JPH0221800B2 - - Google Patents
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- Publication number
- JPH0221800B2 JPH0221800B2 JP8098781A JP8098781A JPH0221800B2 JP H0221800 B2 JPH0221800 B2 JP H0221800B2 JP 8098781 A JP8098781 A JP 8098781A JP 8098781 A JP8098781 A JP 8098781A JP H0221800 B2 JPH0221800 B2 JP H0221800B2
- Authority
- JP
- Japan
- Prior art keywords
- blood cells
- red blood
- haemophilus
- paragarinarum
- strain
- 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.)
- Expired
Links
- 210000003743 erythrocyte Anatomy 0.000 claims description 39
- 101710154606 Hemagglutinin Proteins 0.000 claims description 32
- 101710093908 Outer capsid protein VP4 Proteins 0.000 claims description 32
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 claims description 32
- 101710176177 Protein A56 Proteins 0.000 claims description 32
- 239000000185 hemagglutinin Substances 0.000 claims description 32
- 241000606790 Haemophilus Species 0.000 claims description 21
- 230000004520 agglutination Effects 0.000 claims description 18
- 230000002950 deficient Effects 0.000 claims description 17
- 101710186708 Agglutinin Proteins 0.000 claims description 15
- 101710146024 Horcolin Proteins 0.000 claims description 15
- 101710189395 Lectin Proteins 0.000 claims description 15
- 101710179758 Mannose-specific lectin Proteins 0.000 claims description 15
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 claims description 15
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 claims description 15
- 239000000910 agglutinin Substances 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 9
- 235000002639 sodium chloride Nutrition 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 238000000527 sonication Methods 0.000 claims description 6
- 241000606767 Avibacterium paragallinarum Species 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- NJYFRQQXXXRJHK-UHFFFAOYSA-N (4-aminophenyl) thiocyanate Chemical compound NC1=CC=C(SC#N)C=C1 NJYFRQQXXXRJHK-UHFFFAOYSA-N 0.000 claims 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims 1
- 238000001816 cooling Methods 0.000 claims 1
- 229940072033 potash Drugs 0.000 claims 1
- 235000015320 potassium carbonate Nutrition 0.000 claims 1
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims 1
- 230000035931 haemagglutination Effects 0.000 description 20
- 241000287828 Gallus gallus Species 0.000 description 18
- 235000013330 chicken meat Nutrition 0.000 description 18
- 230000002401 inhibitory effect Effects 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 210000000601 blood cell Anatomy 0.000 description 10
- 208000015181 infectious disease Diseases 0.000 description 9
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 230000002458 infectious effect Effects 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 206010039083 rhinitis Diseases 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 230000000069 prophylactic effect Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Separation Of Suspended Particles By Flocculating Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
