JPS63115899A - Antibody against male specific antigen and production thereof - Google Patents
Antibody against male specific antigen and production thereofInfo
- Publication number
- JPS63115899A JPS63115899A JP17078886A JP17078886A JPS63115899A JP S63115899 A JPS63115899 A JP S63115899A JP 17078886 A JP17078886 A JP 17078886A JP 17078886 A JP17078886 A JP 17078886A JP S63115899 A JPS63115899 A JP S63115899A
- Authority
- JP
- Japan
- Prior art keywords
- male
- sperm
- specific antigen
- female
- antibody against
- 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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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は雄性特異抗原に対する抗体及びその製造方法に
関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an antibody against a male-specific antigen and a method for producing the same.
近文系マウスの皮膚移植実験において、同性間の移植及
び雌から雄への移植においては拒絶は生じないが雄から
雌への移植の場合には免疫反応が誘発されて移植片がす
べて拒絶されることが見出された（Ｅｉｃｈｗａｌｄ、
Ｅ、 Ｊ、及び５１１ｍ５ｅｒ、 Ｃ，Ｒ，。In skin transplant experiments using modern mice, rejection did not occur in same-sex transplants and female-to-male transplants, but in cases of male-to-female transplants, an immune response was induced and all the grafts were rejected. It was found that (Eichwald,
E, J, and 511m5er, C, R,.
１９５５）。そして、これがＹ染色体上にある遺伝子の
発現生成物により惹起されることから、この生成物は組
織適合性Ｙ抗原（Ｈｉｓｔｏｃｏｍｐａｔｉｂｉｌｉｔ
ｙＹ Ｃｈｒｏｍｏｓｏｍｅ １ｉｎｋｅｄ ａｎｔｉ
ｇｅｎ；Ｈ−Ｙ抗原）、又はＨ−Ｙ抗原を含み、他の可
能性を加えて雄性特異抗原と命名されている。1955). Since this is caused by the expression product of a gene on the Y chromosome, this product is associated with histocompatibility Y antigen (Histocompatible Y antigen).
yY Chromosome 1 inked anti
gen; H-Y antigen), or H-Y antigen, along with other possibilities, have been named male-specific antigens.
なお、本発明においては、雄性特異抗原に対する抗体を
「抗雄性特異抗原」又は「雄性特異抗体」と称する。In the present invention, antibodies against male-specific antigens are referred to as "anti-male-specific antigens" or "male-specific antibodies."
この雄性特異抗原に対する抗体は、近文系マウスにおい
て、雄性マウスの肺臓又は精巣により雌性マウスを免疫
感作し、このマウスから抗体を回収することにより調製
することができることが知られている（Ｗａｃｈｔｅｌ
、Ｓ、Ｓ、＋Ｋｏｏ、Ｇ、Ｃ，，Ｚｕｃｋｅｒｍａｎ。It is known that antibodies against this male-specific antigen can be prepared in modern mouse strains by immunizing female mice with the lungs or testes of male mice and collecting the antibodies from the mice (Wachtel
,S,S,+Koo,G,C,,Zuckerman.
Ｅ、Ｅ、 、 Ｈａｍｔｔ＋ｅｒ１ ｉｎｇ＋υ、、５
ｃｈｅｉｄ、Ｍ、Ｐ、及びＢｏｙｓｅ、Ｅ、Ａ。E, E, , Hamtt+er1 ing+υ,,5
cheid, M. P., and Boyse, E. A.
