JPH01245159A - Plate for detecting newcastle disease virus antibody and method for solubilizing antigen virus - Google Patents
Plate for detecting newcastle disease virus antibody and method for solubilizing antigen virusInfo
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- JPH01245159A JPH01245159A JP7392088A JP7392088A JPH01245159A JP H01245159 A JPH01245159 A JP H01245159A JP 7392088 A JP7392088 A JP 7392088A JP 7392088 A JP7392088 A JP 7392088A JP H01245159 A JPH01245159 A JP H01245159A
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- ndv
- plate
- soln
- solubilized
- antigens
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Links
- 241000711404 Avian avulavirus 1 Species 0.000 title claims abstract description 47
- 241000700605 Viruses Species 0.000 title claims description 11
- 238000000034 method Methods 0.000 title claims description 10
- 230000003381 solubilizing effect Effects 0.000 title claims description 4
- 239000000427 antigen Substances 0.000 title abstract description 27
- 102000036639 antigens Human genes 0.000 title abstract description 27
- 108091007433 antigens Proteins 0.000 title abstract description 27
- 239000002245 particle Substances 0.000 claims abstract description 17
- HEGSGKPQLMEBJL-RKQHYHRCSA-N octyl beta-D-glucopyranoside Chemical compound CCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RKQHYHRCSA-N 0.000 claims description 8
- 230000000890 antigenic effect Effects 0.000 claims 1
- 238000002965 ELISA Methods 0.000 abstract description 17
- 239000011248 coating agent Substances 0.000 abstract description 8
- 238000000576 coating method Methods 0.000 abstract description 8
- 239000012530 fluid Substances 0.000 abstract description 5
- 239000006228 supernatant Substances 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 239000000872 buffer Substances 0.000 abstract description 2
- 238000005199 ultracentrifugation Methods 0.000 abstract description 2
- 230000021615 conjugation Effects 0.000 abstract 3
- 241000654838 Exosporium Species 0.000 abstract 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract 1
- 230000015556 catabolic process Effects 0.000 abstract 1
- 238000006731 degradation reaction Methods 0.000 abstract 1
- 230000000593 degrading effect Effects 0.000 abstract 1
- 238000000856 sucrose gradient centrifugation Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 13
- 230000035931 haemagglutination Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 208000010359 Newcastle Disease Diseases 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 230000000415 inactivating effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 230000007928 solubilization Effects 0.000 description 3
- 238000005063 solubilization Methods 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 210000001643 allantois Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 208000011873 mild conjunctivitis Diseases 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は醇素免疫測定法（以下ＥＬＩＳＡと略称する。[Detailed description of the invention] (Industrial application field) The present invention is an immunosorbent immunoassay (hereinafter abbreviated as ELISA).
）によるニューカッスル病の診断に使用するニューカッ
スル病ｒクイルス抗体の検出用ブレ、−ト及び抗原ウィ
ルスの可溶化方法に関するものである。The present invention relates to a test plate for detecting Newcastle disease virus antibodies used in the diagnosis of Newcastle disease according to the present invention, and a method for solubilizing antigen virus.
（従来の技術）
ＥＬＩＳＡは■他の血清反応よりも感度が高い、■迅速
に結果が得られる、■手技が簡単で自動化に適している
、■−度に多数の検体処理が可能である、■抗体の力価
測定に検体の限界希釈の代わりに１点希釈の吸光度（Ｏ
Ｄ値）で代用できる、などの利点から、ウィルス性疾病
、細菌性疾病などの各種疾病についてその診断への応用
が試みられている。そして、一般にＥＬＩＳＡの特異性
及び感度は固相化される抗原によって左右されるので、
抗原作製法はＥＩＩＳΔの最も重要なポイントになる。(Conventional technology) ELISA: ■Higher sensitivity than other serum reactions; ■Results can be obtained quickly; ■The procedure is simple and suitable for automation; ■A large number of specimens can be processed at one time. ■One-point dilution absorbance (O
Due to the advantage that it can be used as a substitute for D value), attempts have been made to apply it to the diagnosis of various diseases such as viral diseases and bacterial diseases. In general, the specificity and sensitivity of ELISA depend on the antigen immobilized, so
The antigen production method is the most important point for EIISΔ.