〔産業上の利用分野〕
本発明はヘモフイルス・パラガリナルムの新鮮
赤血球に対する凝集素欠損株の赤血球凝集素の製
造法に関する。
〔従来の技術〕
ヘモフイルス・パラガリナルム
（Haemophilus Paragallinarum）は、鶏伝染性
コリーザの病原体として知られている。本菌の血
清型は異なつた血清型１および２の二つに区分さ
れ、血清型１に属する菌は鶏などの新鮮赤血球に
対し高い凝集性を有することが知られている
〔Jap.J.Vet.Sci.、40(6)、645〜652（1978）（文献
１）〕。この性質を利用し、血清型１菌株で調整し
た赤血球凝集素と鶏新鮮赤血球を用いて実施する
赤血球凝集抑制抗体の測定法は、血清型１菌株で
調整した予防液の力価測定や感染鶏血清における
診断に広く応用されている。
近年、市販伝染性コリーザワクチンを注射され
た鶏で血清型１菌株の赤血球凝集素に対する赤血
球凝集抑制抗体がその感染を防御するに充分な程
上昇しているにもかかわらず、伝染性コリーザの
発症する例が急増している。これらの鶏から分離
された菌株は、本発明者らによつて血清型２と命
名され、その抗原構造が明らかにされた。〔（文献
１）、Jap.J.Vet.Sci.、40(1)、65〜78（1978）（文献
２）、Am.J.Vet.Res.、40、1450〜1453（1979）
（文献３）〕。血清型１および血清型２菌株は交差
免疫を欠き、そのため、伝染性コリーザを予防す
るには血清型１および２の菌株で調製した予防液
を使用することが必要である。とりわけ血清型２
菌株の予防液は本菌の我が国における広範な分布
とあいまつて、その応用が熱望されている〔文献
２、Am.J.Vet.Res.、41、1900〜1903（1980）（文
献４）〕。
しかるに、血清型２菌株はいずれも新鮮赤血球
に対する凝集性を欠くことに加え、凝集反応用抗
原に対する低い被凝集性とあいまつて、その簡易
な抗原測定法の確立が望まれている。
〔発明が解決しようとする問題点〕
そこで、本発明者らは、上記要望に鑑み、ヘモ
フイルス・パラガリナルム血清型２菌株の赤血球
凝集素について種々の研究を続けた結果、血清型
２菌株の病原性保有株は通常明瞭な莢膜を有する
こと、ならびにこの表面物質を除去することによ
り、グルタルアルデヒド固定血球に対し高い被凝
集性を示す赤血球凝集素が得られることを見出
し、この赤血球凝集素を用いて、血清型２菌株に
対する特異的な赤血球凝集抑制抗体を測定できる
ことを見出した。
本発明は、上記の知見に基づいて完成されたも
のであり、ヘモフイルス・パラガリナルムの新鮮
赤血球に対する凝集素欠損株に対する赤血球凝集
抑制抗体の測定に特異的に使用できる赤血球凝集
素の製造法を提供するものである。
〔問題点を解決するための手段〕
上記の如く問題点を解決するために本発明によ
れば、ヘモフイルス・パラガリナルムの新鮮赤血
球に対する凝集素欠損株を液体培地に培養し、得
られた菌液を食塩含有ロダンカリ水溶液で処理
し、次いで音波処理することを特徴とする新鮮赤
血球に対し低凝集性で、グルタルアルデヒド固定
赤血球に対し高凝集性を有する赤血球凝集素の製
造法を提供するにある。
本発明の赤血球凝集素を製造するに当つては、
先ず、ヘモフイルス・パラガリナルムの新鮮赤血
球に対する凝集素欠損株が適当な液体培地で培養
される。上記新鮮赤血球に対する凝集素欠損株
は、ヘモフイルス・パラガリナルムに属し、新鮮
赤血球に対する凝集性を欠くか、または極めて弱
い菌株を意味する。このような菌株としては、血
清型２菌株、例えば本発明者らが分離固定したＨ
−18株（工業技術院微生物工業技術研究所、受託
書「受託番号FERM Ｐ−6004」）が使用され得
る。また、本発明者らが血清型２菌株と同じ抗原
性を有することを示したPageの血清型Ｃ菌株も
使用され得る〔文献４、Am.J.Vet.Res.、41、
757〜760（1980）〕。さらに、同じく新鮮赤血球に
対する凝集性を欠くか、または極めて弱いPage
の血清株Ｂ菌株も使用され得、本発明は血清型２
菌株に限定されものではなく、新鮮赤血球に対す
る凝集性を欠くか、または極めて弱い菌株に使用
され得る。液体培地としては、通常サワタ培地
（文献２）が使用される。培養条件としては、培
養温度は通常35〜37℃付近、培養時間は通常10〜
24時間程度で振とう培養法により行われる。
次に、培養物から集菌するのであるが、通常遠
心分離により集菌される。遠心分離は通常２〜５
℃前後の低温下で行われ、7000〜10000ｇで20〜
40分程度行うのが好ましい。得られた細胞は、中
性付近のリン酸緩衝食塩水で洗浄され、上記の緩
衝液に懸濁し、再度遠心分離により集菌される。
懸濁は通常細胞濃度が10”個／ml程度となるよう
比濁計（650nｍ）で測定して行われる。
このようにして得られた細胞の表面物質を除去
するのであるが、この除去処理は、食塩含有ロダ
ンカリ水溶液処理および音波処理により行われ
る。
上記の食塩含有ロダンカリ水溶液処理は、集菌
された細胞を食塩含有ロダンカリ水溶液に浮遊さ
せることにより行われる。上記の水溶液はPH5.0
〜7.0の範囲で使用するのが好ましい。食塩の濃
度は0.1〜0.6Mの範囲で使用するのが好ましい。
また、ロダンカリの濃度は0.25〜1.5Mの範囲で
使用するのが好ましい。次いで、音波処理するの
であるが、通常は２〜５℃程度の低温下で行わ
れ、10KCで１〜15分間処理すれば充分である。
上記処理した浮遊液は、再度遠心分離される。
遠心分離の条件は前記と同様にして行われる。集
められた処理細胞は、リン酸緩衝食塩水で１回洗
浄した後、リン酸緩衝食塩水で適当の濃度に懸濁
して赤血球凝集素として使用される。
このようにして得られた赤血球凝集素は、新鮮
赤血球に対してはほとんど凝集性を欠くが、グル
タルアルデヒド固定赤血球に対しては高い凝集性
を有する特徴を有する。この赤血球凝集素は易熱
性、トリプシン感受性、ヒアルロニダーゼ耐性で
ある性質を有する。
本発明の赤血球凝集素を用いることにより、ヘ
モフイルス・パラガリナルムの新鮮赤血球に対す
る凝集素欠損株に対する赤血球凝集抑制抗体を測
定することができる。例えば、抗原として、本発
明の赤血球凝集素を用い、また赤血球浮遊液とし
てグルタルアルデヒド固定赤血球浮遊液を用いる
ことを大きな特色とし、実施にあたつては、ヘモ
フイルス・パラガリナルム血清型１菌株、例えば
No.221株に対する赤血球凝集抑制抗体の測定系を
若干変更しなくてはならない。上記の測定系によ