（１９７４） 、 Ｐｒｏｃ、Ｎａｔｌ、Ａｃａｄ、Ｓ
ｃｉ、Ｕ、Ｓ、Ａ、 Ｖｏｌ ７１，１２１５；Ｕｔｓ
ｕｍｉ、に、＋ Ｓａ ｔｏ ＋ Ｅ、及びＹｕｈａｒ
ａ、 Ｍ、 （１９８３） 、ムｈ肛剋［ｍｍｕｎｏｌ
、Ｖｏｌ、５．（Ｓｕｐ、１）、５９） ｅしかしなが
ら、このような方法によって調製された抗体の特異性に
ついては詳細な検討がなされておらず、特に雄性精子に
結合するか否かについては示唆されていない。しかしな
がら、近文系マウス、そして一般に近交系動物は、マス
・メイティング（ｍａｓｓ ｍａｔｉｎｇ）により育成
、維持されているものであり、インブリード・ストレイ
ン（ｉｎｂｒｅｅｄｉｎｇ ５ｔｒａｉｎ）であり、遺
伝子構成の血縁係数は必ずしも高くはなく、従って、こ
のような近交系動物を用いて調製された抗体の特異性は
あまり畜くないと予想される。(1974), Proc, Natl, Acad, S.
ci, U, S, A, Vol 71, 1215; Uts
umi, ni, + Sa to + E, and Yuhar
A, M. (1983), mmunol
, Vol. 5. (Sup, 1), 59) eHowever, the specificity of antibodies prepared by such a method has not been investigated in detail, and in particular, there is no suggestion as to whether or not they bind to male sperm. However, inbred mice, and inbred animals in general, are raised and maintained by mass mating, and are an inbreeding strain, and the kinship coefficient of genetic makeup is The specificity of antibodies prepared using such inbred animals is therefore not expected to be very high.
Ｐｒｏｃ、 Ｎａ目、 Ａｃａｄ、 Ｓｃｉ、 ＵＳＡ
、Ｖｏｌ １０＋ １１ｈ５．１５０２−１５０５．
１９７３には、雄性マウスからの皮層移植を拒絶した後
の雌性マウスから得られた抗血清が精子の頭部に結合す
ることが記載されている。Proc, Na, Acad, Sci, USA
, Vol 10+ 11h5.1502-1505.
(1973) described that antiserum obtained from female mice after rejecting cortical grafts from male mice binds to sperm heads.
しかしながら、この抗体は雄性精子の中片部に特異的に
結合する本発明の抗体と異る。However, this antibody differs from the antibody of the present invention, which specifically binds to the middle segment of male sperm.
従って本発明は、極めて簡単な方法で特異性の高い抗層
性特異抗原（ａ性特異抗体）を製造することができる方
法、及びこの方法により製造された抗層性特異抗原（雄
性特異抗体）を提供しようとするものである。Therefore, the present invention provides a method for producing a highly specific anti-layer specific antigen (a-specific antibody) by an extremely simple method, and an anti-layer specific antigen (male-specific antibody) produced by this method. This is what we are trying to provide.
前記の本発明の目的は、雄性精子の中片部に特異的に結
合することを特徴とする、雄性特異抗原に対する抗体；
及び雄性動物の細胞、組織又は器官を摘出し、これを雌
性動物に注射し、この雌性動物の血液から抗体を回収す
ることを特徴とする雄性精子の中片部に特異的に結合す
る雄性特異抗原に対する抗体の製造方法を提供すること
により達成される。The object of the present invention is an antibody against a male-specific antigen, which is characterized by specifically binding to the middle segment of male sperm;
and a male-specific antibody that specifically binds to the middle segment of male sperm, which is characterized by extracting cells, tissues, or organs of a male animal, injecting them into a female animal, and collecting antibodies from the female animal's blood. This is achieved by providing a method for producing antibodies against antigens.
一般に、実験動物をベアー・メイティング（Ｐａｉｒｍ
ａｔｉｎｇ）により約２０代継代繁殖せしめた場合、血
縁係数が９９．８％以上となり、この状態においては雄
性動物及び雌性動物は、雄性特異抗原を産生ずる遺伝子
以外のすべての遺伝子については同一の遺伝子構成を有
しており、このような動物集団は純系（アイソジェニッ
ク（ｉｓｏｇｅｎｉｃ））であると称され、マス・メイ
ティングにより継代された近交系動物とは異る。従って
、このような純系動物を一旦樹立すれば、雄性動物の雄
性特異抗原を産生ずる器官、例えば精巣等により雌性動
物を免疫感作するという極めて簡単な方法により、雄性
特異抗原に対する抗体、すなわち抗層性特異抗原（雄性
特異抗体）を該免疫感作された雌性動物の血清中に産生
せしめることができる。Generally, experimental animals are subjected to bear mating (pair mating).
