産生細胞を用意し、そ
のハイブリドーマを確立して目的とする抗ＨＤモノクロ
ナール抗体を得ることは未だ成功していない。and H, old gashi+1983. )langan
utziu-Deicherheterophile
antigen in human retin'o
blastomace11s, Am, J, Ophtha
mol, +96; 321° By the way, in order to measure a trace amount of this HD antigen or to conduct a more detailed study of its properties, it is necessary to prepare a monoclonal antibody. However, this poses an issue that must be resolved. That is, as mentioned above, this HD antigen is normally present in the serum of animals other than humans and chickens, so the monoclonal antibody to be prepared must be cultured in a serum-free medium during its production process. and must be of human or chicken type. For example, when a normal medium containing fetal calf serum is used for cell culture, the HD antigen is already present in the serum, so new monoclonal antibodies cannot be obtained by antigen stimulation. For example, if a mouse is used in a conventional manner to produce antibodies in response to antigen stimulation, the HD antigen is already present in the mouse, so new monoclonal antibodies can be obtained by antigen stimulation in the same manner as above. I can't. Therefore, human cells are first used as the predetermined antibody-producing cells, and are cultured in a serum-free medium.
Next, it must be prepared by stimulating this with HD antigen. However, it has not yet been possible to obtain a desired anti-HD monoclonal antibody by preparing a predetermined antibody-producing cell and establishing a hybridoma thereof while satisfying such difficult conditions.
そこで、本発明者は上記抗体産生細胞の形成を可能にす
るために必要な技術手段の解明を本発明の解決すべき課
題としてとりあげた。Therefore, the present inventors have taken up elucidation of the technical means necessary to enable the formation of the above-mentioned antibody-producing cells as a problem to be solved by the present invention.
種々の検討の結果、まずヒト牌リンパ球を使用し、別に
抗ヒトグロブリンμ鎖抗体およびコンカナバリンＡで刺
激したヒトリンパ球の培養上清の両者を含む無血清培地
を用意し、該培地中で前記ヒト牌リンパ球を培養し、次
にこれをＨＤ抗原によって抗原刺激すれば、前記課題が
解決されることを見出し、本発明を完成するに至った。As a result of various studies, we first used human tile lymphocytes and separately prepared a serum-free medium containing both an anti-human globulin μ chain antibody and a culture supernatant of human lymphocytes stimulated with concanavalin A. The present inventors have discovered that the above-mentioned problems can be solved by culturing human tile lymphocytes and then stimulating them with HD antigens, and have completed the present invention.
コンカナバリンＡで刺激したヒトリンパ球の培養上清（
ＣｏｎＡ培養上清と略記する）で刺激されたＴ細胞がＢ
細胞成長因子、Ｂ細胞分化因子および他の未知の成長因
子を分泌し、コンカナバリンＡ自体にもＢ細胞を活性化
する作用はあるが単独では増殖分化には到らない。文献
１２）〜１７）参照。Culture supernatant of human lymphocytes stimulated with concanavalin A (
T cells stimulated with ConA (abbreviated as culture supernatant) are B
Concanavalin A secretes cell growth factors, B cell differentiation factors, and other unknown growth factors, and concanavalin A itself has the effect of activating B cells, but alone it does not lead to proliferation and differentiation. See references 12) to 17).
１２） Ａｎｄｅｒｓｓｏｎ＋Ｊ、＋Ｇ、Ｍｏｅｌｌｅ
ｒ＋ａｎｄ Ｏ，Ｓｊｏｂｅｒｇ。12) Andersson+J, +G, Moelle
r+and O, Sjoberg.
１９７２、Ｂ ｌｙｗ＋ｐｈｏｃｙｔｅｓ ｃａｎ ｂ
ｅ ｓｔｉｍｕｌａｔｅｄｂｙ ｃｏｎｃａｎａｖａｌ
ｉｎ Ａ ｉｎ ｔｈｅ ｐｒｅｓｅｎｃｅ ｏｆｈｕ
ｍｏｒａｌ ｆａｃｔｏｒｓ ｒｅｌｅａｓｅｄ ｂｙ
Ｔ ｃｅｌｌｓ。1972, B lyw+phocytes can b
e stimulated by concanaval
in A in the presence ofhu
moral factors released by
T cells.
Ｅｕｒ、Ｊ、Ｉｍｍｕｎｏｌ、＋２：９９１３） Ａｎ
ｄｅｒｓｓｏｎ、Ｊ、、Ｇ、Ｍ、Ｅｄｅ１ｍａｎ＋Ｇ、
Ｍｏｅｌｌｅｒ＋ａｎｄＯ，Ｓｊｏｂｅｒｇ、１９７２
．Ａｃｔｉｖａｔｉｏｎ ｏｆ Ｂ ｌｙｍｐｈｏｃｙ
ｔｅｓｂｙ １ｏｃａｌｌｙ ｃｏｎｃｅｎｔｒａｔｅ
ｄ ｃｏｎｃａｎａｖａｌｉｎ Ａ。Eur, J. Immunol, +2:9913) An
dersson, J., G.M., Ede1man+G.
Moeller+andO, Sjoberg, 1972
．． Activation of B lymphocy
tesby 1locally concentrate
d concanavalin A.
Ｅｕｒ、Ｊ、 Ｉｍｍｕｎｏｌ、、２：２３３１４）
Ｋｅｈｒｌ、Ｊ、Ｈ，＋Ａ、Ｍｕｒａｇｕｃｈｉ、Ｊ、
Ｌ、８ｕｔｌｅｒ、Ｒ，Ｊ。Eur, J. Immunol, 2:23314)
Kehrl, J., H. +A., Muraguchi, J.
L, 8utler, R,J.
Ｍ、Ｆａｌｋｏｆｆ、ａｎｄ Ａ、Ｓ、Ｆａｕｃｉ、１
９８４．Ｈｕｍａｎ Ｂｃｅｌｌ ａｃｔｉｖａｔｉｏ
ｎ、ｐｒｏｌｉｆｅｒａｔｉｏｎ ａｎｄｄｉｆｆｅｒ
ｅｎｔｉａｔｉｏｎ、Ｉｍｍｕｎｏｌｏｇｉｃａｌ Ｒ
ｅｖｉｅｗｓ。M, Falkoff, and A, S, Fauci, 1
984. Human Bcell activation
n,proliferation and different
entiation, Immunological R
videos.