り伝染性コリーザの防御と関連するヘモフイル
ス・パラガリナルム血清型２菌株に対する特異的
な赤血球凝集抑制抗体を測定することができ、本
病予防のため、血清型２菌株で調製した予防液の
効果を知ることができ、有効である。
次に、実施例および参考例を挙げて本発明を具
体的に説明するが、これらによつて本発明を制限
するものではない。
実施例
２容振とうコルベンフラスコに鶏肉浸出液20
％、カザアミノ酸（Difco）0.5％、ポリプトンＳ
（ダイゴ社製）0.5％、食塩0.5％、鶏血清（56℃、
30分間熱処理）５％、乾燥酵母エキス（25％ｗ／
ｖ、日本 甜菜糖社製）５％、NADH0.00025％
を含むサワタ液体培地（PH7.4）700mlを仕込み、
これに伝染性コリーザ羅患鶏の鼻腔から分離した
ヘモフイルス・パラガリナルムＨ−18株（血清型
２）接種し、37℃で12時間振とう培養する。培養
物を４℃で30分間8000Gの遠心分離により集菌す
る。得られた細胞を、0.02Mリン酸緩衝食塩水
（0.15M、PH7.0）（PBSI）で１回洗浄し、細胞濃
度を650nｍの比濁計により10”細胞／mlと成る
ようPBSIに懸濁し、４℃で遠心分離により洗浄
細胞を集める。これを上記懸濁で用いたPBSIと
同量の0.425M食塩含有1Mロダンカリ水溶液（PH
6.3）に浮遊させる。この浮遊液を４℃で10KCの
音波処理を10分間行つた後、４℃で遠心分離す
る。得られた処理細胞をPBSIで１回洗浄し、適
量のPBSIに浮遊し、赤血球凝集素とした。
この赤血球凝集素は、新鮮血球に対し著しく凝
集性を欠くが、グルタルアルデヒド固定赤血球に
対し高い凝集性を有する特徴を示す。このような
特徴は次の実験の測定結果から明らかである。
(1) 各動物の赤血球を用いた新鮮血球およびグル
タルアルデヒド固定血球に対する赤血球凝集力
価の測定結果は第１表の通りである。
[Industrial Application Field] The present invention relates to a method for producing hemagglutinin from an agglutinin-deficient strain of Haemophilus paragarinarum for fresh red blood cells. [Prior Art] Haemophilus Paragallinarum is known as a pathogen of chicken infectious coryza. The serotypes of this bacterium are divided into two different serotypes, serotypes 1 and 2, and bacteria belonging to serotype 1 are known to have a high agglutination property against fresh red blood cells from chickens and other sources [Jap.J. Vet.Sci., 40 (6), 645-652 (1978) (Reference 1)]. Taking advantage of this property, methods for measuring hemagglutination-inhibiting antibodies using hemagglutinin prepared with a serotype 1 strain and fresh chicken red blood cells include titration of a prophylactic solution prepared with a serotype 1 strain and methods for measuring hemagglutination-inhibiting antibodies in infected chickens. It is widely applied to diagnosis in serum. In recent years, the development of infectious coryza in chickens injected with commercially available infectious coryza vaccines, despite the hemagglutination-inhibiting antibodies against hemagglutinin of serotype 1 strains being sufficiently elevated to protect against infection. The number of cases of this is rapidly increasing. The strain isolated from these chickens was named serotype 2 by the present inventors, and its antigenic structure was clarified. [(Reference 1), Jap.J.Vet.Sci., 40 (1), 65-78 (1978) (Reference 2), Am.J.Vet.Res., 40 , 1450-1453 (1979)
(Reference 3)]. Serotype 1 and 2 strains lack cross-immunity, so it is necessary to use prophylactic solutions prepared with serotype 1 and 2 strains to prevent infectious coryza. especially serotype 2