When the kinship coefficient is 99.8% or more when the breeding is carried out for about 20 generations by sub-aging), in this state male and female animals are identical in all genes other than the gene that produces male-specific antigens. Such a population of animals is said to be isogenic and distinct from inbred animals that have been passed through mass mating. Therefore, once such pure-bred animals are established, antibodies against male-specific antigens, that is, antibodies against male-specific antigens, can be produced by immunizing female animals with organs that produce male-specific antigens, such as testis. Layer specific antigens (male specific antibodies) can be produced in the serum of the immunized female animal.
しかしながら、前記のような純系を樹立するには長い年
月と厳重な管理を必要とし、極めて困難な作業である。However, establishing such a pure line as described above requires a long period of time and strict control, and is an extremely difficult task.
すなわち、純系を樹立するためには２０代以上にわたる
ペアー・メイティングを行わなければならず、ここでペ
アー・メイティングとは共通の親動物から生まれた手動
物の内の雌雄一対のみを他の動物から隔離して繁殖せし
め、これを反復する育成方法であり、複数対の親動物を
一緒にして繁殖せしめるマス・メイティングと異り、こ
れを反復継続するには極めて厳重な管理を必要とする。In other words, in order to establish a pure lineage, pair mating must be carried out for over 20 generations, and pair mating is defined as pair mating, in which only one male and female pair of hand animals born from a common parent animal is separated from another. This is a breeding method in which animals are isolated from other animals and bred repeatedly, and unlike mass mating, in which multiple pairs of parent animals are bred together, extremely strict management is required to continue this process repeatedly. do.
このため、純系（ｉｓｏＨｅｎｉｃ ５ｔｒａｉｎ）が
樹立された動物種は非常に少ない。For this reason, there are very few animal species for which a pure strain (isoHenic 5 train) has been established.
本発明者は、長年にわたりベアー・メイティングを反復
することにより、ハムスターの純系を樹立することに成
功し、これを基礎として本発明を完成したものである。The present inventor succeeded in establishing a pure line of hamsters by repeating bear mating over many years, and completed the present invention based on this.
本発明の方法は、純系が確立されるすべての動物種につ
いて適用することができる。このような動物として、マ
ウス、ラット、ハムスター、ラビット、ブタ、モルモッ
ト、ウシ等各種の動物種が挙げられる。本発明において
は、本発明者により始めて樹立されたハムスターの純系
を用いて具体的に説明する。この純系は８年にわたる２
０代のペアー・メイティングにより確立されたものであ
って９９．８％以上の血縁係数を有する。The method of the invention can be applied to all animal species for which pure lines can be established. Examples of such animals include various animal species such as mice, rats, hamsters, rabbits, pigs, guinea pigs, and cows. The present invention will be specifically explained using a pure strain of hamster that was first established by the present inventor. This pure line has been running for 8 years 2
It was established through pair mating between teenagers and has a kinship coefficient of 99.8% or more.
本発明の方法によれば、雄性特異抗原を含有する雄性動
物由来の免疫原調製物により雌性マウスを免疫感作する
。この免疫原調製物は、精巣、肺臓、応等雄性特異抗原
を発現している細胞、組織又は器官から調製することが
できる。精巣を用いるのが特に好ましい。これらの細胞
、組織又は器官の処理は、−ｉにホモシネ−ジョンによ
り行うのが便利である。雌性動物の免疫感作に当っては
、免疫化補助剤として、フロイントの完全アジュバント
（Ｃｏｍｐｌｅｔｅ Ｆｒｅｕｎｄ’ｓ Ａｄｊｕｖａ
ｎｔ）等を、例えばホモジネートと同容量で使用するの
が好ましい。According to the method of the invention, female mice are immunized with an immunogen preparation from a male animal containing a male-specific antigen. The immunogenic preparation can be prepared from testis, lung, cells, tissues or organs expressing iso-male-specific antigens. Particular preference is given to using testicles. The treatment of these cells, tissues or organs is conveniently carried out by homocynation. For immunization of female animals, complete Freund's adjuvant is used as an immunization adjuvant.
nt) etc., for example in the same volume as the homogenate.