従来、ニューカッスル病の診断についてもＥｌ−ＩＳＡ
の適用が検討されており、ＥＬＩＳＡの抗原作製法とし
て、ニューカッスル病ウィルス（以下ＮＤＶと略称する
。）を接種した発育鶏卵の尿膜膣液そのものを抗原どす
る方法、これから精製したウィルスを熱又はホルマリン
で不活化して抗原とする方法などがある。（Ｖｅｔ、
Ｂｕｌｌ 、 ５６゜３３７〜３４３ （１９８６））
（発明が解決しようとする課題）
ＮＤＶは感染力が強く、ときには人間にも伝染して軽い
結膜炎の症状を現わすので、生きたＮＯＶを抗原として
ＥＬＩＳＡに使用する場合にはその取扱いが面倒になる
。Conventionally, El-ISA was also used for the diagnosis of Newcastle disease.
The application of ELISA is being considered, and methods for preparing antigens for ELISA include using the allantoic vaginal fluid of embryonated chicken eggs that have been inoculated with Newcastle disease virus (NDV) as an antigen, and using the virus purified from this as an antigen using heat or heat treatment. There are methods such as inactivating it with formalin and using it as an antigen. (Vet,
Bull, 56° 337-343 (1986)) (Problem to be solved by the invention) NDV is highly infectious and can sometimes be transmitted to humans, causing symptoms of mild conjunctivitis. When used for this purpose, handling becomes troublesome.
一方、ＮＤＶを熱又はホルマリン処理で不活化した場合
には、ウィルスの抗体との結合に関与づる部分が変性し
て抗体との結合性が低下する。従って、ＥＬＩＳＡ用抗
原として使用する際には、固相化するウィルスの量を多
く必要とするという問題がある。On the other hand, when NDV is inactivated by heat or formalin treatment, the portions of the virus that are involved in binding to antibodies are denatured, resulting in decreased binding to antibodies. Therefore, when used as an antigen for ELISA, there is a problem in that a large amount of virus is required to be solidified.
又、ウィルスの不活化方法としてノニデツ１〜Ｐ−４０
、トライトンＸ−１００（いずれも商品名）などの界面
活性剤を用いてウィルスを可溶化する方法も知られてい
るが、これらを用いて再啓化したＮＯＶをＥＬＩＳＡの
抗原として使用した場合には、その感度が低下するとい
う問題がある。In addition, as a method for inactivating viruses, Nonidetsu 1 to P-40
It is also known to solubilize viruses using surfactants such as , Triton has the problem that its sensitivity decreases.
又、ＥＬＩＳＡにおいては、固相に対する抗原の結合状
態のバラツキが測定結果の正確性を左右する重要な因子
となる。従って、養鶏場等で多量の検体をＥＬＩＳＡで
処理する場合、固相に対する抗原の結合作業〈コーティ
ング処理）からＥ ＬＩＳＡの術式を行うことは、作業
に１，１間がかかるばかりでなく測定結果にバラツキを
生ずる原因となる。Furthermore, in ELISA, variations in the binding state of the antigen to the solid phase are an important factor that influences the accuracy of the measurement results. Therefore, when processing a large number of samples using ELISA at a poultry farm, etc., performing the ELISA procedure from the binding of antigen to the solid phase (coating treatment) not only takes time, but also makes the measurement difficult. This causes variations in results.
本発明は従来技術の有するこのような問題点に鑑みでな
されたものであり、その目的は抗体に対する結合能が低
下Ｊることなく不活化された安全なＮＤＶ抗原が固相化
されたＥＬＩＳＡ用プレートを提供することＣあり、さ
らには、抗体に対ηる結合能を低下させることなくＮＤ
Ｖを不活化ザる方法を提供することである。The present invention was made in view of the problems of the prior art, and its purpose is to provide a safe NDV antigen for use in ELISA that is immobilized on a safe inactivated NDV antigen without reducing its binding ability to antibodies. It is possible to provide a plate, and furthermore, it is possible to provide a ND plate without reducing the binding ability to the antibody.