Due to the widespread distribution of this bacterium in Japan, the application of preventive solutions for this strain is eagerly anticipated [Reference 2, Am.J.Vet.Res., 41 , 1900-1903 (1980) (Reference 4)] . However, all serotype 2 bacterial strains lack agglutination properties for fresh red blood cells, and in addition to their low agglutinability to antigens for agglutination reactions, it is desired to establish a simple method for measuring the antigen. [Problems to be Solved by the Invention] Therefore, in view of the above request, the present inventors continued various studies on the hemagglutinin of Haemophilus paragarinarum serotype 2 strains, and as a result, the pathogenicity of serotype 2 strains was determined. We found that the strain usually has a clear capsule, and that by removing this surface material, we could obtain hemagglutinin that showed high agglutination to glutaraldehyde-fixed blood cells. We found that it was possible to measure specific hemagglutination-inhibiting antibodies against serotype 2 bacterial strains. The present invention was completed based on the above findings, and provides a method for producing hemagglutinin that can be specifically used to measure hemagglutination-inhibiting antibodies against an agglutinin-deficient strain of Haemophilus paragarinarum against fresh red blood cells. It is something. [Means for Solving the Problems] In order to solve the problems as described above, according to the present invention, an agglutinin-deficient strain of Haemophilus paragarinarum for fresh red blood cells is cultured in a liquid medium, and the resulting bacterial liquid is cultured. The present invention provides a method for producing hemagglutinin that has low agglutination properties for fresh red blood cells and high agglutination properties for glutaraldehyde-fixed red blood cells, which comprises treating the red blood cells with a salt-containing Rhodanpotash aqueous solution and then subjecting them to sonication. In producing the hemagglutinin of the present invention,
First, an agglutinin-deficient strain of Haemophilus paragarinarum for fresh red blood cells is cultured in a suitable liquid medium. The agglutinin-deficient strain for fresh red blood cells belongs to Haemophilus paragarinarum, and refers to a strain that lacks or has very weak agglutination ability for fresh red blood cells. Such strains include serotype 2 strains, such as H
-18 strain (National Institute of Microbial Technology, Agency of Industrial Science and Technology, accession number FERM P-6004) can be used. In addition, Page's serotype C strain, which the present inventors have shown to have the same antigenicity as serotype 2 strains, can also be used [Reference 4, Am.J.Vet.Res., 41 ,
757-760 (1980)]. In addition, Page also lacks or has very weak agglutination properties for fresh red blood cells.
A serotype B strain of serotype 2 can also be used, and the present invention
The present invention is not limited to any particular bacterial strain, and can be used for bacterial strains that lack or have extremely weak agglutination properties for fresh red blood cells. As the liquid medium, Sawata medium (Reference 2) is usually used. As for the culture conditions, the culture temperature is usually around 35-37℃, and the culture time is usually around 10-37℃.