免疫感作は雌性動物への皮下注射、皮肉注射、腹腔内注
射又は肺臓への直接注入等により行う。この免疫感作は
およそ１週間の間隔で５回程度行う。Immunization is carried out by subcutaneous injection, subcutaneous injection, intraperitoneal injection, or direct injection into the lungs of female animals. This immunization is carried out about five times at intervals of approximately one week.
最終免疫感作のおよそ１０日後に動物から血液を採取し
、血液から抗体を回収するための常法に従って目的とす
る抗体を分離する。Approximately 10 days after the final immunization, blood is collected from the animal and the antibody of interest is isolated according to conventional methods for recovering antibodies from blood.
このようにして調製された本発明の抗雄性特異抗原（雄
性特異抗体）は種々の哺乳類動物、例えばハムスター、
マウス、ラット、ブタ、ヒト等の雄性精子と結合するこ
とができる。例えば、試験しようとする精子を遠心洗浄
した後、緩衝液、例えば５％ウシ血清アルブミンを含む
タイロード民法又は等張のリン酸緩衝液中で本発明の抗
体と共にインキュベートし、次に再度洗浄して、未結合
抗体及び非特異的に結合した抗体を精子から分離除去す
る０次にこの精子をＦＩＴＣ（Ｆｌｕｏｒｅｓｃｅｉｎ
ｉｓｏｔｈｉｏｃｙａｎａｔｅ）で標識したウサギ抗−
ハムスターＩｇＧと共にインキュベートし、再度洗浄す
る。The anti-male-specific antigen (male-specific antibody) of the present invention thus prepared can be used in various mammals, such as hamsters,
It can bind to male sperm of mice, rats, pigs, humans, etc. For example, the spermatozoa to be tested are centrifugally washed, then incubated with antibodies of the invention in a buffer, such as Tyrode's or isotonic phosphate buffer containing 5% bovine serum albumin, and then washed again. Then, unbound antibodies and non-specifically bound antibodies are separated and removed from the spermatozoa.Next, the spermatozoa are treated with FITC (fluorescein
isothiocyanate)-labeled rabbit anti-
Incubate with hamster IgG and wash again.
こうして調製した精子を螢光顕微鏡下で観察する。The spermatozoa thus prepared are observed under a fluorescence microscope.
こうして観察する場合、本発明の抗雄性特異抗原（ａ性
特異抗体）はハムスターのみならず、マウス、ラット、
ブタ、ウシ及びヒトの精子の約半数に結合する。また、
ヒト精子の中でＹ染色体を持つものすなわち雄性精子が
キナクリンマスタードにより染色され、Ｆ−ボディーと
呼ばれる輝点を現わすことを利用しくＢａｒｌｏ−等１
９７０）キナクリンマスタードならびに抗雄性特異抗原
を用いた螢光免疫染色による二重染色をすると、キナク
リンマスタード染色によりＦ−ボディーを示す精子は、
螢光免疫染色によっても染色される。この結果から、本
発明の抗体は、ハムスターのみならず、マウス、ラット
、ブタ、ヒト等広範囲の種類の雄性精子に結合すること
ができる抗雄性特異抗原（雄性特異抗体）であることが
合理的に推定され、又ＩｇＧクラスであることが決定さ
れる。When observed in this way, the anti-male-specific antigen (a-specific antibody) of the present invention is applicable not only to hamsters but also to mice, rats,
Binds to about half of pig, cow, and human sperm. Also,
Taking advantage of the fact that among human spermatozoa that have a Y chromosome, that is, male spermatozoa, are stained with quinacrine mustard and reveal bright spots called F-bodies, Barlo et al.