The object of the present invention is to provide a method for inactivating V.
（課題を解決するための手段及び作用）前記の目的を達
成するため、本発明はＥＬＩＳＡによるニューカッスル
病診断に使用するニューカッスル病ウィルス抗体の検出
用プレートどして、プレートに形成された複数個のつ１
ルを、オクチルグルコシド溶液処理で可溶化したニュー
カッスル病ウィルス粒子溶液でコーティングした。(Means and effects for solving the problem) In order to achieve the above object, the present invention provides a plate for detecting Newcastle disease virus antibodies used for diagnosis of Newcastle disease by ELISA. 1
The cells were coated with a solution of Newcastle disease virus particles solubilized by treatment with octyl glucoside solution.
又、精製されたニューカッスル病ウィルス粒子をオクチ
ルグルコシド溶液で処理して可溶化することにより、Ｎ
ＤＶの抗体に対する結合能を低下させることなくＮＤＶ
が不活化される。In addition, by treating and solubilizing purified Newcastle disease virus particles with an octyl glucoside solution, N
NDV without reducing the binding ability of DV to antibodies.
is inactivated.
精製ＮＤＶ粒子は、ＮＤＶを接挿した発育鶏卵から採取
した尿膜膣液を遠心分離して得た上清を、ショ糖密度勾
配遠心にかけた後、Ｐｕｓ （リン酸緩衝液）に再浮遊
して超遠心にかけることにより１ｑられる。ＮＤＶ粒子
の可溶化は、ＮＤＶ粒子をＰＢＳに浮遊させた溶液に、
オクチルグルコシドのＰＢＳ溶液を加えることにより室
温で行われる。Purified NDV particles are obtained by centrifuging the allantoic vaginal fluid collected from embryonated chicken eggs that have been injected with NDV.The supernatant obtained is subjected to sucrose density gradient centrifugation, and then resuspended in PuS (phosphate buffer). 1q is obtained by ultracentrifugation. To solubilize NDV particles, add NDV particles to a solution suspended in PBS.
This is done at room temperature by adding a PBS solution of octyl glucoside.
可溶化されたＮＤＶのＨＡ活性（赤血球凝集性）は精製
ＮＤＶよりも多くなり、従来の不活化と異なり抗体に対
する結合能の低下はない。これは第１図に示すように、
ＮＤＶはその外膜（エンベロープ〉１に抗原２が多数存
在しており、可溶化していないＮＤＶを固相化した場合
には、第２図に示すようにプレート３と対応する側の抗
原２が抗体との結合に関与できなくなるのに対し、本発
明による可溶化ではＮＯＶの外膜１が第１図のＸ印部性
で切断されることにより、プレート３に固相化された際
、多くの抗原２が抗体との結合に関与できるようになる
ためと考えられる。The HA activity (hemagglutination) of solubilized NDV is higher than that of purified NDV, and unlike conventional inactivation, there is no decrease in the binding ability to antibodies. As shown in Figure 1, this
NDV has a large amount of antigen 2 in its outer membrane (envelope) 1, and when unsolubilized NDV is immobilized, antigen 2 is present on the side corresponding to plate 3 as shown in Figure 2. In contrast, in the solubilization according to the present invention, the outer membrane 1 of NOV is cut at the X mark in FIG. 1, so that when it is immobilized on the plate 3, This is thought to be because a large amount of antigen 2 becomes able to participate in binding with the antibody.
可溶化されたＮＤＶ粒子溶液をコーティングするプレー
トは、ＥＬＩＳＡで一般に使用されるプレートが使用さ
れる。コーティングに使用する可溶化ＮＤＶ粒子溶液は
、ＨＡ活性（赤血球凝集性）が１＝３０〜５０となるよ
うに緩衝液で希釈調整して使用する（実用化の点では安
定的に反応を促進するため１００ＨＡとする。）。A plate commonly used in ELISA is used for coating the solubilized NDV particle solution. The solubilized NDV particle solution used for coating is diluted with a buffer solution so that the HA activity (hemagglutination) is 1 = 30 to 50. 100HA).
