TW202313673A - 抗諾羅病毒抗體 - Google Patents
抗諾羅病毒抗體 Download PDFInfo
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- TW202313673A TW202313673A TW111118835A TW111118835A TW202313673A TW 202313673 A TW202313673 A TW 202313673A TW 111118835 A TW111118835 A TW 111118835A TW 111118835 A TW111118835 A TW 111118835A TW 202313673 A TW202313673 A TW 202313673A
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Classifications
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
課題係提供一種抗諾羅病毒抗體,其會對許多基因型的諾羅病毒反應,並能夠全面性地檢測諾羅病毒。
解決手段係提供了一種抗諾羅病毒抗體或其抗原結合片段,其會與由序列識別號1及序列識別號2各自所示之胺基酸序列構成的各肽進行抗原抗體反應。
效果係提供了一種新穎的抗諾羅病毒抗體以及使用其之諾羅病毒的免疫測定方法及免疫測定器具,其會廣泛地與諾羅病毒結合,並能夠廣泛且專一性並高靈敏度地檢測人類諾羅病毒。
Description
本發明關於一種抗諾羅病毒抗體以及使用其之諾羅病毒的免疫測定方法及免疫測定器具。
諾羅病毒對人類進行經口感染而在十二指腸到小腸上部增殖,引起感染性胃腸炎。使十二指腸附近的小腸上皮細胞脫落,並引起嘔吐、腹瀉、腹痛等症狀。感染到發病的潛伏期係12小時~72小時(平均1~2日),且症狀消退後,病毒排出至糞便也會持續1~3週左右,也有報告超過7週的排出。通報食物中毒患者中約7成係諾羅病毒感染症。
諾羅病毒係不具有套膜且於基因體具有約7,500鹼基的正單股RNA之病毒。諾羅病毒的基因體存在3個蛋白質編碼區(ORF),報告了ORF1係編碼與病毒複製有關的非結構蛋白質、ORF2編碼殼體(capsid)結構蛋白質(VP1)、ORF3編碼次要結構蛋白質(VP2)。此外,基於殼體基因序列的類似性,諾羅病毒被分類於基因群(genogroup)I~V(GI~GV)的5個群,其中,GI、GII、GIV係人類感染的主流。其中,基因群I(GI)與基因群II(GII)富有遺傳多樣性,從源自人類的試料來說,檢測到進化系統不同的各種各樣的病毒,認為:基因群I能夠分類為9種或更多的基因型(genotype)、基因群II能夠分類為22種或更多的基因型。
諾羅病毒的檢測係使用抗體透過酵素免疫分析(EIA)法(參照非專利文獻1)或透過免疫層析(immunochromatography)法(參照非專利文獻2)來檢測殼體結構蛋白質而進行。因而為了正確地檢測諾羅病毒抗原,會需要會對全部基因型反應的抗體。
不過,辨識抗原共同區域而反應的抗體不容易取得,迄今為止進行了製作組合了複數個對於具有特定胺基酸序列的諾羅病毒抗原肽或其片段之抗體(例如:參照專利文獻1)的試劑,並將不同的基因型的諾羅病毒個別地檢測。
例如:為了獲得基因群GII的諾羅病毒共通反應的抗體而進行探討時,與屬於GII之諾羅病毒殼體之突出結構域的特定部位結合的抗體,被報告會廣泛地與GII的諾羅病毒結合,且會專一性地檢測屬於該GII之基因型(GII/1~GII/17)大致全部的諾羅病毒(參照專利文獻2)。