970) When double-stained with fluorescent immunostaining using quinacrine mustard and anti-male-specific antigen, spermatozoa showing F-bodies by quinacrine mustard staining
It is also stained by fluorescent immunostaining. From this result, it is reasonable that the antibody of the present invention is an anti-male-specific antigen (male-specific antibody) that can bind not only to hamsters but also to a wide variety of male sperm such as mice, rats, pigs, and humans. It was estimated to be of the IgG class.
次に、この発明を実施例によりさらに具体的に説明する
。Next, the present invention will be explained in more detail with reference to Examples.
動車ニー−゛（）の
裂盈
２０代以上ペアー・メイティングを行った純系雄性ハム
スターの精巣を摘出し、これを市販のガラスホモジナイ
ザーによりホモジホイズし、フロイントの完全アジュバ
ントの等容量と混合してエマルジョンを調製した。その
１ｍｌずつを２匹の純系雌性ハムスターに皮下注射する
ことにより免疫感作した。これを１週間の間隔で５回行
い、最後の注射の後１０日回定頚椎脱臼法により動物を
殺し、開腹し、心臓から血液を採取した。これにより１
頭の動物より約４ｍｌの血液を得ることができ、２頭の
動物から約８ｍｌの血液を得ることができた。The testicles of purebred male hamsters that have been pair-mated for over 20 years are removed, homogenized using a commercially available glass homogenizer, and mixed with an equal volume of Freund's complete adjuvant to form an emulsion. was prepared. Two purebred female hamsters were immunized by subcutaneous injection of 1 ml each. This was done five times at one-week intervals, and ten days after the last injection, the animals were sacrificed by fixed cervical dislocation, laparotomy was performed, and blood was collected from the heart. This results in 1
Approximately 4 ml of blood could be obtained from one animal and approximately 8 ml from two animals.
この血液を４℃にて一晩放置することにより血液を凝固
せしめ、０℃にて２０．ＯＯＯＸｇで１０分間遠心分離
することにより２頭の動物より抗血清４ｍｌを得た。こ
うして得られた抗血清を５６℃にて３０分間熱処理し、
抗雄性特異抗原（雄性特異抗体）を含む抗血清を得た。The blood was allowed to coagulate by standing at 4°C overnight, and then at 0°C for 20 minutes. 4 ml of antiserum was obtained from two animals by centrifugation in OOOXg for 10 minutes. The antiserum thus obtained was heat-treated at 56°C for 30 minutes,
Antiserum containing anti-male-specific antigen (male-specific antibody) was obtained.
ノＬ応」生−」し の のｄ ラ°ｙ１ハ
ムスター、マウス、ラット、ウシ、ブタ、及びヒトの精
子をそれぞれ別々に５％ウシ血清アルブミンを含むタイ
ロード民法又は等張の緩衝液により２〜３回遠心洗浄（
２５０〜３００ Ｘ ｇにて１０分間）し、タイロード
民法又は等張のリン酸緩衝液中精子懸濁液を調製した。Sperm from hamster, mouse, rat, cow, pig, and human were separately cultured in Tyrode's or isotonic buffer containing 5% bovine serum albumin. ~3 times centrifugal washing (
(250-300 x g for 10 min) to prepare a sperm suspension in Tyrode's Civil Law or isotonic phosphate buffer.