７８ニア５
１５） Ｍｕｒａｇｕｃｈｉ、＾、 、 Ｔ、Ｋａｓａ
ｈａｒａ、 Ｊ、Ｊ、Ｏｐｐｅｎｈｅｉｍ。78 Near 5 15) Muraguchi, ^, , T, Kasa
hara, J. J., Oppenheim.
ａｎｄ Ａ、Ｓ、Ｆａｕｃｉ、１９Ｂ２．Ｂ ｃｅｌｌ
ｇｒｏｗｔｈ ｆａｃｔｏｒａｎｄ Ｔ ｃｅｌｌ
ｇｒｏｗｔｈ ｆａｃｔｏｒ ｐｒｏｄｕｃｅｄ ｂｙ
ｍｉｔｏｇｅｎ−ｓｔｉｓ＋ｕｌａｔｅｄ ｎｏｒｍａ
ｌ ｈｕｍａｎ ｐｅｒｉｐｈｅｒａｌｂｌｏｏｄ Ｔ
ｌｙｍｐｈｏｃｙｔｅｓ ａｒｅ ｄｉｓｔｉｎｃｔ
＋ｌ１ｏｌｅｃｕｌｅｓ。and A.S., Fauci, 19B2. B cell
growth factor and T cell
growth factor produced by
mitogen-stis + lated norma
l human peripheral blood T
lymphocytes are distinct
+l1olecules.
Ｊ、 ｒｍｍｕｎｏｌ、 、 １２９：２４８６゜１６
） Ｍｉｃｈａｅｌ、Ｊ、Ｈ，Ｒ，、ａｎｄ Ｈ，Ｊ、
Ｍｉｃｈａｅｌ、１９８４．Ｔ−ｄｅｐｅｎｄｅｎｔ
ａｃｔｉｖａｔｉｏｎ ｏｆ ｒｅｓｔｉｎｇ Ｂ ｃ
ｅｌｌｓｍｅｄｉａｔｅｄ ｂｙ ｃｏｎｃａｎａｖａ
ｌｉｎ Ａ、Ｅｕｒ、Ｊ、Ｉｍｍｕｎｏｌ、。J, rmmunol, 129:2486°16
) Michael, J.H.R., and H.J.
Michael, 1984. T-dependent
activation of resting B c
ellsmediated by concanava
lin A, Eur, J, Immunol.
１４：２８０゜
１７） Ｒａｗｒｙｌｏｗｉｃｚ、Ｃ，Ｍ、＋Ｇ、Ｇ
、Ｂ、Ｋｌａｕｓ、１９８４゜Ａｃｔｉｖａｔｉｏｎ
ａｎｄ ｐｒｏｌｉｆｅｒａｔｉｏｎ ｓｉｇｎａｌｓ
ｉｎｍｏｕｓｅ Ｂ ｃｅｌｌｓ ＩＶ、ｃｏｎ
ｃａｎａｖａｌｉｎ Ａ ｓｔｉｍｕｌａｔｅｓＢ
ｃｅｌｌｓ ｔｏ １ｅａｖｅ Ｇ６．ｂｕｔ ｎ
ｏｔ ｔｏ ｐｒｏｌｉｆｅｒａｔｅ。14:280゜17) Rawrylowicz, C, M, +G, G
, B. Klaus, 1984゜Activation
and proliferation signals
inmouse B cells IV, con
canavalin A stimulatesB
cells to 1 leave G6. but n
ot to proliferate.
Ｉｍｍｕｎｏｌｏｇｙ、５３ニア０３
またＢ細胞の増殖分化の第一段階は抗原がＢ細胞表面の
レセプターに結合することであるが、抗ヒトグロブリン
μ鎖抗体がこの効果を代行することが知られている。文
献１Ｂ）、 １９）参照。Immunology, 53 Near 03 Furthermore, the first step in the proliferation and differentiation of B cells is for antigen to bind to receptors on the surface of B cells, and it is known that anti-human globulin μ chain antibodies act on behalf of this effect. See References 1B) and 19).
１Ｂ） ＤｅＦｒａｎｃｏ＋Ａ、Ｌ、＋Ｅ、Ｓ、Ｒａｖ
ｅｃｈｅ＋Ｒ，Ａａｏｆｓｋｙ、ａｎｄＷＪ、Ｐａｕｌ
、１９Ｂ２．Ｆｒｅｑｕｅｎｃｙ ｏｆ Ｂ Ｉｙｍｐ
ｈｏｃｙｔｅｓｒｅｓｐｏｎｓｉｖｅ ｔｏ ａｎｔｉ
−ｉ＋ｗｍｕｎｏｇｌｏｂｕｌｉｎ、Ｊ、Ｅｘｐ。1B) DeFranco+A,L,+E,S,Rav
eche+R, Aaofsky, and W.J., Paul.
, 19B2. Frequency of B Iymp
responsive to anti
−i+wmunoglobulin, J, Exp.
Ｍｅｄ、、１５５：１５２３
１９） Ｍｕｒａｇｕｃｈｉ、Ａ、＋Ｊ、Ｌ、Ｂｕｔｌ
ｅｒ、Ｊ、Ｈ，Ｋｅｈｒ１．ａｎｄＡ、Ｓ、Ｆａｕｃｉ
、１９Ｂ３．Ｄｉｆｆｅｒｅｎｔｉａｌ ５ｅｎｓｉｔ
ｉｖｉｔｙｏｆ ｈｕｏ＋ａｎ Ｂ ｃｅｌｌ
５ｕｂｓｅｔｓ ｔｏ ａｃｔｉｖａｔｉｏｎｓｉ
ｇｎａｌｓ ｄｅｌｉｖｅｒｅｄ ｂｙ ａｎｔｉ−μ
ａｎｔｉｂｏｄｙａｎｄ ｐｒｏｌｉｆｅｒａｔｉｖｅ
ｓｉｇｎａｌｓ ｄｅｌｉｖｅｒｅｄ ｂｙａ ｍｏ
ｎｏｃｌｏｎａｌ Ｂ ｃｅｌｌ ｇｒｏｗｔｈ ｆａ
ｃｔｏｒ、Ｊ、Ｅｘｐ。Med, 155:1523 19) Muraguchi, A., +J.L., Butl.
er, J.H., Kehr1. and A, S, Fauci.
