We appreciate the colleagues in Taiwan Cetacean Society, Marine Biology and Cetacean Research Center of National Cheng Kung Univerisy, Farglory Ocean Park, and Animal Disease Diagnostic Center of National Chiayi Universiy for sample collection. This project was funded by grant to WCY from the Council of Agriculture of Taiwan (100AS-02.1-FB99).

Etik Beyanı: Çalışma Ulusal Chiayi Üniversitesi, onay numarası Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından uluslararası kurallarına uygun olarak yapılan ve kabul edildi: cetacean örnek kullanımı Tayvan Tarım Konseyi (Araştırma İzni tarafından izin verildi 99022. 100M-02.1-Cı-99). 1. Kas Numune Hazırlama ve SDS-PAGE Not: Deniz memelilerinin 16 türün, karasal memeli 5 türler de dahil olmak üzere 23 türe kas örnekleri, ton balığı ve tavuk Bu çalışmada (Tablo 1) kullanılmıştır. cetacean kas örnekleri mahsur kişiler, balıkçılık hedef dışı av ve müsadere elde edilmiştir. Tavşan, fare, köpek ve tavuk kas dokuları Ulusal Chiayi Üniversitesi Hayvan Hastalıkları Teşhis Merkezi&#39;nden elde edilmiştir. sığır, domuz, kuzu ve orkinos örnekleri yerel bir süpermarketten satın alındı. Liman foku (Phoca vitulina kas örneği </em&gt;) Farglory Ocean Park tarafından sağlandı. Sodyum dodesil sülfat poliakrilamit jel elektroforezi (SDS-PAGE), kas örnekleri, farklı moleküler ağırlıklara sahip çözünebilir proteinlerin ayrılması için kullanılmıştır. <ol><li> kullanılana kadar -20 ° C&#39;de tüm örnekler saklayın. </li><li> -20 ° C&#39;de ön-serin harcı. Sonra içine donmuş kas örnek 3 g koydu. </li><li> Bir doku homojenleştirici kullanarak soğuk fosfat tamponlu salin içinde 10 ml (PBS) ile örnek homojenize edilir. </li><li> 4 ° C&#39;de 10 dakika boyunca 10,000 x g&#39;de homojenize santrifüjleyin. süpernatantlar toplamak ve kullanılana kadar -20 ° C&#39;de saklayın. </li><li> (5 mi,% istifleme jeli (3.07 ml damıtılmış suya, pH 6.8,% 40 akrilamid 625 ul,% 10 amonyum persülfat (APS), 50 | il ve tetrametiletilendiamin 5 ul 4x üst jel tamponu 1.25 ml 5 hazırlanması TEMED)) ve 10 ml% 15 ayırma jeli (damıtılmış su 3.65 mi, 4 kere pH 8.8 alt jel tamponu, 2.5 ml% 40 akrilamid 3.75 mi,% 10, 100 ul APSve TEMED 4 ul). <ol><li> Elektroforez hücresinde, ikinci katılaşmasından sonra ayırma jeli (% 15 akrilamid) üzerine jel (% 5 akrilamid) istifleme dökün. istifleme jel jel tarak yerleştirin. </li></ol></li><li> Aşağıdaki koşullara göre PAGE gerçekleştirin: İlk çalışma koşulu: 100 volt, 20 dakika ve son durum: 120 volt, 40 dk. </li><li> Jel renk eşit mavi olana kadar 30 dakika oda sıcaklığında Coomassie parlak mavisi ile jel leke. Not: Jel artık boya çözeltisi görünür olduğunda Boyama tamamlanır. </li><li> 1 saat süre ile, oda sıcaklığında asetik asit çözeltisi (% 10) ile destain. Bantları görünmeye başlayacaktır. Arka plan temizlenene kadar bir gecede 4 ° C&#39;de lekenin devam edin. </li></ol> 2. Peptid Sentezi ve Tek klonlu Antikor Üretimi <ol><li> ton balığı, tavuk, devekuşu, evcil hayvanlar, mühür ve 18 tür dahil olmak üzere, GenBank&#39;tan Mb amino asit sekanslarını almakdeniz memelileri (Tablo 2). </li><li> Uygun yazılım 16 kullanarak dizileri hizalayın: <ol><li> Hizala seçerek Hizalama Explorer&#39;ı başlatın: başlatmak çubuğunda Hizalama kurmak / Edit; Yeni Hizalama Oluştur&#39;u seçin ve Tamam&#39;a tıklayın. Bir iletişim soran görünecektir &quot;Bir DNA veya protein dizi hizalama oluşturuyor musunuz?