この精子懸濁液と、実施例１において調製した抗腫性特
異抗原（雄性特異抗体）含有血清とを混合し、３７℃に
て３０分間インキュベートした。次に、再びタイロード
民法又は等張のリン酸緩衝液により精子を１〜２回、２
５０〜３００ Ｘ ｇにて１０分間洗浄することにより
未結合抗体及び非特異的に吸着した抗体を除去した。次
に、この精子をＦＩＴＣで標識したウサギ抗−ハムスタ
ーＩｇＧにより３７℃にて３０分間処理した。次に５％
ウシ血清アルブミンを含むタイロード民法又は等張のリ
ン酸緩衝液により、２５０〜３００ ｘｇにて１０分間
遠心洗浄し、無螢光スライドガラス上にマウントし、カ
バーグラスで覆った後、透明なマニキュアで封じ、螢光
観察用プレパラートとした。こうして８用型したプレパ
ラートを螢光顕微鏡により観察したところ、プレパラー
ト上に存在する精子の約半数に抗体が結合することが明
らかとなった。This sperm suspension and the antitumor-specific antigen (male-specific antibody)-containing serum prepared in Example 1 were mixed and incubated at 37°C for 30 minutes. Next, the spermatozoa are washed once or twice again with Tyrode's civil method or isotonic phosphate buffer.
Unbound and non-specifically adsorbed antibodies were removed by washing at 50-300 x g for 10 minutes. The sperm were then treated with FITC-labeled rabbit anti-hamster IgG for 30 minutes at 37°C. then 5%
Centrifuged for 10 minutes at 250-300 x g with Tyrode's or isotonic phosphate buffer containing bovine serum albumin, mounted on non-fluorescent glass slides, covered with a coverslip, and then covered with clear nail polish. The sample was sealed with plastic wrapper and used as a preparation for fluorescence observation. When the 8-sized preparation was observed under a fluorescence microscope, it was revealed that the antibody bound to about half of the spermatozoa present on the preparation.
また、この実験において、抗体がウサギ抗−ハムスター
１ｇＧ抗体により検出されることから、前記のようにし
てｉｌｌ製した抗体がＩｇＧクラスに属することが明ら
かになった。In addition, in this experiment, the antibody was detected using a rabbit anti-hamster 1gG antibody, which revealed that the antibody produced in the ill-produced manner described above belongs to the IgG class.
この結果を第１図〜第４図に示す。この図は上記の様に
して調製したプレパラートの同じ部分を螢光顕微鏡を用
いて可視光線観察をした場合（Ａ）、及び螢光観察をし
た場合（Ｂ）の顕微鏡写真である。この写真から、視野
に存在する複数の精子の内、一部の精子の中片部に螢光
の分布が認められ、本発明の抗体が特異的に結合したこ
とが推定される。The results are shown in FIGS. 1 to 4. These figures are micrographs of the same part of the preparation prepared as described above, observed under visible light using a fluorescence microscope (A) and (B) when observed under fluorescence. From this photograph, a distribution of fluorescence was observed in the midsections of some of the spermatozoa present in the field of view, and it is presumed that the antibody of the present invention specifically bound thereto.
次Ｗ−盃
ヒト精子を上記の通り洗浄した後、２％パラホルムアル
デヒド、７５ｍＭリジン、１０ｍＭメタ過ヨウ素酸ナト
リウム及び３２．５ｍＭリン酸緩衝液を含むＩ）Ｈ６，
２の溶液中、４℃で１時間固定し、等張リン酸緩衝液で
２回上記の通り遠心洗浄し、スライドグラス上に塗抹乾
燥し、その後スライドグラスに付着した精子を次のよう
に処理した。I) H6 containing 2% paraformaldehyde, 75mM lysine, 10mM sodium metaperiodate and 32.5mM phosphate buffer after washing the human spermatozoa as above.
Fix in solution of 2 for 1 hour at 4°C, wash by centrifugation twice in isotonic phosphate buffer as described above, smear onto a glass slide and dry, then process the sperm attached to the glass slide as follows. did.