This is done using the shaking culture method in about 24 hours. Next, bacteria are collected from the culture, which is usually done by centrifugation. Centrifugation usually takes 2 to 5
It is carried out at a low temperature around ℃, and 7000~10000g is 20 ~
It is preferable to do this for about 40 minutes. The obtained cells are washed with near-neutral phosphate buffered saline, suspended in the above buffer, and collected again by centrifugation.
Suspension is usually carried out by measuring with a nephelometer (650 nm) so that the cell concentration is about 10" cells/ml. The surface material of the cells obtained in this way is removed, and this removal process is carried out by treatment with an aqueous solution of Rodanpotash containing salt and sonication. The above treatment with an aqueous solution of Rodanpotash containing salt is carried out by suspending the collected cells in an aqueous solution of Rodanpotash containing salt. The above aqueous solution has a pH of 5.0.
It is preferable to use it in the range of ~7.0. It is preferable to use the concentration of common salt in the range of 0.1 to 0.6M.
Moreover, it is preferable to use the concentration of Rhodankali in the range of 0.25 to 1.5M. Next, sonication is carried out, which is usually carried out at a low temperature of about 2 to 5° C., and treatment at 10 KC for 1 to 15 minutes is sufficient. The treated suspension is centrifuged again.
The conditions for centrifugation are the same as described above. The collected treated cells are washed once with phosphate buffered saline, suspended in phosphate buffered saline at an appropriate concentration, and used as hemagglutinin. The hemagglutinin thus obtained has a characteristic that it has almost no agglutination to fresh red blood cells, but has high agglutination to glutaraldehyde-fixed red blood cells. This hemagglutinin has the properties of being thermolabile, sensitive to trypsin, and resistant to hyaluronidase. By using the hemagglutinin of the present invention, it is possible to measure hemagglutination-inhibiting antibodies against an agglutinin-deficient strain of Haemophilus paragarinarum against fresh red blood cells. For example, the major feature is that the hemagglutinin of the present invention is used as the antigen and a glutaraldehyde-fixed red blood cell suspension is used as the red blood cell suspension.
The measurement system for the hemagglutination-inhibiting antibody against strain No. 221 must be slightly changed. The above measurement system enables the measurement of specific hemagglutination-inhibiting antibodies against Haemophilus paragarinarum serotype 2 strains, which are associated with protection against infectious coryza, and preventive solutions prepared with serotype 2 strains to prevent this disease. It is effective because the effects can be known. Next, the present invention will be specifically explained with reference to examples and reference examples, but the present invention is not limited by these. Example 20 minutes of chicken infusion in a 2 volume shaker Kolben flask
%, Kazaamino acid (Difco) 0.5%, Polypton S
(manufactured by Daigo) 0.5%, salt 0.5%, chicken serum (56℃,
Heat treatment for 30 minutes) 5%, dried yeast extract (25% w/
v, made by Japanese Beetroot Co., Ltd.) 5%, NADH 0.00025%
Prepare 700ml of Sawata liquid medium (PH7.4) containing
This is inoculated with Haemophilus paragarinarum H-18 strain (serotype 2) isolated from the nasal cavity of a chicken affected by infectious coryza, and cultured with shaking at 37°C for 12 hours. The culture is harvested by centrifugation at 8000 G for 30 minutes at 4°C. The obtained cells were washed once with 0.02M phosphate buffered saline (0.15M, PH7.0) (PBSI) and suspended in PBSI to a cell concentration of 10” cells/ml using a nephelometer at 650 nm. The washed cells were collected by centrifugation at 4°C.These were mixed with a 1M Rodankali aqueous solution (PH
6.3) Float. This suspension was subjected to sonication at 10 KC for 10 minutes at 4°C, and then centrifuged at 4°C. The obtained treated cells were washed once with PBSI and suspended in an appropriate amount of PBSI to obtain hemagglutinin. This hemagglutinin exhibits a characteristic in that it has a marked lack of agglutination with fresh blood cells, but a high agglutination with glutaraldehyde-fixed red blood cells. These characteristics are clear from the measurement results of the following experiment. (1) Table 1 shows the measurement results of hemagglutination titer for fresh blood cells and glutaraldehyde-fixed blood cells using red blood cells from each animal.