&quot; </li><li> &quot;Protein&quot; etiketli düğmesini tıklayın; Açık: Verileri seçmek Dosya seçin ve dizi dosyasından dizileri Al; Düzenle&#39;yi seçin: Bir çoklu dizi hizalaması oluşturmak için veri seti her dizisi için tüm siteleri seçmek için Tüm menü komutunu seçin. </li><li> Seç Hizalama: ClustalW algoritması kullanılarak seçilen dizileri veri hizalamak için ana menüden ClustalW tarafından hizalayın; Tamam düğmesini tıklatın Protein Kilo Matrix olarak &quot;BLOSUM&quot; i seçin. </li></ol></li><li> dizi hizalama analiz: <ol><li> 5 antijenik reaktif si odaklanıntes 17: Alanı 1 (AKVEADVA, 15-22), Alanı 2 (KASEDLK, 56-62), Alanı 3 (ATKHKI, 94-99), Alanı 4 (HVLHSRH, 113-119) ve site 5 (KYKELGY, 145- 151) ve deniz memelileri arasında muhafaza fragmanı bulmak. Bir * (yıldız; hizada konsensüs sembolü (Protokol 2.2.3)) tek, tamamen korunmuş kalıntısı pozisyonları gösterir. deniz memelileri aşağıdaki korunmuş parçaları bulundu: (siteyi 2 dahil) dizisi KASEDLKKHG ve dizi HVLHSRHP (hangi siteyi 4 içerir). </li></ol></li><li> ticari hizmetlerini kullanarak taşıyıcı protein olarak ovalbümin proteininin (OVA) ile sekans analizi ve konjugat göre aday dizisi fragmanları sentezlemek. <ol><li> Ayrışma peptidin önlemek için antijenik reaktif site N-terminali hidrofobik amino asitler (örneğin, metionin) ilave (örneğin, E-KASEDLKKHG). </li><li> immünosit çekirdek bir antijenik yeri sergilemek için antijenik reaktif sitenin C-terminal uzatır. Bundan başka, konjugatKapı Cı-terminal sistein (Cys; C) eklenerek OVA peptid (örneğin, E-KASEDLKKHG-NTVL-C). </li></ol></li><li> imünojen 1 ya da PBS içinde, her sentetik peptit, eşit hacimde (3 mL, nihai konsantrasyon 30-50 g / 100 ul) ile imünojen 2 tam olmayan Freund katkı maddesi için tam Freund katkı maddesi emülsiyon. </li><li> deri altından 5 dişi BALB / c farelerinin her birine imünojen 1 (0.1 mg), inoküle. </li><li> İki haftalık aralıklarla subkutan güçlendirici enjeksiyonları immünojen 2 kullanılarak beş kez gerçekleştirin ve her güçlendirici önce farelerden alınan kuyruk klip örnekleme test serumları toplamak. </li><li> İlk tarama için, sera titresi belirlenir. <ol><li> 96 oyuklu bir plakanın her bir oyuğuna, reaksiyon tamponu ve kısım çözeltisi 50 ul 25 ml serbest peptidin 100 ug çözündürülür. Her kuyuya 10 ul birleştirme reajanı solüsyonu eklenir ve plaka karıştırın. Oda sıcaklığında 2 saat boyunca inkübasyona bırakılır. </li><li> , Kuyunun içeriğini kaldırdamıtılmış su ile her bir oyuğa 3 kez yıkama ve bloke etme çözeltisi 200 ul ilave edilerek plaka engeller. Oda sıcaklığında 1 saat inkübe edin. içeriğini çıkarmak ve damıtılmış su ile her bir oyuğa 3 kez yıkayın. </li><li> İlk tarama için sera titresinin belirlenmesi için dolaylı ELISA (Protokol 5,1-5,7) gerçekleştirin. dalak koleksiyonu için en yüksek titre (en yüksek optik yoğunluk) ile fare seçin. </li></ol></li><li> Üç gün önce dalak koleksiyonuna, deri altı yüksek titreye sunan fare içine immünojen 1 0.1 mg aşılamak. </li><li> MAb nesil 18 için hibridoma hücreleri elde etmek için fare miyelom hücreleri F0 (sp2 / 0-Ag14) ile seçilmiş aşılanmış farelerde ve sigorta dalak hücreleri toplamak. </li><li> 14 gün füzyon sonra, dolaylı ELISA kullanılarak içermeyen sentetik peptid doğru hibridom üst fazları (Protokol 5,1-5,7) reaktivitesini tarama ile pozitif klonlar seçin. </li><li> maximiz için oyuk başına uygun sayıda hücreleri sulandırmaksadece tek bir klon (seyreltme klonlama) içeren havuzlara oranını ing. <ol><li> Boş kalan de A1 hariç 96 oyuklu plaka içerisinde tüm oyuklara hücre kültür ortamının 100 ul ekle. </li><li> de A1 hücre süspansiyonu 200 ul ekle. Sonra hızlı bir şekilde B1 A1 100 ul transferi ve hafifçe pipetleme karıştırın. Tüm sütun boyunca 2 dilüsyonları, ve bunun