まず５％ウシ血清アルブミンを含む等張リン酸緩衝液中
で３７℃１時間インキュベートし、抗腫性特異抗原（雄
性特異抗体）を含む血清中で３７”Ｃ３０分間、ビオチ
ン化抗ハムスター１ｇＧ中で３７℃３０分間、ローダミ
ン標識アビジン中で３７℃３０分間、ついでｏ、ｏｏｓ
％キナクリンマスタード中室温で３０分間インキュベー
トした。この過程で次のインキュベートに移る場合、等
張のリン酸緩衝液で洗浄した。最終洗浄の後、カバーグ
ラスで覆い観察に供した。First, incubate in isotonic phosphate buffer containing 5% bovine serum albumin at 37°C for 1 hour, then in serum containing antitumor-specific antigen (male-specific antibody) for 30 minutes at 37°C, and then in biotinylated anti-hamster 1gG. 30 min at 37°C, 30 min at 37°C in rhodamine-labeled avidin, then o, oos.
% quinacrine mustard for 30 minutes at room temperature. When moving on to the next incubation during this process, wash with isotonic phosphate buffer. After final washing, it was covered with a cover glass and subjected to observation.
観察の結果は表１に示すとうりである。３人の異なるヒ
トから提供された精子を用いた３回の異なる実験をカイ
二乗法により検定すると、精子の提供者による実験結果
の存意差はない。また、３回の異なる実験の総計を用い
てカイ二乗検定したところ、０．１％以上の精度で、免
疫染色された精子はキナクリンマスタードによっても染
色されていることが認められた。したがって本発明の抗
腫性特異抗原はＹ染色体を持つ精子、すなわち雄性精子
のみを認識していると結論できる。The results of the observation are shown in Table 1. When testing three different experiments using sperm donated by three different people using the chi-square method, there is no significant difference in the experimental results depending on the sperm donor. Furthermore, when a chi-square test was performed using the total of three different experiments, it was observed with an accuracy of 0.1% or higher that immunostained spermatozoa were also stained by quinacrine mustard. Therefore, it can be concluded that the antitumor-specific antigen of the present invention recognizes only spermatozoa having a Y chromosome, that is, male spermatozoa.
第１表
染色した時に染色された場合を＋、されない場合を−で
表わし、その組み合わせの結果を示す。When the first table was stained, the case where the sample was stained is indicated by +, and the case where it was not stained is indicated by -, and the result of the combination is shown.
表中の括弧内の数値は実験ごとの全体での百分率を示す
。各実験は異なるヒトから提供された精子を用いての独
立した実験である。The numbers in parentheses in the table indicate the overall percentage for each experiment. Each experiment is an independent experiment using sperm donated by a different person.
結果の１例を第５図に示す。この図に頭部のキナクリン
螢光部位及び抗体が結合した中片部を示す。精子（Ａ）
及び（Ｃ）は雄性精子であり、精子（Ｂ）は雌性精子で
あると推定される。An example of the results is shown in FIG. This figure shows the quinacrine fluorescent site on the head and the middle piece to which the antibody was bound. Sperm (A)
and (C) are male spermatozoa, and spermatozoa (B) is presumed to be female spermatozoa.
以上の結果から明らかなように、本発明の抗雄性特異抗
原（相性特異抗体）は簡単な方法で製造することができ
、この抗体を用いることにより、１種類の抗体を用いて
複数の哺乳動物種の精子の雌雄識別を行うことができ、
さらには、本発明の抗体の雄性精子への特異的結合を利
用して、精子の雌雄分画を行うこともできる。As is clear from the above results, the anti-male specific antigen (phasic specific antibody) of the present invention can be produced by a simple method, and by using this antibody, multiple mammals can be treated with one type of antibody. It is possible to identify the sexes of sperm of a species,
Furthermore, the specific binding of the antibody of the present invention to male sperm can be used to perform sex fractionation of sperm.