【表】【table】
【表】
(2) 鶏赤血球を用い製造ロツトの異なる赤血球凝
集素の新鮮赤血球およびグルタルアルデヒド固
定血球に対する赤血球凝集力価を測定した結果
は第２表の通りである。[Table] (2) Table 2 shows the results of measuring the hemagglutination titer of hemagglutinin from different production lots using chicken red blood cells against fresh red blood cells and glutaraldehyde-fixed blood cells.
【表】
(3) 赤血球凝集素の各製造工程における鶏新鮮血
球とグルタルアルデヒド固定血球に対する赤血
球凝集力価を測定した結果は第３表の通りであ
る。[Table] (3) Table 3 shows the results of measuring the hemagglutination titer for fresh chicken blood cells and glutaraldehyde-fixed blood cells in each production process of hemagglutinin.
【表】
前記の赤血球凝集力価は次の方法で測定する。
＜赤血球凝集力価（抗原力価）の測定法＞
抗原をPBSIIで５倍から始めてて２倍段階希釈
列を調製する（各管0.4ml）。これに等量のPBS
（牛血清アルブミン0.1％およびゲラチン0.001％
を含むPBSI）で浮遊した１％グルタルアルデヒ
ド固定鶏血球浮遊液を加えて十分混和し、室温で
２時間静置する。管底像を観察して管底に赤血球
が一面に拡がつているものを凝集反応陽性とし、
この試験管の最高希釈倍数を凝集力価とする。
参考例
被検血清はあらかじめ10％鶏グルタルアルデヒ
ド固定血球で５倍に希釈し、１夜４℃に静置す
る。。翌日、低速遠心（1500rpm、10分間）で血
球を落とし、その上清を検体として用いる。最初
の管には上清を0.2ml入れ（５倍）、第２管目から
PBSを用いて２倍段階希釈する（各管0.2ml）。
あらかじめ使用する赤血球凝集素の力価を上記測
定法で測定しておき、各試験管に４単位に調整し
た赤血球凝集素0.2ml宛加える。15〜20分間室温
で感作した後、各試験管にPBSで浮遊した１
％グルタルアルデヒド固定鶏赤血球浮遊液を0.4
ml宛加えてよく混和し、室温に２時間静置する。
管底像観察により判定する。判定は完全に赤血球
凝集抑制を示した血清の最高希釈倍数を被検血清
の赤血球凝集抑制抗体価（HI抗体価）とする。
このHI抗体価は被検血清の希釈倍数で表し、HI
抗体価５倍以上をもつてHI抗体陽性とする。
上記の測定系を用いてヘモフイルス・パラガリ
ナルム血清型２菌株に対する特異的な赤血球凝集
抑制抗体を測定すると、第４表の測定の通り有効
に測定できることが判明した。なお、HI抗体価
は同じ赤血球凝集素を用い、抗原力価を測定後い
わゆるマイクロタイター法でも測定できる。[Table] The hemagglutination titer described above is measured by the following method. <Method for measuring hemagglutination titer (antigen titer)> Prepare a series of 2-fold serial dilutions of the antigen in PBSII starting from 5-fold (each tube 0.4 ml). equal amount of PBS
(Bovine serum albumin 0.1% and gelatin 0.001%
Add a 1% glutaraldehyde-fixed chicken blood cell suspension suspended in PBSI (including PBSI), mix well, and let stand at room temperature for 2 hours. Observe the image of the tube bottom, and if red blood cells are spread all over the tube bottom, the agglutination reaction is positive.
The highest dilution ratio of this test tube is taken as the agglutination titer. Reference example: Test serum is diluted 5 times in advance with 10% chicken glutaraldehyde-fixed blood cells and left overnight at 4°C. . The next day, the blood cells are removed by low-speed centrifugation (1500 rpm, 10 minutes), and the supernatant is used as the sample. Pour 0.2 ml of supernatant (5x) into the first tube, and from the second tube.