, 19B3. Differential 5ensit
ivity of huo+an B cell
5ubsets to activation
gnals delivered by anti-μ
antibody and proliferative
signals delivered by mo
noclonal B cell growth fa
ctor, J., Exp.
Ｍｅｄｏ、１５７：５３０
すなわち抗ヒトグロブリンμ鎖抗体による刺激によりＢ
細胞はその半径を増大し、Ｇ０相がらＧ、相へと移行す
る。Medo, 157:530 that is, stimulation with anti-human globulin μ chain antibody
The cell increases its radius and transitions from the G0 phase to the G, phase.
しかしＣｏｎＡ培養上清と抗ヒトグロブリンμ鎖抗体と
の両者を組合せて刺激した場合に、Ｂ細胞の増殖分化が
促進され、リンパ球が抗原、例えばＨＤ抗原によってｉ
ｎ ｖｉｔｒｏで抗原刺激を受けるのに存効に作用し、
該ｉｎ ｖｉｔｒｏの抗原刺激の効果を増大することは
知られていなかった。本発明者はこの新規な組合せを最
初に見い出し本発明の課題への適用を試み、本発明を完
成するに到ったものである。However, when stimulated with a combination of ConA culture supernatant and anti-human globulin μ chain antibody, proliferation and differentiation of B cells is promoted, and lymphocytes are stimulated by antigens, such as HD antigens.
n Effective against antigenic stimulation in vitro,
It was not known to increase the effect of the in vitro antigen stimulation. The present inventor first discovered this novel combination, attempted to apply it to the problem of the present invention, and completed the present invention.
即ち、本発明は、ヒト牌リンパ球とミエローマ細胞との
ハイブリドーマより製造される抗ＨＤモノクロナール抗
体であり、該ヒト牌リンパ球が、抗ｌトゲロブリンμ鎖
抗体およびコンカナバリンＡで刺激されたヒトリンパ球
の培養上清の両者を含む無血清培地中で培養され、更に
ＨＤ抗原によって抗原刺激を受けたヒト牌リンパ球であ
ることを特徴とする抗ＨＤモノクロナール抗体、及びそ
の製法を提供するものである。That is, the present invention is an anti-HD monoclonal antibody produced from a hybridoma of human tile lymphocytes and myeloma cells, and the human tile lymphocytes are human lymphocytes stimulated with anti-l togelobin μ chain antibody and concanavalin A. The present invention provides an anti-HD monoclonal antibody characterized by being human tile lymphocytes cultured in a serum-free medium containing both culture supernatants and further stimulated with an HD antigen, and a method for producing the same. be.
以下に本発明の詳細な説明する。The present invention will be explained in detail below.
本発明においてＨＤ抗原としてはＨＤ３ 、ＨＤ５、Ｈ
Ｄ７あるいはこれらの化学的誘導体が自由に使用される
が、臨床的な診断上の意義から求めるならば、特に）１
０３が使用される。ＨＤ３は前記文献７）記載の方法に
よってウマ赤血球から単離精製したものを使用すればよ
い。またＨＤ５ 、ＨＤ７は前記文献７）および下記文
献２０）記載の方法によりウシ赤血球から得たものを使
用すればよい。In the present invention, HD antigens include HD3, HD5, H
D7 or their chemical derivatives may be used freely, but especially if clinical diagnostic significance demands
03 is used. HD3 isolated and purified from horse red blood cells by the method described in the above-mentioned document 7) may be used. HD5 and HD7 may be obtained from bovine red blood cells by the methods described in the above-mentioned document 7) and the following document 20).
また例えばＨＤ３の諸種の化学的誘導体は下記文献２１
） 、 ２２） 、 ２３）によって製造し、使用すれ
ばよい。これらのＨＤ抗原を抗原刺激に使用する場合に
は、あらかじめこれらをオクチルセファロースに結合さ
せたものを用意して加えるようにする。For example, various chemical derivatives of HD3 are described in the following document 21.
), 22), 23) and used. When these HD antigens are used for antigen stimulation, they are bound to octyl sepharose and prepared in advance.
２０） Ｈｉｇａｓｈｉ、Ｈ，、に、Ｉｋｕｔａ＋Ｓ、
Ｕｅｄａ、Ｓ、Ｋａｔｏ、Ｙ。20) Higashi, H., Ikuta+S.
Ueda, S., Kato, Y.
旧ｒａｂａｙａｓｈｉ＋Ｍ、Ｍａｔｓｕｍｏｔｏ、ａｎ
ｄ Ｍ、Ｎａ１ｋｉ。Former rabayashi+M, Matsumoto, an
d M, Na1ki.
１９８４、Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ
Ｎ−ｇｌｙｃｏｌｙｌｎｅｕｒａｍｉｎｉｃａｃｉｄ−
ｃｏｎｔａｉｎｉｎｇ ｇｌｙｃｏｓｐｈｉｎｇｏｌｉ
ｐｉｄｓ ｆｒｏｍａ Ｍａｒｅｋ’ｓ ｄｔｓｅａｓ
ｅ ｌｙｍｐｈｏｍａ ｄｅｒｉｖｅｄ ｃｂｉｃｋｅ
ｎｃｅｌｌ １ｉｎｅ、ＭｓＢｌ、ａｓ ｔｕｍｏ
ｒ−ａｓｓｏｃｉａｔｅｄｈｅｔｅｒｏｐｈｉｌｅ Ｈ
ａｎｇａｎｕｔｚｉｕ−Ｄｅｉｃｈｅｒ ａｎｔｉｇｅ
ｎｓ。1984, Characterization of
N-glycolylneuraminicacid-
containing glycosphingoli
pids from Marek's dtseas
e lymphoma derived cbicke
ncell 1ine, MsBl, as tumo
r-associatedheterophile H
anganutziu-Deicher antige
ns.
Ｊ、Ｂｉｏｃｈｅｎ＋、、９５ニア８５゜２１） Ｖ
ｅｌｌ、Ｒ，Ｗ、＋Ａ、Ｐ、Ｃｏｒｆｉｅｌｄ、Ｍ、５
ａｎｄｅｒ、ａｎｄ Ｒ。J, Biochen+, 95 near 85° 21) V
ell, R, W, +A, P, Corfield, M, 5
Ander, and R.