（実施例） 以下、本発明についてより具体的に説明する。(Example) The present invention will be explained in more detail below.
（１’）ＮＤＶの精製
ＮＤＶを１０〜１１日齢の発育鶏卵に接種し、２４〜４
８時間後に尿膜膣液を採取した。細胞成分除去のため５
ｏｏｏ〜１００００ｇで２時間遠心し、その上清を１０
０００００で２時間遠心してペレット状の沈澱層を回収
した。この沈澱層をＰＢＳに浮遊し、この浮遊液を２０
％及び５０％の不連続密度勾配を右するショ糖溶液層上
に重層して１０００００ｏで９０分遠心した。２０％と
５０％の間のバンドを採取して５倍♀のＰＢＳに再浮遊
し、さらに連続密度でショ糖密度勾配遠心く２０〜５０
％、１０００００ｇ、１時間）を行なった ５ｏ％領域
に近い部分に生じたバンドを採取し、シヨ砂除去のため
Ｐ［３Ｓに再浮遊して１０００００（］で１１時間心を
行った。ペレット状の沈澱物を回収し、ＰＢＳに浮遊後
液体窒素中に保存した。この精製ウィルスのＨＡ活性は
１：２０００以上、タンパク量は１０００ＨＡ／ｍｌ当
たり１００μ９以下であった。(1') Purification of NDV NDV is inoculated into 10-11 day old embryonated chicken eggs,
Eight hours later, allantois vaginal fluid was collected. 5 for cell component removal
ooo~ Centrifuge at 10,000g for 2 hours, and then centrifuge the supernatant at 10000g.
The mixture was centrifuged at 00,000 for 2 hours to collect a pelleted precipitate layer. This precipitated layer was suspended in PBS, and this suspension was
% and 50% discontinuous density gradients were overlaid on the right sucrose solution layer and centrifuged at 100,000° for 90 minutes. Bands between 20% and 50% were collected, resuspended in 5x ♀ PBS, and further centrifuged on a sucrose density gradient at continuous density for 20 to 50 min.
%, 100,000 g, 1 hour).The band that appeared near the 5o% region was collected, resuspended in P[3S to remove sand, and subjected to heart testing at 100,000 g for 11 hours. The precipitate was collected, suspended in PBS, and then stored in liquid nitrogen.The HA activity of this purified virus was 1:2000 or more, and the protein amount was 100 μ9 or less per 1000 HA/ml.
（２）精製ＮＤＶ粒子の可溶化
精ＷＡＮＤＶ粒子を浮遊したＰＦ３５溶液に４％オクヂ
ルグルコシド液を等量加え、室温で１時間放置すること
にＪ：すＮＤＶ粒子を可溶化した。(2) Solubilization of purified NDV particles An equal volume of 4% ocdyl glucoside solution was added to a PF35 solution in which purified WANDV particles were suspended, and the mixture was left to stand at room temperature for 1 hour to solubilize the J:su NDV particles.
比較試験として０．１％のトライトンｘ−ｉ。0.1% Triton x-i as a comparative test.
Ｏ及びノニデットＰ−４０（いずれも非イオン性界面活
性剤の商品名）で、ＮＤＶ粒子を可溶化した。NDV particles were solubilized with O and Nonidet P-40 (both are trade names of nonionic surfactants).
精’ＩＮＤＶ、オクチルグルコシドによる可溶化ＮＤＶ
、トライトンＸ−１００による可溶化ＮＤ■、ノニデッ
トＰ−４０による可溶化ＮＤＶについて、０．５％鶏赤
血球液を使用して一般的に用いられるｍ１ｃｒｏ法に従
って赤血球凝集（ＨＡ）試験を２倍希釈法で行った。又
、抗原中に残留する可溶化物質の溶血性試験をも行った
。結果を表に示す。Semen'INDV, solubilized NDV with octyl glucoside
, ND solubilized with Triton X-100■, NDV solubilized with Nonidet P-40, 2-fold dilution of hemagglutination (HA) test according to the commonly used M1CRO method using 0.5% chicken red blood cell fluid I went by law. We also conducted a hemolytic test of the solubilized substance remaining in the antigen. The results are shown in the table.