不過,諾羅病毒係易突變性病毒,露出在殼體表面之突出結構域的突變多,即便可暫時製作出透過在突出結構域進行抗原抗體反應而與廣泛的基因型反應之抗體,也會有這樣的問題:若發生突變株則變得無法進行反應。
因此,期望能夠首創一種抗體,其能夠全面性地檢測基因型不同的多數諾羅病毒。
另一方面,在非專利文獻3記載有一種單株抗體,其會對諾羅病毒殼體的殼結構域(shell domain)進行抗原抗體反應。
[先前技術文獻]
[專利文獻]
專利文獻1:日本特表2009-542715號公報
專利文獻2:國際公報WO13/039165
[非專利文獻]
非專利文獻1:「改良諾羅病毒抗原檢測EIA套組的評價」,月刊醫學與藥學 Vol.61 No.1 Page.93-98 (2009.01.25)
非專利文獻2:「諾羅病毒抗原快速診斷用藥諾羅病毒快篩(QuickNavi-Norovirus)的評價」,月刊醫學與藥學 Vol.61 No.5 Page.779-785 (2009.05.25)
非專利文獻3:Gabriel I. Parra et al.,PLOS ONE,June 2013,Volume 8,Issue 6,e67592
[發明欲解決之課題]
本發明係提供一種抗諾羅病毒抗體,其能對許多基因型的諾羅病毒反應,而全面性地檢測諾羅病毒。
[用以解決課題之手段]
本發明有鑒於上述課題,在為了獲得諾羅病毒共通反應的抗體進行探討時,並非著眼於露出在諾羅病毒殼體表面的突出結構域,而是著眼於為內部結構的殼結構域,發現到:與諾羅病毒殼體的殼結構域的特定部位結合的抗體係會廣泛地與諾羅病毒結合,並能夠廣泛且專一性地檢測人類諾羅病毒。
即本發明係提供以下者。
(1)一種抗諾羅病毒單株抗體或其抗原結合片段,其會與由序列識別號1及序列識別號2各自所示之胺基酸序列構成的各肽進行抗原抗體反應。
(2)如(1)記載之抗諾羅病毒抗體或其抗原結合片段,其係將序列識別號3所示之胺基酸序列的整體或一部分區域設為表位(epitope)。
(3)如(1)或(2)記載之抗諾羅病毒抗體或其抗原結合片段,其中前述抗諾羅病毒抗體為單株抗體。
(4)一種諾羅病毒之免疫測定方法,其利用了檢體中的諾羅病毒、與如(1)~(3)中任一項記載之抗諾羅病毒單株抗體或其抗原結合片段的抗原抗體反應。
(5)一種諾羅病毒之免疫測定器具,其包含如(1)~(3)中任一項記載之抗諾羅病毒單株抗體或其抗原結合片段。
[發明之效果]
依據本發明,提供一種新穎的抗諾羅病毒單株抗體以及使用其之諾羅病毒的免疫測定方法及免疫測定器具,其中,該抗諾羅病毒單株抗體會廣泛地與諾羅病毒結合,並且能夠廣泛且專一性並且高靈敏度地檢測人類諾羅病毒。
[用以實施發明的形態]
本發明之抗諾羅病毒抗體係會與由MLYTPLRTGGGSGGTDPFVVAGR(序列識別號1)及RLVAMLYTPLRANGSGDDVFTVS(序列識別號2)所示之胺基酸序列構成的各肽進行抗原抗體反應的抗體,且係會結合到存在於諾羅病毒殼體結構蛋白的殼結構域之表位的抗體。由序列識別號1及序列識別號2各自所示之胺基酸序列的共同性來看,茲認為MLYTPLR(序列識別號3)的整體或一部分區域係為表位。再者,該區域與非專利文獻3所記載之單株抗體的表位係為不同的區域。
諾羅病毒的殼體結構蛋白(VP1)已知係由殼結構域(S結構域)與突出結構域(P結構域)構成。茲認為S結構域係掌管VP1的組裝。另一方面,P結構域進一步被分為P1與P2次結構域,P1次結構域會與S結構域相互作用並增強殼體的物理穩定性,而P2次結構域係位置於病毒粒子最外圍,在小鼠諾羅病毒來說被報告係中和抗體的標的。