５ｃｈａｕｅｒ、１９７７、Ｎｅｕｒａｍｉｎｉｃ ａ
ｃｉｄ−ｓｐｅｃｉｆｉｃｍｏｄｉｆｉｃａｔｉｏｎ
ａｎｄ ｔｒｉｔｉｕｍ ｌａｂｅｌｌｉｎｇ ｏ
ｆｇａｎｇｌｉｏｓｉｄｅｓ、Ｂｉｏｃｈｅｍ、 ｂｉ
ｏｐｈｙｓ、Ａｃｔａ、４８６：１４５゜
２２） Ｈａｎａｄａ、Ｓ、＋ａｎｄ Ｋ、Ｎａｋａｍ
ｕｒａ、１９８４．Ｍｏｄｔｆｉｃａｔｉｏｎｏｆ ５
ｉａｌｉｃ ａｃｉｄ ｃａｒｂｏｘｙｌ ｇｒｏｕ
ｐ ｏｆｇａｎｇｌｉｏｓｉｄｅ、Ｊ、Ｂｉｏｃｈｅｍ
、９５：１３２３゜２３） Ｍｕｋｕｒｉａ＋Ｊ、’Ｃ
，＋Ｍ、Ｎａ１ｋｉ、ａｎｄ Ｓ、Ｋａｔｏ、１９８６
゜Ｍｉｃｒｏｓｔｒｕｃｔｕｒｅ ｏｆ ｔｈｅ ５
ｉａｌｉｃ ａｃｉｄ ｍｏｉｅｔｙｏｆ Ｎ−ｇｌｙ
ｃｏｌｙｌｎｅｕｒａｍｉｎｙｌｌａｃｔｏｓｙｌｃｅ
ｒａｍｉｄｅａｎｄ ｔｈｅ ｅｌｕｃｉｄａｔｉｏ
ｎ ｏｆ Ｈａｎｇａｎｕｔｚｉｕ ａｎｄＤｅｉｃｈ
ｅｒ （ＨＤ） ａｎｔｉｇｅｎｉｃｉｔｙ、Ｉｍｍｕ
ｎｏｌ、Ｌｅｔｔｅｒｓ。5chauer, 1977, Neuraminic a
cid-specific modification
and tritium labeling o
fgangliosides, Biochem, bi
ophys, Acta, 486:145゜22) Hanada, S, + and K, Nakam
ura, 1984. Modification of 5
ialic acid carboxyl grou
pofganglioside, J.Biochem.
, 95:1323゜23) Mukuria+J,'C
, +M, Na1ki, and S, Kato, 1986
゜Microstructure of the 5
ialic acid moiety of N-gly
colylneuraminyllactosylce
ramide and the elucidation
of Hanganutziu and Deich
er (HD) antigenicity, Immu
nol, Letters.
１２：１６５゜
ヒト牌リンパ球は適当に用意すればよいが、例えば肝硬
変合併食道静脈層患者の開腹手術時に得られた摘出牌よ
りリンパ球を分離して使用すればよい。さらに具体的に
は下記文献２４）記載の方法が参照される。12:165° Human tile lymphocytes may be prepared as appropriate, but for example, lymphocytes may be separated and used from a tile removed from a spleen obtained during laparotomy of a patient with esophageal vein layer associated with liver cirrhosis. More specifically, reference is made to the method described in Document 24) below.
２４） Ｍｉｓｈｅｌｌ、Ｂ、Ｂ、、ａｎｄ Ｓ、Ｍ、
Ｓｈｉｉｇｉ、（ｅｄ、）１９８０゜５ｅｌｅｃｔｅｄ
Ｍｅｔｈｏｄｓ ｉｎ Ｃｅ１ｌｕｌａｒ ｉ＋ｎ＋
ｗｕｎｏｌｏｇｙ。24) Michelle, B.B., and S.M.
Shiigi, (ed,) 1980゜5elected
Methods in Ce1lular i+n+
Wunology.
Ｗ、Ｈ，Ｆｒｅｅｎａｎ Ｃｏ、＋Ｓａｎ Ｆｒａｎｃ
ｉｓｃｏ ｐｐ、４゜抗ヒトグロブリンμ鎖抗体は例え
ばＣａｐｐｅｌ。W, H, Freenan Co, + San Franc
isco pp, 4° anti-human globulin μ chain antibody, such as Cappel.
Ｃｏｃｈｒａｎｖｉ ｌｉｅ、 ＰＡより入手されるヒ
ツジの抗ヒトＩｇμ鎖抗体のＦ（ａｂ’）、画分を使用
すればよい。The F(ab') fraction of a sheep anti-human Igμ chain antibody obtained from Cochranvilie, PA may be used.
またコンカナバリンＡは例えばＰｈａｒｍａｃｉａ Ｆ
ｉｎｅＣｈｅｍｉｃａｌｓ、 Ｕｐｐｓａｌａ、 Ｓｗ
ｅｄｅｎより入手されるＣｏｎＡＳｅｐｈａｒｏｓｅ
４Ｂ （５０μｇ ｐｒｏｔｅｉｎ／ｍｌ）を使用すれ
ばよい、また無血清培地は例えばＨａｎａ Ｍｅｄｉａ
。Also, concanavalin A is available from Pharmacia F, for example.
ine Chemicals, Uppsala, Sw
ConASepharose obtained from eden
4B (50 μg protein/ml) may be used, and a serum-free medium such as Hana Media
.
Ｂａｒｋｅｌｅｙ、 ＣＡより入手されるＨＢ１０４を
用意し、ヒト血清アルブミン、インシュリン、トラスフ
ェリン、エタノールアミン、ピルビン酸ナトリウム（例
えばＩＩＩＩＭ）、Ｌ−グルタミン（例えば５００μｇ
／ａｄ）をそれぞれ細胞生長に必要な量だけ補充し、さ
らにストレプトマイシンおよびペニシリンをそれぞれ１
００μｇ／ｍ！、 １００ ［０／ａＺだけ加えて使用
すればよい。HB104 (obtained from Berkeley, CA) was prepared and supplemented with human serum albumin, insulin, transferrin, ethanolamine, sodium pyruvate (e.g. IIIM), L-glutamine (e.g. 500 μg).