抗体との結合能を表すＨＡ活性は、オクチルグルコシド
処理ＮＤＶではＩｔ製ＮＤＶより２３高くなったのに対
して、トライトンＸ−１００処理ＮＤＶ及びノニデット
Ｐ−４０処理ＮＤＶでは精製ＮＯＶより２１以上低くな
った。The HA activity, which indicates the ability to bind to antibodies, was 23 higher in octyl glucoside-treated NDV than It-made NDV, whereas it was more than 21 lower in Triton X-100-treated NDV and Nonidet P-40-treated NDV than purified NOV. Ta.
（３〉可溶化処理ＮＤＶのコーティングオクチルグルコ
シドで処理した可溶化ＮＯＶを透析ののち溶液（１−Ｉ
Ａ活性１：１６０００以上）を炭酸緩衝液（ｐ Ｈ９
，６，Ｎａ ＮＪ ０．０２％含右）でｌ−Ｉ Ａ活
性１：１００となるように希釈調整したものをコーティ
ング用液とした。一般のＥＬＩＳＡ用プレー上プレート
多数（例えば９６個）のウェルを有するプレートの各ウ
ェルに、前記コーティング用液を１００μｍずつ入れ、
４℃で４８時間放置した。コーティング用液を捨て、１
％アルブミン加ＰＢＳを１００μｍずつ各ウェルに入れ
、３７℃で３０分反応させた。次に０．１％トウィーン
２０加ＰＢＳで各ウェルを２回洗浄した後、プレートを
よく乾燥した。コーテイング後のプレートは４℃では長
期保存後も規準陽性血清に対するＥＬＩＳＡのＯＤ値は
０．５前後を保持したが、３７℃では１週間で０．２ま
で低下した。(3> Coating of solubilized NDV After dialysis of the solubilized NOV treated with octyl glucoside, a solution (1-I
A activity of 1:16,000 or more) in carbonate buffer (pH 9
, 6, containing 0.02% NaNJ) to give a l-IA activity of 1:100, which was used as a coating solution. Pour 100 μm of the coating solution into each well of a general ELISA plate having a large number of wells (for example, 96 wells),
It was left at 4°C for 48 hours. Discard the coating liquid and
100 μm of PBS supplemented with % albumin was added to each well and allowed to react at 37° C. for 30 minutes. Next, each well was washed twice with PBS supplemented with 0.1% Tween, and then the plate was thoroughly dried. Even after long-term storage of the coated plate at 4°C, the ELISA OD value for standard positive serum remained around 0.5, but at 37°C it decreased to 0.2 in one week.
すなわち、プレートの保存は低温（４℃）で行う必要が
ある。That is, the plate must be stored at a low temperature (4°C).
（４）ＥＬＩＳＡのＯＤ値と中和抗体価との関係前記の
ようにして可溶化ＮＤＶ抗原をコーティングしたプレー
トを使用するとともに、酵素標識抗体としペルオキシダ
ーゼで標識したウサギ抗ニワトリＩａＧを使用して定法
に従いＥＬＳＩＡを行った。又、定法に従い赤血球凝集
抑制（ト１１）試験及び中和試験を行った。(4) Relationship between ELISA OD value and neutralizing antibody titer Using a plate coated with the solubilized NDV antigen as described above, and using a standard method using rabbit anti-chicken IaG labeled with peroxidase as an enzyme-labeled antibody. ELSIA was performed according to the following. Further, hemagglutination inhibition (T11) test and neutralization test were conducted according to standard methods.
次式で定義するＳ／Ｐ比とＨ１価及び中和指数の相関性
はいずれもγ＝０．７であった。The correlation between the S/P ratio defined by the following formula, the H1 value, and the neutralization index was γ=0.7.