本發明之抗諾羅病毒抗體能夠係IgG、IgA、IgY、IgD、IgM、IgE或者該等之1個以上的一部分,例如:被任意要求的類型如重鏈、輕鏈、Fc或者F(ab)一部分。
本發明所使用的抗諾羅病毒抗體能夠使用已知手段而以多株或單株抗體的形式獲得。就源自哺乳動物的單株抗體而言,包含:由融合瘤所產生者;及經基因工程手法,由利用包含抗體基因的表現載體進行轉形的宿主所產生者。
產生抗諾羅病毒單株抗體的融合瘤基本上係能夠使用已知的技術,而如以下般進行而製作。即,可藉由如下製作:使用基因重組諾羅病毒樣顆粒(VLP)作為致敏抗原,並按照一般的免疫方法將其進行免疫,並透過一般的細胞融合法使所獲得之免疫細胞與已知的母細胞融合,並透過一般的篩選(screening)法來篩選產生單株的抗體之細胞,並從所獲得的單株抗體中,選擇會與由序列識別號1及序列識別號2各自所示之胺基酸序列構成的各肽進行抗原抗體反應的單株抗體。此外,多株抗體能夠藉由如下製作:將由序列識別號1及序列識別號2各自所示之胺基酸序列構成之各肽的至少任一者,直接固定化、或是固定於常用載體,而對小鼠、大鼠、倉鼠等動物進行免疫,並從其血清回收多株抗體。
基因重組諾羅病毒VLP能夠將諾羅病毒殼體的基因序列插入至轉移載體(transfer vector),並將桿狀病毒(baculovirus)DNA、與前述的質體同時地轉染到Sf9細胞,利用同源重組(homologous recombination),製作重組病毒並使增殖而獲得種子病毒,其後透過在Tn5細胞進行蛋白質的表現,而利用已知方法從細胞中或培養上清液中將目標的重組病毒VLP進行精製而藉以獲得。再者,基因重組諾羅病毒VLP本身眾所周知,係廣為使用來用以創製抗諾羅病毒抗體等。
就利用致敏抗原所免疫的哺乳動物而言,並非被特別限定者,但較佳係考慮與使用在細胞融合之母細胞的適合性來選擇,一般來說是使用齧齒類的動物,例如:小鼠、大鼠、倉鼠等。
要使致敏抗原對動物進行免疫來說,係按照已知方法進行。例如:透過對哺乳動物的腹腔內或皮下注射致敏抗原而進行。具體地說,能夠利用PBS(磷酸鹽緩衝生理鹽水,Phosphate-Buffered Saline)或生理食鹽水等將致敏抗原稀釋為適當量並懸浮者,依期望適量混合一般的佐劑,例如:弗氏完全佐劑(Freund’s complete adjuvant),並乳化後,對動物的皮下、皮內、腹腔等進行投予而予以暫時刺激後,因應需要重複進行同樣的操作。抗原的投予量係因應投予途徑、動物物種而適宜決定,一般投予量較佳係每1次10μg~1mg左右。如此進行免疫並確認了血清中所期望的抗體濃度(antibody level)上升後,從哺乳動物採血,並精製血清成分而藉以獲得多株抗體。在精製血清成分之際來說,能夠使用固定有致敏抗原的親和管柱等。
此外,製作單株抗體之際來說,從抗體濃度已上升的哺乳動物取出免疫細胞,並進行細胞融合。就進行細胞融合之際較佳的免疫細胞而言,特別可舉脾細胞。
作為與前述免疫細胞所融合的另一個母細胞之哺乳動物的骨髓瘤細胞,適宜使用早就已知的各種細胞株,例如:P3X63、NS-1、MPC-11、SP2/0等。
前述免疫細胞與骨髓瘤細胞的細胞融合能夠按照已知的方法,例如:柯勒等人的方法(Kohler et al.,Nature,vol,256,p495-497(1975))等而進行。即,在聚乙二醇(平均分子量1000~6000的PEG,30~60%濃度)、仙台病毒(Sendai virus)(HVJ)等細胞融合促進劑的存在下,依期望添加二甲亞碸等輔助劑,並在RPMI1640培養液、MEM培養液等營養培養液中,混合免疫細胞與骨髓瘤細胞而藉以進行融合細胞(融合瘤)的形成。