/ad) in the amount necessary for cell growth, and further added 1 portion each of streptomycin and penicillin.
00μg/m! , 100 [0/aZ only needs to be added and used.
増殖因子として補充用ヒトリンパ球はヘパリン処理した
正常人血液をＦｉｃｏｌｌ−Ｈｙｐａｑｕｅ ｇｒａ−
ｄｉｅｎｔｓにより遠心分離して末梢血の単核細胞を用
意し、次にこれを細胞培養皿（たとえばＢｅｃ ｔｏｎ
Ｄｉｃｋｉｎｓｏｎ ａｎｄ Ｃｏ−＋ ０ｘｎａｒｄ
、 ＣＡより入手される３００３ ）で３７°Ｃ，２時
間インキュベートし、非吸着の単核細胞を集めて使用す
ればよい。Human lymphocytes for supplementation as growth factors are prepared by using heparin-treated normal human blood as Ficoll-Hypaque gra-
Peripheral blood mononuclear cells are prepared by centrifugation with dients, and then placed in a cell culture dish (e.g., Bec ton
Dickinson and Co-+ 0xnard
3003 (available from CA) for 2 hours at 37°C, and non-adsorbed mononuclear cells may be collected and used.
抗原刺激は以下のごとく行なう。まず、ＨＢ１０４培地
にヒトリンパ球（５Ｘ １０’／ｒｎ１）およびＣｏｎ
ＡＳｅｐｈａｒｏｓｅ ４Ｂを入れ、Ｃ０，７％、空気
９３％の気流下で３７°Ｃ４Ｂ時間インキュベートし、
遠心分離してその培養上清をとり、ＣｏｎＡ培養上清と
して一７０°Ｃで冷凍保存しておく。Antigen stimulation is performed as follows. First, human lymphocytes (5X 10'/rn1) and Con
Add ASepharose 4B and incubate for 4 hours at 37°C under an air flow of 7% CO and 93% air.
The culture supernatant was collected by centrifugation and stored frozen at -70°C as a ConA culture supernatant.
次に無血清培地にヒト牌リンパ球をとり、前記ＣｏｎＡ
培養上清および抗ヒトグロブリンμ鎖抗体をそれぞれ加
えてインキュベートする。本発明において、抗ヒトグロ
ブリンμ鎖抗体およびＣｏｎＡ培養上清、すなおちコン
カナバリンＡで刺激したヒトリンパ球の培養上清の両者
を組合わせることは必須であり、組合せによる抗原刺激
によりヒト牌リンパ球の抗体産生能は増大する。Next, human tile lymphocytes were placed in a serum-free medium, and the ConA
Culture supernatant and anti-human globulin μ chain antibody are respectively added and incubated. In the present invention, it is essential to combine both the anti-human globulin μ chain antibody and the ConA culture supernatant, that is, the culture supernatant of human lymphocytes stimulated with concanavalin A. Antibody production capacity increases.
すなわちそれぞれ単独で使用した場合に比較して、組合
せ使用の場合にはイムノグロブリンの生産量が高くなる
。That is, when used in combination, the production amount of immunoglobulin is higher than when each is used alone.
次にオクチルセファロースに結合させたＨＤ抗原を加え
、ヒト牌リンパ球にインビトロでの抗原刺激を行ない、
抗ＨＤ抗体産生細胞とする。Next, HD antigen bound to octyl sepharose was added to perform in vitro antigen stimulation of human tile lymphocytes.
Anti-HD antibody producing cells.
ミエローマ細胞は適宜選択して使用すればよいが、例え
ばチオグアニン耐性ヒト雑種骨髄腫株であるＫＲ−１２
を使用すればよい。抗ＨＤ抗体産生細胞とミエローマ細
胞との融合は常法によって行えばよいが、例えばＳｈｉ
ｍａｄｚｕ、 Ｋｙｏｔｏ、 Ｊａｐａｎより入手され
るＳＨＩＭＡＤＺＵ、 ５ＳＨ−１を用いて電気融合を
行なえばよい。Myeloma cells may be selected and used as appropriate; for example, KR-12, a thioguanine-resistant human hybrid myeloma cell line,
You can use . Fusion of anti-HD antibody-producing cells and myeloma cells may be performed by a conventional method, but for example, Shi
Electrofusion may be performed using SHIMADZU, 5SH-1 available from Madzu, Kyoto, Japan.
融合後の選択培地は使用したミエローマ細胞に応じて使
用すればよいが、例えばＣｏｎＡ培養上清、すなわちコ
ンカナバリン＾で刺激したヒトリンパ球の培養上清を加
えたＨＡＴ培地を使用すればよい。The selection medium after fusion may be used depending on the myeloma cells used; for example, a HAT medium to which a ConA culture supernatant, that is, a culture supernatant of human lymphocytes stimulated with concanavalin^, may be used.
抗体産生陽性ハイブリドーマのスクリーニングのために
は適当な方法を考案して行なえばよいが、例えばＨＤ抗
原をソフトプレート上に固相化し、−次抗体と反応させ
、二次抗体として１２％１ラベル抗ヒト！ｇを用いると
か、あるいは無標識の抗ヒトＩｇを二次抗体とし、三次
抗体としてＩｔＪラベルのプロティン＾を用いるとかす
ればよい。詳細に一例を示せば次のごとくである。Screening for antibody-producing positive hybridomas can be carried out by devising an appropriate method. For example, HD antigen is immobilized on a soft plate, reacted with a secondary antibody, and 12% 1-labeled antibody is added as a secondary antibody. Human! Alternatively, unlabeled anti-human Ig may be used as the secondary antibody and ItJ-labeled protein^ may be used as the tertiary antibody. A detailed example is as follows.