Make 2-fold serial dilutions using PBS (0.2 ml each tube).
Measure the titer of the hemagglutinin to be used in advance using the above method, and add 0.2 ml of hemagglutinin adjusted to 4 units to each test tube. After sensitization at room temperature for 15-20 min, each test tube was incubated with PBS-suspended
0.4% glutaraldehyde-fixed chicken red blood cell suspension
ml, mix well, and leave at room temperature for 2 hours.
Judgment is made by observing the bottom image of the tube. For determination, the highest dilution of serum that completely inhibits hemagglutination is determined as the hemagglutination-inhibiting antibody titer (HI antibody titer) of the test serum.
This HI antibody titer is expressed as the dilution factor of the test serum, and the HI antibody titer is
An antibody titer of 5 times or more is considered positive for HI antibody. When the hemagglutination-inhibiting antibody specific to Haemophilus paragarinarum serotype 2 strain was measured using the above measurement system, it was found that it could be measured effectively as shown in Table 4. Note that the HI antibody titer can also be measured by the so-called microtiter method after measuring the antigen titer using the same hemagglutinin.
【表】【table】
【表】
本赤血球凝集素を用いて測定された赤血球凝集
抑制抗体価と感染防御との関係を検討してみる
と、第５表に示す通り血清型２菌株であるＨ−18
株で調製した予防液を注射された鶏では、本赤血
球凝集素に対する赤血球凝集抑制抗体の産生が認
められるが、血清型１菌株であるNo.221株で調製
した予防液を注射された鶏では認められなかつ
た。[Table] When examining the relationship between the hemagglutination-inhibiting antibody titer measured using this hemagglutinin and infection protection, we found that H-18, a serotype 2 bacterial strain, is shown in Table 5.
Production of hemagglutination-inhibiting antibodies against this hemagglutinin was observed in chickens injected with the prophylactic solution prepared with the serotype 1 strain No. 221, but in chickens injected with the prophylactic solution prepared with the serotype 1 strain No. It was not recognized.
【表】
個々の鶏における赤血球凝集抗体価と感染防御
との関係は第６表に示す通り、両者はおおむね一
致し、とりわけ赤血球凝集抗体価５倍以上の鶏で
は発症を見るものがなかつた。[Table] As shown in Table 6, the relationship between the hemagglutination antibody titer and infection protection in individual chickens is generally consistent, and in particular, no chickens with a hemagglutination antibody titer of 5 times or more developed symptoms.
【表】【table】
Claims (1)
に対する凝集素欠損株を液体培地に培養し、得ら
れた菌液を食塩含有ロダンカリ水溶液で処理し、
次いで音波処理することを特徴とする新鮮赤血球
に対し低凝集性で、グルタルアルデヒド固定赤血
球に対し高凝集性を有するヘモフイルス・パラガ
リナルムの新鮮赤血球に対する凝集素欠損株の赤
血球凝集素の製造法。 ２ ヘモフイルス・パラガリナルムの新鮮赤血球
に対する凝集素欠損株が新鮮赤血球に対し高凝集
性を有するヘモフイルス・パラガリナルム血清型
１またはPage血清型１以外の株である特許請求
の範囲第１項記載のヘモフイルス・パラガリナル
ムの新鮮赤血球に対する凝集素欠損株の赤血球凝
集素の製造法。 ３ ヘモフイルス・パラガリナルムの新鮮赤血球
に対する凝集素欠損株がヘモフイルス・パラガリ
ナルムＨ−18株である特許請求の範囲第２項記載
のヘモフイルス・パラガリナルムの新鮮赤血球に
対する凝集素欠損株の赤血球凝集素の製造法。 ４ 食塩含有ロダンカリ水溶液が5.0〜7.0の範囲
PHである特許請求の範囲第１項記載のヘモフイル
ス・パラガリナルムの新鮮赤血球に対する凝集素
欠損株の赤血球凝集素の製造法。 ５ 食塩の濃度が0.1〜0.6Mの範囲である特許請
求の範囲第３項記載のヘモフイルス・パラガリナ
ルムの新鮮赤血球に対する凝集素欠損株の赤血球
凝集素の製造法。 ６ ロダンカリの濃度が0.25〜1.5Mの範囲であ
る特許請求の範囲第３項記載のヘモフイルス・パ
ラガリナルムの新鮮赤血球に対する凝集素欠損株
の赤血球凝集素の製造法。 ７ 音波処理を10KCで１〜15分間の条件で行う
特許請求の範囲第１項記載のヘモフイルス・パラ
ガリナルムの新鮮赤血球に対する凝集素欠損株の
赤血球凝集素の製造法。 ８ 音波処理を氷冷下で行う特許請求の範囲第６
項記載のヘモフイルス・パラガリナルムの新鮮赤
血球に対する凝集素欠損株の赤血球凝集素の製造
法。[Scope of Claims] 1. A strain of Haemophilus paragarinarum deficient in agglutinin against fresh red blood cells is cultured in a liquid medium, and the resulting bacterial liquid is treated with a Rodankali aqueous solution containing salt,