Ｓ／Ｐ 比＝ （Ｓ−Ｎ＞／ （Ｐ−Ｎ＞Ｓ・・・
被検血清ＯＤ値、Ｎ・・・対照陰性血清ＯＤ値、Ｐ・・
・対照陽性血清００１直
（発明の効果）
以上詳述したように本発明のプレートは、ＮＤ■の抗体
に対する結合能が低下することなく不活化されて、各ウ
ェルに抗原として結合されているので安全かつ簡単にＥ
ＬＩＳＡを行うことができ、作業者が異なっても測定結
果のバラツキが小さくなる。又、可溶化処理によりＥＬ
ＩＳＡ抗原として精製ＮＤＶより高感度となるため、結
果的に固相生に必要な抗原の間が少なくて済み′！！Ａ
造コス１〜が低くなる。S/P ratio = (S-N>/ (P-N>S...
Test serum OD value, N... Control negative serum OD value, P...
- Control positive serum 001 direct (effects of the invention) As detailed above, in the plate of the present invention, ND■ is inactivated without decreasing its ability to bind to antibodies, and is bound to each well as an antigen. Safely and easily
LISA can be performed, and variations in measurement results are reduced even if different operators are used. In addition, EL can be improved by solubilization treatment.
Since it is more sensitive than purified NDV as an ISA antigen, fewer antigen gaps are required for solid-phase growth! ! A
The construction cost 1~ becomes lower.
第１図はニューカッスルウィルスの模式図、第２図はニ
ューカッスルウィルスを同相に付性させた状態を示す概
略図である。
外膜〈エンベロープ〉１、抗原２、プレート３゜特許出
願人 株式会社ゲン・〕−ポレーション代 理 人
弁理士 恩１）博宣FIG. 1 is a schematic diagram of Newcastle virus, and FIG. 2 is a schematic diagram showing a state where Newcastle virus is attached to the same phase. Outer membrane (envelope) 1, antigen 2, plate 3゜Patent applicant Gen Co., Ltd. - Poration agent
Patent Attorney On 1) Hironobu
Claims (1)
グルコシド溶液処理で可溶化したニユーカッスル病ウィ
ルス粒子溶液でコーティングしたことを特徴とするニユ
ーカッスル病ウィルス抗体の検出用プレート。 ２、精製されたニユーカッスル病ウィルス粒子をオクチ
ルグルコシド溶液で処理して可溶化することを特徴とす
る抗原ウィルスの可溶化方法。[Claims] 1. A plate for detecting Newcastle disease virus antibodies, characterized in that a plurality of wells formed on the plate are coated with a solution of Newcastle disease virus particles solubilized by treatment with an octyl glucoside solution. 2. A method for solubilizing an antigenic virus, which comprises treating purified Newcastle disease virus particles with an octyl glucoside solution to solubilize them.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7392088A JPH01245159A (en) | 1988-03-28 | 1988-03-28 | Plate for detecting newcastle disease virus antibody and method for solubilizing antigen virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7392088A JPH01245159A (en) | 1988-03-28 | 1988-03-28 | Plate for detecting newcastle disease virus antibody and method for solubilizing antigen virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01245159A true JPH01245159A (en) | 1989-09-29 |
Family
ID=13532076
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7392088A Pending JPH01245159A (en) | 1988-03-28 | 1988-03-28 | Plate for detecting newcastle disease virus antibody and method for solubilizing antigen virus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01245159A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004060303A3 (en) * | 2002-12-26 | 2005-11-24 | Cell Genesys Inc | Methods and reagents for the enhancement of virus transduction in the bladder epithelium |
| WO2017009926A1 (en) * | 2015-07-13 | 2017-01-19 | 和光純薬工業株式会社 | Fixing method for specific binding substance |
-
1988
- 1988-03-28 JP JP7392088A patent/JPH01245159A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004060303A3 (en) * | 2002-12-26 | 2005-11-24 | Cell Genesys Inc | Methods and reagents for the enhancement of virus transduction in the bladder epithelium |
| US7267815B2 (en) | 2002-12-26 | 2007-09-11 | Cell Genesys, Inc. | Methods and reagents for the enhancement of virus transduction in the bladder epithelium |
| US7459154B2 (en) * | 2002-12-26 | 2008-12-02 | Cell Genesys, Inc. | Methods and reagents for the enhancement of virus transduction in the bladder epithelium |
| WO2017009926A1 (en) * | 2015-07-13 | 2017-01-19 | 和光純薬工業株式会社 | Fixing method for specific binding substance |
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