ＨＤ抗原のエタノール溶液（５０μｇ／ａＺ）の２５Ｉ
ｌｌを固相プレートのウェルに入れ、乾燥してコート化
する。非特異結合サイトをブロックするために１％卵ア
ルブミン／ＰＢＳの２００１Ｊ１を加えて３７°Ｃ２時
間インキエベートし０．０５％Ｔｗｅｅｎ２０／ＰＢＳ
で４回洗浄する。ここにハイブリドーマの培養上清２５
ｐ１を加え、４℃２時間インキュベートし、同様に洗浄
する。無標識のヒツジ抗ヒトＩｇ （ＴＡＧＯＩｎｃ、
、Ｂｕｒｌｉｎｇａｓ＋ａ、 ＣＡ）を１％卵アルブミ
ン／ＰＢＳで３．２μｇ／ａＺの濃度となるように調製
した液２５ｕ１を加え、４°Ｃ２時間インキュベートシ
、同様に洗浄する。次に約Ｉ Ｘ１０’ｃｐｍの１１５
１−プロティン＾（Ａｍｅｒｓｈａｍ、 Ｂｕｃｋｓ、
ＵＫ）を含む１％卵アルブミン／ＰＢＳ液２５ＪＩＩ
を加え、水浴上に１時間放置し、同様に洗浄する。各ウ
ェルについてガンマカウンターで放射活性を測定する。25I of HD antigen ethanol solution (50μg/aZ)
11 into the wells of a solid phase plate and dry to coat. Incubate at 37°C for 2 hours with 1% ovalbumin/PBS 2001J1 to block non-specific binding sites and 0.05% Tween20/PBS.
Wash 4 times with Here is hybridoma culture supernatant 25
Add p1, incubate at 4°C for 2 hours, and wash in the same manner. Unlabeled sheep anti-human Ig (TAGO Inc,
Add 25ul of a solution prepared with 1% ovalbumin/PBS to a concentration of 3.2 μg/aZ, incubate at 4°C for 2 hours, and wash in the same manner. Then approximately I x 115 of 10'cpm
1-Protein^ (Amersham, Bucks,
1% egg albumin/PBS solution 25JII containing UK)
was added, left on a water bath for 1 hour, and washed in the same manner. Measure radioactivity for each well with a gamma counter.
また産生イムノグロブリンの測定のためには次のように
行えばよい。すなわち培養上清２５ＩＪ１をウェルにと
り、室温で２時間インキュベートし、１％卵アルブミン
／ＰＢＳで２時間インキュベートし、１％卵アルブミン
２５ＩＪ１中に約ｌＸｌ０’ｃｐｒａのＩｔＪ−抗ヒト
ＩｇＳ （Ａｍｅｒｓｈａｏ＋）を含む液を加え、水浴
上に１時間放置し、洗浄して放射活性を測定する。Furthermore, the production immunoglobulin can be measured as follows. That is, culture supernatant 25IJ1 was placed in a well, incubated at room temperature for 2 hours, and incubated with 1% ovalbumin/PBS for 2 hours, containing about 1X10'cpra of ItJ-anti-human IgS (Amershao+) in 1% ovalbumin 25IJ1. Add the solution, leave on a water bath for 1 hour, wash, and measure radioactivity.
融合後、常法により目的のハイブリドーマを選択する。After fusion, the desired hybridoma is selected by a conventional method.
すなわち、融合細胞の形成されているウェルを選択し、
さらにその中から上記検出方法によりイムノグロブリン
の産生じているウェルを選択し、最後に抗体産生陽性ハ
イブリドーマをスクリーニングする。その培養上清中に
抗ＨＤモノクロナール抗体を得る。得られた抗ＨＤモノ
クロナール抗体を使用して上記スクリーニング方法と同
様の操作を行なえば逆にＨＤ抗原を測定することができ
ることが予想される。従って本発明方法で製造される抗
１１０モノクロナール抗体を使用してＨＤ抗原の測定方
法および測定試薬を提供することが可能となることが考
えられる。That is, select the well in which fused cells are formed,
Furthermore, wells in which immunoglobulin is produced are selected from among them by the above-mentioned detection method, and finally antibody-producing positive hybridomas are screened. Anti-HD monoclonal antibodies are obtained in the culture supernatant. It is expected that if the obtained anti-HD monoclonal antibody is used in the same manner as in the above-mentioned screening method, HD antigen can be measured conversely. Therefore, it is possible to provide a method and reagent for measuring HD antigen using the anti-110 monoclonal antibody produced by the method of the present invention.
以下に記載する実施例によって本発明をさらに具体的に
説明する。The present invention will be explained in more detail with reference to Examples described below.
実施例１
（ａ） 細胞の調製
ヒトリンパ球として正常人の末梢血から非吸着単核細胞
を調製した。また肝硬変合併食道静脈瘤患者の開腹手術
時に得られた摘出牌よりリンパ球を分離し、抗原刺激用
ヒト牌すンパ球とした。Example 1 (a) Preparation of cells Non-adsorbed mononuclear cells were prepared as human lymphocytes from the peripheral blood of a normal person. In addition, lymphocytes were isolated from the extirpated tile obtained during laparotomy of a patient with liver cirrhosis and esophageal varices, and used as human tile lymphocytes for antigen stimulation.
（ｂ） 培地
ＨＢ１０４培地に人血清アルブミン、インシュリン、ト
ランスフェリン、エタノールアミン、ピルピン酸ナトリ
ウム、し−グルタミン、ストレプトマイシン、ペニシリ
ンを加えて無血清培地とした。(b) Medium Human serum albumin, insulin, transferrin, ethanolamine, sodium pyruvate, diglutamine, streptomycin, and penicillin were added to HB104 medium to make a serum-free medium.
（Ｃ） 抗原刺激
ヒトリンパ球をＣｏｎＡ ５ｅｐｈａｒｏｓｅ ４Ｂと
共にＨＢ１０４培地中で７％Ｃ０ｚｔ９３％空気の気流
下で３７°Ｃ４８時間インキュベートした。遠心分離し
て培養上清を濾取し、ＣｏｎＡ培養上清とした。無血清
培地にヒト牌リンパ球をとり、ヒツジ抗ヒトグロブリン
μ鎖抗体のＦ（ａｂ’）ｚ画分およびＣｏｎＡ培養上清
を加え、次にオクチルセファローズにＨＤ３抗原を結合
させた液を加えインキュベートし、リンパ球刺激を行っ
た。(C) Antigen-stimulated human lymphocytes were incubated with ConA 5epharose 4B in HB104 medium at 37°C under a flow of 7% COzt93% air for 48 hours. After centrifugation, the culture supernatant was collected by filtration and used as a ConA culture supernatant. Take human tile lymphocytes in a serum-free medium, add the F(ab')z fraction of sheep anti-human globulin μ chain antibody and ConA culture supernatant, and then add a solution containing HD3 antigen bound to Octyl Sepharose. After incubation, lymphocyte stimulation was performed.