1. A method for producing hemagglutinin of a hemagglutinin-deficient strain of Haemophilus paragarinarum for fresh red blood cells, which has low agglutination properties for fresh red blood cells and high agglutination properties for glutaraldehyde-fixed red blood cells, the method comprising then sonicating. 2. Haemophilus paragarinarum as claimed in claim 1, wherein the agglutinin-deficient strain of Haemophilus paragarinarum against fresh red blood cells is a strain other than Haemophilus paragarinarum serotype 1 or Page serotype 1, which has high agglutination properties against fresh red blood cells. A method for producing hemagglutinin using an agglutinin-deficient strain for fresh red blood cells. 3. The method for producing hemagglutinin from a strain of Haemophilus paragarinarum deficient in agglutinin against fresh red blood cells according to claim 2, wherein the strain deficient in agglutinin against fresh red blood cells of Haemophilus paragallinarum is Haemophilus paragallinarum strain H-18. 4 Rodan potash aqueous solution containing salt ranges from 5.0 to 7.0
A method for producing hemagglutinin from a hemagglutinin-deficient strain of Haemophilus paragarinarum against fresh red blood cells according to claim 1, which is PH. 5. The method for producing hemagglutinin from an agglutinin-deficient strain of Haemophilus paragarinarum against fresh red blood cells according to claim 3, wherein the concentration of common salt is in the range of 0.1 to 0.6M. 6. The method for producing hemagglutinin of an agglutinin-deficient strain of Haemophilus paragarinarum against fresh red blood cells according to claim 3, wherein the concentration of Rhodankali is in the range of 0.25 to 1.5M. 7. A method for producing hemagglutinin from an agglutinin-deficient strain of Haemophilus paragarinarum on fresh red blood cells according to claim 1, wherein sonication is performed at 10 KC for 1 to 15 minutes. 8 Claim 6 in which sonication is performed under ice cooling
A method for producing hemagglutinin from an agglutinin-deficient strain of Haemophilus paragarinarum against fresh red blood cells as described in 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8098781A JPS57198089A (en) | 1981-05-29 | 1981-05-29 | Preparation of hemagglutinin of fresh erythrocyte- agglutininless strain of haemophilus paragallinarum, and determination system using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8098781A JPS57198089A (en) | 1981-05-29 | 1981-05-29 | Preparation of hemagglutinin of fresh erythrocyte- agglutininless strain of haemophilus paragallinarum, and determination system using the same |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP29327487A Division JPS64467A (en) | 1987-11-20 | 1987-11-20 | Measuring system using hemagglutinin of agglutinin deficient strain to fresh red blood cells of haemophilus paragallinarum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS57198089A JPS57198089A (en) | 1982-12-04 |
| JPH0221800B2 true JPH0221800B2 (en) | 1990-05-16 |
Family
ID=13733844
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8098781A Granted JPS57198089A (en) | 1981-05-29 | 1981-05-29 | Preparation of hemagglutinin of fresh erythrocyte- agglutininless strain of haemophilus paragallinarum, and determination system using the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS57198089A (en) |
-
1981
- 1981-05-29 JP JP8098781A patent/JPS57198089A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS57198089A (en) | 1982-12-04 |
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