【図面の簡単な説明】
第１図〜第４図は、いずれも実施例２において螢光染色
された各種精子の顕微鏡写真であって、生物の形態を表
わす図面に代る写真である。第１図はハムスター、第２
図はマウス、第３図はラット、第４図はウシの精子をそ
れぞれ表わし、（Ａ）は可視光線観察した場合、（Ｂ）
は螢光観察した場合をそれぞれ示す。
第５図は実施例３における顕微鏡観察のスケッチである
。
第４面
第５回BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 to 4 are microscopic photographs of various spermatozoa that were fluorescently stained in Example 2, and are photographs in place of drawings showing the morphology of living organisms. Figure 1 is a hamster, Figure 2
The figure shows mouse sperm, Figure 3 shows rat sperm, and Figure 4 shows cow sperm.
1 shows the case of fluorescence observation. FIG. 5 is a sketch of microscopic observation in Example 3. 4th page 5th episode
Claims (1)
する、雄性特異抗原に対する抗体。 ２、雄性動物の細胞、組織又は器官を摘出し、これを雌
性動物に注射し、この雌性動物の血液から抗体を回収す
ることを特徴とする、雄性精子の中片部に特異的に結合
する雄性特異抗原に対する抗体の製造方法。 ３、純系（アイソジェニック）雄性動物の細胞、組織又
は器官を摘出し、これをホモジホイズし、このホモジネ
ートを所望によりアジュバントと共に純系雌性動物に注
射し、この純系雌性動物の血液から抗体を回収すること
を特徴とする特許請求の範囲第２項に記載の方法。[Scope of Claims] 1. An antibody against a male-specific antigen, which is characterized by specifically binding to the middle segment of male sperm. 2. Extracting cells, tissues or organs from a male animal, injecting them into a female animal, and collecting antibodies from the female animal's blood, which specifically bind to the middle part of the male sperm A method for producing an antibody against a male-specific antigen. 3. Extracting cells, tissues, or organs from a purebred (isogenic) male animal, homogenizing the same, injecting this homogenate together with an adjuvant if desired into a purebred female animal, and recovering antibodies from the blood of the purebred female animal. A method according to claim 2, characterized in that:
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU74532/87A AU7453287A (en) | 1986-06-26 | 1987-06-19 | Antibody to middle piece of y chromosome bearing spermatozoon |
| NZ22079787A NZ220797A (en) | 1986-06-26 | 1987-06-22 | Male specific antibody against male specific antigen and its production |
| CN198787104368A CN87104368A (en) | 1986-06-26 | 1987-06-23 | Antibody of anti-male-specific antigen and preparation method thereof |
| EP87305671A EP0251710A3 (en) | 1986-06-26 | 1987-06-25 | Antibody against male-specific antigen and process for production thereof |
| DK324287A DK324287A (en) | 1986-06-26 | 1987-06-25 | ANTIBODY AGAINST COUNTRY SPECIFIC ANTIGEN |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61-148072 | 1986-06-26 | ||
| JP14807286 | 1986-06-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63115899A true JPS63115899A (en) | 1988-05-20 |
Family
ID=15444588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17078886A Pending JPS63115899A (en) | 1986-06-26 | 1986-07-22 | Antibody against male specific antigen and production thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63115899A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018523490A (en) * | 2015-07-13 | 2018-08-23 | キム、トン クKIM, Dong Ku | Sperm sex discrimination antibody and its use |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56118019A (en) * | 1980-02-15 | 1981-09-16 | Bryant Bernard | Manufacture of male idiosyncrasy antibody to single idiosyncrasy and its use for raising either sexual birth rate of mammal |
-
1986
- 1986-07-22 JP JP17078886A patent/JPS63115899A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56118019A (en) * | 1980-02-15 | 1981-09-16 | Bryant Bernard | Manufacture of male idiosyncrasy antibody to single idiosyncrasy and its use for raising either sexual birth rate of mammal |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018523490A (en) * | 2015-07-13 | 2018-08-23 | キム、トン クKIM, Dong Ku | Sperm sex discrimination antibody and its use |
| US10550177B2 (en) | 2015-07-13 | 2020-02-04 | Nuriscience Co., Ltd. | Antibody for determining sex of sperm, and use thereof |
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