利用包含次黃嘌呤(hypoxanthine)、胸苷(thymidine)及胺基喋呤(aminopterin)的培養基(HAT培養基)等選擇培養基將透過融合所形成的融合瘤培養1日~7日,並與未融合細胞分離。所獲得之融合瘤透過其產生的抗體來進一步選擇。按照已知的極限稀釋法將選擇出之融合瘤進行單一殖株化,而建立為產生單一殖株性抗體的融合瘤。
檢測融合瘤產生之抗體活性的方法能夠使用已知的方法。可舉,例如:ELISA法、凝聚反應法、放射免疫分析(radioimmunoassay)法。
為了從所獲得之融合瘤取得單株抗體,可採用:按照一般的方法培養該融合瘤,並以其培養上清液的形式獲得的方法、或者將融合瘤投予至與其具有適合性的哺乳動物並使增殖,而以其之腹水的形式獲得的方法等。
抗體的精製能夠使用鹽析(salting-out)法、凝膠過濾法、離子交換層析法或者親和層析法等已知的精製手段而進行。
透過將本發明之抗諾羅病毒抗體應用於任意的免疫測定方法,而能夠專一性地測定・檢測檢體中的諾羅病毒。
免疫測定方法本身眾所周知,本發明之免疫測定除使用上述本發明之抗諾羅病毒抗體或其抗原結合片段來作為抗體或其抗原結合片段以外,係眾所周知之免疫測定方法的任一者均能夠採用。因此,免疫測定方法未被特別限制,三明治(sandwich)法、免疫凝聚法、競爭(competitive)法等眾所周知的免疫測定方法之任一者均能夠採用。該等之中較佳係使用了抗諾羅病毒抗體與標識抗諾羅病毒抗體的三明治法,進一步較佳係使用固定化抗諾羅病毒抗體與標識抗諾羅病毒抗體的方法。
就固定抗諾羅病毒抗體的支撐體而言,較佳為聚苯乙烯板、乳膠粒子、磁性粒子、玻璃纖維膜、耐綸膜、硝化纖維素膜、乙酸纖維素膜等不溶性支撐體。抗體的固定方法係眾所周知的。
此外,就標識抗諾羅病毒抗體而言,能夠使用已知的標識物,例如:放射性同位素(例如:
32P、
35S、
3H)、酵素(例如:過氧化酶、鹼性磷酸酶、螢光素酶)、蛋白(例如:卵白素(avidin))、低分子化合物(例如:生物素)、螢光物質(例如:FITC)、化學發光物質(例如:吖啶(acridinium))、乳膠粒子(例如:著色乳膠粒子、螢光乳膠粒子)、金屬(例如:金、銀、鉑等貴金屬)膠體粒子、碳原子等。
檢體中諾羅病毒的檢測係透過使檢體中的諾羅病毒與固定化抗諾羅病毒抗體反應而進行,在利用三明治法的情況下,能夠使含檢體之液與固定化抗諾羅病毒抗體反應,然後藉著使前述標識抗體反應、或使固定化抗諾羅病毒抗體與標識抗體同時地反應而進行。反應結束後,若測定由檢體中的諾羅病毒、固定化抗諾羅病毒抗體以及標識抗體所形成的複合物中的標識量,則能夠測定檢體中的諾羅病毒量。標識量的測定能夠利用因應於標識物種類的手段來進行。例如:當使用了酵素、卵白素作為標識的情況下,反應後,添加基質,並測定酵素活性。此外,當使用了螢光(包含螢光乳膠粒子)或化學發光物質作為標識的情況下,在不引起消光的條件下測定信號。著色乳膠粒子、金屬膠體粒子、及碳粒子等係利用目視或反射光等來測定信號。
上述免疫測定方法之中,更佳係ELISA及免疫層析術法。
使用有本發明之抗諾羅病毒抗體的免疫測定器具,包含固定化之本發明的抗諾羅病毒抗體。亦可為套組的形態,該套組在固定化抗諾羅病毒抗體之外,係由例如:檢體用稀釋液、標識抗諾羅病毒抗體、反應基質等所構成。
[實施例]