（ｄ） 融合
融合用緩衝液（グルコース０．２５Ｍ、塩化カルシウム
０．１ｍＭ、塩化マグネシウム０．１ｍＭ、ｐ）１７．
３のトリス塩酸緩衝液０．２ｍＭ含有＞ ４ｏｏ ｍに
上記の抗ＨＤ抗体産生細胞１×１０７およびＫＲ−１２
細胞（１×１０６）を懸濁し、その懸濁液２０Ｊ１１を
電気融合器５Ｓ）１−Ｃ１２に入れ、室温で融合した。(d) Fusion fusion buffer (glucose 0.25M, calcium chloride 0.1mM, magnesium chloride 0.1mM, p)17.
3 containing 0.2 mM of Tris-HCl buffer> 1 x 107 of the above anti-HD antibody producing cells and KR-12
Cells (1×10 6 ) were suspended, and the suspension 20J11 was placed in an electric fusion device 5S) 1-C12 and fused at room temperature.
４０Ｖ／ｃｍ、１．０ＭＨｚで１０秒間通電するとバー
ル状に混合細胞が形成された。さらに３．ＯＫＶ／ｃｍ
で１０Ｍ秒だけ２回１秒間隔で通電した。融合後５０倍
容のＨＢ１０４培地に移し、５分間３７°Ｃを保った。When electricity was applied at 40 V/cm and 1.0 MHz for 10 seconds, mixed cells were formed in a crowbar shape. Furthermore, 3. OKV/cm
The current was applied twice for 10M seconds at 1 second intervals. After fusion, the cells were transferred to 50 times the volume of HB104 medium and kept at 37°C for 5 minutes.
１００ ｊｉｌの融合液（５Ｘ１０’細胞）をウェルに
入れ、−日培養し、培地の半量を新鮮なＨＡＴ培地で交
換した。融合処理した１０４のウェルの中で６６のウェ
ルにおいてハイブリドーマが生長しており゛、さらに６
６のウェルの中の２６のウェルにおいて産生イムノグロ
ブリンが検出され、さらにその中の６ウエルにおいてＨ
Ｄ３抗原と反応する抗体の産生が確認された。６ウエル
のうちＩＦ４ と命名したクローンについてリミティン
グダイリューションによりさらにクローン化し、６クロ
ーンを得た。いづれのクローンもＩｇＭ （λ−鎖）ク
ラスであり、７２０〜２４００ｎｇ／ｍ／であった。そ
のうちＩＰ４２Ｆ３１Ｇ７と命名したクローンについて
、ＨＯ２、ＨＯ２，０Ｍ３の各抗原との反応性を検討し
た。結果を図２に示す。図中、・、■、ムの各町で示さ
れる線はそれぞれＨＯ２、ＨＯ２，０Ｍ３での結果を示
す。図２よりＨＯ２と特異的に反応することが知られる
。100 jil of fusion solution (5X10' cells) was placed in the wells and cultured for - days, and half of the medium was replaced with fresh HAT medium. Hybridomas were growing in 66 of the 104 wells that underwent fusion treatment, and in an additional 6 wells.
Produced immunoglobulin was detected in 26 of the 6 wells, and H
Production of antibodies that react with the D3 antigen was confirmed. Among the 6 wells, the clone named IF4 was further cloned by limiting dilution to obtain 6 clones. All clones were of the IgM (λ-chain) class and were 720 to 2400 ng/m/. Among them, the clone named IP42F31G7 was examined for reactivity with each antigen of HO2, HO2, and 0M3. The results are shown in Figure 2. In the figure, the lines indicated by ., ■, and mu indicate the results for HO2, HO2, and 0M3, respectively. It is known from FIG. 2 that it reacts specifically with HO2.
同様に図３は各抗原との反応性の結果を示すグラフであ
り、図中、還元（Ｒｅｄｕｃｅｄ） ＨＯ２、メチルエ
ステル（Ｍｅｔｈｙｌ ｅｓｔｅｒ）化ＨＤ３ 、酸化
及び還元（Ｏｘｉｄｉｚｅｄ ａｎｄ ｒｅｄｕｃｅｄ
）ＨＯ２はＨＯ２のＮ−グリコリルノイラミン酸残基を
化学的に修飾した誘導体である。なおＨＯ２、ＨＯ２、
ＨＯ２，０Ｍ３およびＨＯ２の３種の誘導体の入手はそ
れぞれ前記文献７）、 ７）、 ２０）、下記文献２５
）、前記文献２１）、 ２２）。Similarly, FIG. 3 is a graph showing the results of reactivity with each antigen. In the figure, reduced HO2, methyl esterified HD3, oxidized and reduced
) HO2 is a derivative obtained by chemically modifying the N-glycolylneuraminic acid residue of HO2. Note that HO2, HO2,
The three derivatives of HO2, 0M3 and HO2 are obtained from the above-mentioned documents 7), 7), 20) and the following document 25, respectively.
), References 21), 22).
２３）が参照される。図３よりＨＤ３抗原への特異性が
確認された。23) is referred to. From FIG. 3, specificity to HD3 antigen was confirmed.
２５） Ｎａ１ｋｉＪ、、Ｊ、Ｆｏｎｇ、Ｒ，Ｌｅｄｅ
ｅｎ、ａｎｄ、Ｄ、Ｍ、Ｍａｒｃｕｓ。25) Na1kiJ, J.Fong, R.Lede
en, and D. M. Marcus.
１９７５＋Ｔｈｅ ５ｔｒｕｃ、ｔｕｒｅ ｏｆ ｈｕ
ｍａｎ ｅｒｙｔｈｒｏｃｙｔｅｂｌｏｏｄ ｇｒｏｕ
ｐ Ｐｉ ｇｌｙｃｏｓｐｈｉｎｇｏｌｉｐｉｄ。1975+The 5truc, true of hu
man erythrocyte blood grow
p Pi glycosphingolipid.
Ｂｉｏｃｈｅｍｉｓｔｒｙ １４：４８３１゜〔発明の
効果〕
以下、実験例により本発明の詳細な説明する。Biochemistry 14:4831° [Effects of the Invention] The present invention will be described in detail below using experimental examples.