實施例1 取得抗諾羅病毒單株抗體
將藉由常用方法所製作出之諾羅病毒樣顆粒(VLPs)對BALB/c小鼠進行免疫,並從已經飼育一定期間的小鼠摘除脾臓,透過柯勒等人的方法(Kohler et al.,Nature,vol,256,p495-497(1975))來與小鼠骨髓瘤細胞融合。將所獲得之融合細胞(融合瘤)維持在37℃培養箱中,並使用其培養上清液來調查了對諾羅病毒產生抗體的反應性。將VLPs以140mM NaCl、2.7mM KCl、10mM Na
2HPO
4、1.8mM KH
2PO
4、pH7.3(以下簡記為PBS)稀釋為0.1μg/mL。將經稀釋之VLPs以每1孔100μL的VLPs/PBS,pH7.3溶液添加至塑料製微量滴定盤(Nunc-Immuno Module F8 Maxisorp Surface plate,商品名,Nalgen Nunc International公司製)的孔,並於4℃、12小時的條件下將VLPs於微量滴定盤上進行了固相化。12小時後,透過傾析除去已加入至孔的VLPs/PBS溶液,並以200μL/孔將PBS、0.05%(v/v)Tween20(以下簡記為PBS-T,Tween係商品名))添加至該微量滴定盤的孔,透過傾析進行了PBS-T的除去。該洗淨步驟合計進行了3次。
其後,以200μL/孔來添加1%PerfectBlock (商品名,Funakoshi公司製阻斷劑)/PBS pH7.3,以4℃、12小時的條件下進行了VLPs固相化微量滴定盤的孔內阻斷。以經過12小時後,4℃的狀態設為保存狀態。為了確認培養上清液中抗諾羅病毒單株抗體的反應性,以培養上清液100μL/孔添加至VLPs固相化微量滴定盤,進行了37℃、1小時加溫。其後,透過傾析而除去已加入至孔的培養上清液,以200μL/孔添加PBS-T,並透過傾析進行了PBS-T的除去,而進行孔內的洗淨。該洗淨步驟合計進行了3次。
其後,以100μl/孔(稀釋1萬倍)將過氧化酶結合山羊抗小鼠免疫球蛋白(Peroxidase-Conjugated Goat Anti-Mouse Immunoglobulins,安捷倫科技(Agilent Technologies)公司製)(以下稱為酵素標識抗體)添加至孔,並進行37℃、1小時加溫。其後,透過傾析而除去已加入至孔的酵素標識抗體,並以200μL/孔來添加PBS-T,並透過傾析進行PBS-T的除去,而進行了孔內的洗淨。該洗淨步驟合計進行了3次。其後,以100μL/孔將3,3’,5,5’-四甲基聯苯胺(3,3’,5,5’-tetramethylbenzidine)(以下簡記為TMB)溶液作為過氧化酶酵素反應基質溶液來添加於孔,並以25℃、30分鐘、遮光下進行了靜置。其後,立即以100μL/孔來添加313mM H
2SO
4溶液,並使酵素反應停止。
其後,測定此孔的吸光度,從450nm的吸光度減去630nm的吸光度,並將該數值設為反應性評價的指標。(Josephy P.D.,Mason R.P.et al.(1982) J. Biol. Chem. 257,3669-3675)。一邊確認上清液的抗體活性,一邊進行細胞的純化(單株化)。
其結果,獲得了會產生表1所示之抗諾羅病毒單株抗體的細胞株。
將取得之細胞株對經姥鮫烷(pristane)處理過的BALB/c小鼠進行腹腔投予,約2週後,採取了含有抗體的腹水。透過使用蛋白A管柱的親和層析法從所獲得之腹水精製IgG,獲得了精製抗諾羅病毒殼結構域抗體(SMoAb)。
實施例2 確認抗諾羅病毒抗體表位
針對實施例1製作出之抗諾羅病毒殼結構域抗體(SMoAb),鑑定諾羅病毒基因群共同的胺基酸序列,以鑑定出的胺基酸序列為基礎進行肽合成,而製作出具有所鑑定之胺基酸序列的肽。利用PBS將製作出的肽稀釋為0.3μg/mL。將經稀釋之肽,以每1孔100μL的諾羅病毒肽/PBS,pH7.3溶液添加於塑料製微量滴定盤(Nunc-Immuno Module F8 Maxisorp Surface plate,商品名,Nalgen Nunc International公司製)的孔中,於4℃、12小時的條件下將諾羅病毒肽於微量滴定盤上進行了固相化。12小時後,透過傾析而除去已加入於孔的諾羅病毒肽/PBS溶液,以200μL/孔將PBS-T添加至該微量滴定盤的孔,並透過傾析進行了PBS-T的除去。該洗淨步驟合計進行了3次。
其後,以200μL/孔添加1%PerfectBlock (商品名,Funakoshi公司製阻斷劑)/PBS pH7.3,並於4℃、12小時的條件下進行了肽固相化微量滴定盤的孔內阻斷。以經過12小時後,4℃的狀態設為保存狀態。為了確認抗諾羅病毒單株抗體的反應性,將1μg/mL的抗諾羅病毒單株抗體以100μL/孔加入於諾羅病毒肽固相化微量滴定盤,並進行了37℃、1小時加溫。其後,透過傾析而除去已加入於孔的抗體液,並以200μL/孔來添加PBS-T,透過傾析進行了PBS-T的除去,進行了孔內的洗淨。該洗淨步驟合計進行了3次。
其後,以100μl/孔(稀釋1萬倍)將酵素標識抗體添加至孔,進行了37℃、1小時加溫。其後,透過傾析而除去已加入於孔的酵素標識抗體,以200μL/孔來添加PBS-T,並透過傾析進行了PBS-T的除去,進行了孔內的洗淨。該洗淨步驟合計進行了3次。其後,將TMB溶液作為過氧化酶酵素反應基質溶液以100μL/孔來加入至孔,於25℃、30分鐘、遮光下進行了靜置。其後,立即以100μL/孔來添加313mM H
2SO
4溶液使酵素反應停止。
其後,測定此孔的吸光度,從450nm的吸光度減去630nm的吸光度,並將該數值設為反應性評價的指標(Josephy P.D.,Mason R.P.et al.(1982) J. Biol. Chem. 257,3669-3675)。使實施例1所獲得之SMoAb 2殖株及抗諾羅病毒多株抗體(PoAb)與微量滴定盤上的肽反應,並使用酵素標識抗體、過氧化酶酵素反應基質溶液來確認反應。可確認到反應之肽的胺基酸序列係顯示於表1。
[表1]
固相肽 | SMoAb① | SMoAb② | PoAb | |
基因群 | 序列 | |||
GⅠ | MLYTPLRTGGGSGGTDPFVVAGR(序列識別號1) | 0.513 | 0.644 | 0.046 |
GⅡ | RLVAMLYTPLRANGSGDDVFTVS(序列識別號2) | 1.819 | 0.641 | 0.046 |
茲認為本發明之SoMb抗體係會辨識包含許多GI與GII共同的序列的MLYTPLR(序列識別號3)。
實施例3 在免疫層析(IC)法中之抗諾羅病毒單株抗體的反應性
1.抗諾羅病毒抗體固定化到硝化纖維素膜
準備以精製水稀釋會對諾羅病毒GI之殼體的突出結構域反應的單株抗體(GIPMoAb)及會對諾羅病毒GII之殼體的突出結構域反應的單株抗體(GIIPMoAb)使其成為1.0mg/mL之液、及抗小鼠IgG抗體,並以1μL/cm的量線狀地塗布於已以PET薄膜襯裡的硝化纖維素膜,並設為試驗線(test line)。對照線(control line)係與上述同樣地塗布抗小鼠球蛋白抗體。在本實施例中,稱為抗體固定化膜。