実験例１
実施例１の（Ｃ）抗原刺激において下記（イ）〜（ニ）
でリンパ球刺激をおこなった４種、および（ホ）リンパ
球刺激を行わなかったコントロールの計５種の培養上清
を試料として用意し、各試料について実施例１と同様に
融合し、ＨＡＴ培地で選択し、経口的に２５μづつサン
プリングして、産生イムノグロブリン量を放射活性によ
って測定した。Experimental Example 1 In (C) antigen stimulation of Example 1, the following (a) to (d)
A total of 5 types of culture supernatants were prepared as samples: 4 types in which lymphocyte stimulation was performed and (v) a control in which lymphocyte stimulation was not performed. The immunoglobulin produced was measured by radioactivity by orally sampling 25 μm each.
（イ）１％ＣｏｎＡ培養上清と１５Ｍｇ／−抗ヒトゲロ
ブリンμ鎖抗体
（ロ）１０％ＣｏｎＡ培養上清と１５ ｕ ｇ／ｃｄ抗
ヒトグロブリンμ鎖抗体
（ハ）１５Ｍｇ／−抗ヒトゲロブリンμ鎖抗体（ニ）
１０＃ｇ／＠ＺｃｏｎＡコート化セファロース（ホ）リ
ンパ球刺激を行わなかったコントロール
結果を図１に示す。図中、・、ム、■、口および○の各
町で示される線はそれぞれ（イ）〜（ホ）についての結
果を示す。図１より抗ヒトグロブリンμ鎖抗体およびＣ
ｏｎＡ培養上清で抗原刺激された場合（イ及び口）に、
高い生産量が得られることが知られる。(B) 1% ConA culture supernatant and 15Mg/- anti-human gellobin μ chain antibody (B) 10% ConA culture supernatant and 15 μg/cd anti-human globulin μ chain antibody (c) 15Mg/- anti-human gellobin μ chain antibody (d)
10#g/@ZconA-coated Sepharose (e) Control results in which lymphocyte stimulation was not performed are shown in FIG. In the figure, the lines indicated by ., MU, ■, mouth, and ○ indicate the results for (a) to (e), respectively. From Figure 1, anti-human globulin μ chain antibody and C
When antigen stimulated with onA culture supernatant (A and mouth),
It is known that a high production amount can be obtained.
図１は培養日数と放射活性の関係を示すグラフであり、
各種のリンパ球刺激を行った場合における結果を示す。
図２は抗＋１０モノクロナール抗体の希釈濃度と放射活
性の関係を示すグラフであり、各種の抗原に対する結果
を示す。
図３は抗１１０モノクロナール抗体の各種の抗原に対す
る反応特異性を示すグラフである。Figure 1 is a graph showing the relationship between culture days and radioactivity.
The results obtained when various types of lymphocyte stimulation were performed are shown. FIG. 2 is a graph showing the relationship between dilution concentration and radioactivity of anti-+10 monoclonal antibody, and shows the results for various antigens. FIG. 3 is a graph showing the reaction specificity of anti-110 monoclonal antibody against various antigens.
Claims (1)
マより製造される抗ＨＤモノクロナール抗体であり、該
ヒト脾リンパ球が、抗ヒトグロブリンμ鎖抗体およびコ
ンカナバリンＡで刺激されたヒトリンパ球の培養上清の
両者を含む無血清培地中で培養され、更にＨＤ抗原によ
って抗原刺激を受けたヒト脾リンパ球であることを特徴
とする抗ＨＤモノクロナール抗体。 ２、ヒト脾リンパ球とミエローマ細胞とのハイブリドー
マより抗ＨＤモノクロナール抗体を製造するにあたり、
該ヒト脾リンパ球として、抗ヒトグロブリンμ鎖抗体お
よびコンカナバリンＡで刺激されたヒトリンパ球の培養
上清の両者を含む無血清培地中で培養され、更にＨＤ抗
原によって抗原刺激を受けたヒト脾リンパ球を使用する
ことを特徴とする抗ＨＤモノクロナール抗体の製法。[Claims] 1. An anti-HD monoclonal antibody produced from a hybridoma of human splenic lymphocytes and myeloma cells, wherein the human splenic lymphocytes are stimulated with an anti-human globulin μ chain antibody and concanavalin A. An anti-HD monoclonal antibody characterized in that it is a human splenic lymphocyte cultured in a serum-free medium containing both human lymphocyte culture supernatant and stimulated with an HD antigen. 2. In producing an anti-HD monoclonal antibody from a hybridoma of human splenic lymphocytes and myeloma cells,
The human splenic lymphocytes are human splenic lymphocytes cultured in a serum-free medium containing both an anti-human globulin μ chain antibody and a culture supernatant of human lymphocytes stimulated with concanavalin A, and further stimulated with HD antigen. A method for producing an anti-HD monoclonal antibody characterized by using a sphere.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63025507A JPH01202295A (en) | 1988-02-05 | 1988-02-05 | Anti-hd monoclonal antibody and its production |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63025507A JPH01202295A (en) | 1988-02-05 | 1988-02-05 | Anti-hd monoclonal antibody and its production |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01202295A true JPH01202295A (en) | 1989-08-15 |
Family
ID=12167979
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63025507A Pending JPH01202295A (en) | 1988-02-05 | 1988-02-05 | Anti-hd monoclonal antibody and its production |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01202295A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0539970A2 (en) * | 1991-10-30 | 1993-05-05 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
| US20100029002A1 (en) * | 2006-10-13 | 2010-02-04 | Jill Giles-Komar | Enhancement of hybridoma fusion efficiencies through cell synchronization |
-
1988
- 1988-02-05 JP JP63025507A patent/JPH01202295A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0539970A2 (en) * | 1991-10-30 | 1993-05-05 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
| EP0539970A3 (en) * | 1991-10-30 | 1994-05-25 | Idemitsu Kosan Co | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
| US5681729A (en) * | 1991-10-30 | 1997-10-28 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
| US5869268A (en) * | 1991-10-30 | 1999-02-09 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
| US20100029002A1 (en) * | 2006-10-13 | 2010-02-04 | Jill Giles-Komar | Enhancement of hybridoma fusion efficiencies through cell synchronization |
| US8216842B2 (en) * | 2006-10-13 | 2012-07-10 | Centocor Ortho Biotech Inc. | Enhancement of hybridoma fusion efficiencies through cell synchronization |
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