2.抗諾羅病毒抗體固定化於著色聚苯乙烯粒子
以精製水稀釋實施例1中所製作出之抗諾羅病毒殼結構域抗體(SMoAb)使其成為0.5mg/ml,並於其加入著色聚苯乙烯粒子使其成為0.1%,攪拌後,加入碳二亞胺使其成為1%,並進一步進行攪拌。透過離心操作而除去上清液,並且再懸浮於50mM Tris(pH9.0)、3.0%BSA,獲得了抗諾羅病毒抗體結合著色聚苯乙烯粒子。在本實施例中,稱為抗體固定化粒子。
3.製作試驗片
貼合抗體固定化膜與其它構件(襯板紙(backing sheet)、吸收帶、樣本墊)而切斷為5mm寬,設為諾羅病毒試驗片。在本實施例中將該等稱為試驗片。
4.免疫測定
對各試驗片滴加100μL包含經任意地稀釋之諾羅病毒VLPs與抗體固定化粒子的檢體懸浮液,並靜置15分鐘。
在抗小鼠IgG抗體及抗諾羅病毒抗體兩者的塗布位置能夠以目視確認到顯色的情況下判定為+。僅在抗小鼠IgG抗體的塗布位置能夠以目視確認到顯色、且在抗諾羅病毒抗體的塗布位置無法以目視確認到顯色的情況下判定為-。此外,在抗小鼠IgG抗體的塗布位置無法以目視確認到顯色的情況下判定為無效。此外,依線的顯色強度記載為+++>++>+。
比較本發明之試驗片、與習知品之試驗片的判定結果,該習知產品之試驗片係使用了會對諾羅病毒殼體之突出結構域反應的單株抗體之試驗片。
[表2-1]
諾羅病毒 | 本發明 | 習知品 | |
基因型 | GI.1 | + | + |
GI.3 | ++ | + | |
GI.4 | + | - | |
GI.5 | ++ | + | |
GI.6 | + | - | |
GI.7 | ++ | - | |
GI.8 | ++ | - | |
GI.9 | ++ | + |
[表2-2]
諾羅病毒 | 本發明 | 習知品 | |
基因型 | GII.2 | + | - |
GII.3 | + | + | |
GII.4 | ++ | + | |
GII.5 | + | + | |
GII.6 | ++ | ++ | |
GII.7 | ++ | ++ | |
GII.9 | + | + | |
GII.10 | + | + | |
GII.11 | + | + | |
GII.12 | + | + | |
GII.13 | + | - | |
GII.14 | + | - | |
GII.15 | + | + | |
GII.16 | + | - | |
GII.20 | + | - | |
GII.21 | + | + |
依表2-1、2-2,與習知品相比較,本發明之試驗片會與試驗之GI基因群、GII基因群的全部反應。進一步,係較習知品還更強烈地反應,確認到抗體對於諾羅病毒抗原的結合親和性高,且係能夠更高靈敏度地檢測諾羅病毒。
無
無。
無。
Claims (5)
- 一種抗諾羅病毒抗體或其抗原結合片段,其會與由序列識別號1及序列識別號2各自所示之胺基酸序列構成的各肽進行抗原抗體反應。
- 如請求項1之抗諾羅病毒抗體或其抗原結合片段,其將序列識別號3所示之胺基酸序列之整體或一部分區域設為表位。
- 如請求項1或2之抗諾羅病毒抗體或其抗原結合片段,其中該抗諾羅病毒抗體為單株抗體。
- 一種諾羅病毒之免疫測定方法,其利用了檢體中的諾羅病毒、與如請求項1或2之抗諾羅病毒抗體或其抗原結合片段的抗原抗體反應。
- 一種諾羅病毒之免疫測定器具,其包含如請求項1或2之抗諾羅病毒抗體或其抗原結合片段。
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