üstünde kuyuları ile aynı hacmi ile biter böylece daha sonra H1 100 ul atmak: Bu 1 tekrarlayın. </li><li> 8 kanallı mikropipet ile sütun 1, orta ve ek bir 100 ul ekle. Daha sonra hızlı bir şekilde, aynı pipet kullanarak sütun 2&#39;de kişilerce sütun 1 kuyuların herbirinden 100 ul transfer ve yavaşça pipetleme karıştırın. </li><li> tüm plaka üzerinde 2 dilüsyonları: Aynı ipuçlarını kullanarak, bu 1 tekrarlayın. son sütunda kuyuların her birinden 100 ul atın. </li><li> Her bir oyuğa 100 ul ortamı eklenerek 200 ul tüm oyuklara nihai hacim getirin. pla inkübeTe nemlendirilmiş CO2 kuluçka makinesi içinde 37 ° C ısıda. </li><li> Her çukurun kontrol edin ve sadece tek bir koloni içeren tüm kuyuları işaretlemek. Tek klonlar içeren kuyuların&gt;% 90 antikor üretimi için pozitif olana kadar iki veya daha fazla klonlama yürütmek. </li></ol></li><li> Western blot ve Dot blot (protokol 3,1-4,6) ile klonları taramak. Sonra daha büyük kaplara kuyulardan alt kültür koloniler mAb elde etmek için hücreleri genişletmek için. Genellikle, her klon 12 veya 24 oyuklu bir plaka içerisinde, tek bir kuyunun içine aktarılır. </li><li> mAbs ve inek, keçi, domuz, köpek, tavşan, ton balığı, tavuk, mühür kas özleri ve western blot ve dot blot (protokol 3,1-4,6) dört temsilci cetacean türler arasındaki yakınlık ölçün. </li><li> Assitler ikna etmek için farelerin periton içine (en fazla 3 x 10 6) seçilen hibridoma hücreleri aşılamak. Karın şişliği 7-10 gün hibridoma enjeksiyon sonrası içinde genellikle açıktır. bir hipodermik bir iğne kullanılarak sıvının toplama(Az 20 ölçü). </li><li> Santrifüj asit sıvısı (10 dakika 10.000 xg) hücreleri ve enkaz kaldırmak için. 0.45 um&#39;lik bir filtre ile filtrelenir. Protein G Sefaroz kolonuna örnek 1 ila 20 ml bağlama tamponu 15 mi, ve elüsyon tamponu 3 ila 5 ml ilave edilir. farenin asit sıvısı arıtılmış antikor içeren ayırma fraksiyonu toplamak. </li><li> Üreticinin talimatlarını kullanarak Antikor İzotipleme Kiti ile antikor izotipini belirlemek. </li></ol> 3. Western Blot <ol><li> BME 50 ul 2x Laemmli numune tamponu 950 ul karıştırılarak β-merkaptoetanol (BME) ihtiva eden 2 x yükleme tamponu hazırlayın. Poliklonal tavşan anti-insan Mb antikoru kullanıldığında 5 (evcil hayvanlar ve ton balığı) ve: 01:50 (, deniz memelileri ve mühür) ve 1: iyi sinyaller elde etmek için uygun oranda yükleme tamponu kas üst fazlar (protokol 1.1-1.4) seyreltin 1: 1 (domuz, tavşan, tavuk ve ton balığı), 1: 5 (inek, keçi ve köpek) ve 01:25 (deniz memelileri ve mühür) zamanntibody Hibridom yüzey arasındadır. </li><li> 5 dakika boyunca 95 ° C&#39;de ısı örneği. moleküler ağırlık belirteçleri ile birlikte SDS-PAGE jel (% 4 akrilamit yığınlama ve% 12 akrilamid ayırma) oyuklarına Numuneleri. V sonra yaklaşık 1 saat içinde koşmak bitirmek için 150 V gerilimi artırmak 50 ° C&#39;de 5 dakika boyunca jel çalıştırın. </li><li> 15 dakika boyunca 1 x transfer tampon maddesi içinde jel yerleştirin. nitroselüloza ayrılan protein aktarın (NC) membranlar da PAGE ile ayrıldıktan sonra. Aktarım 60-90 dakika boyunca 100 V&#39;ta yapılabilir. </li><li> engelleme çözeltisi hazırlayın: 1X PBS,% 5 yağsız kuru süt ile% 0.1 Tween 20 ihtiva etmektedir. 1 saat boyunca oda sıcaklığında bloke etme çözeltisi 25 ml Carolina membran engeller. Tween 20 (PBST), 15 ml fosfat tamponlu tuzlu su ile her biri 5 dakika için üç kez yıkayın. </li><li> Antikor Seyreltme tampon maddesi gece boyunca 4 ° C &#39;de% 5 engelleme çözeltisi ile seyreltildi, 10 ml membran ve primer antikor (assit sıvısı veya hibridom üst faz) inkübe edin. </li><li> zarı yıkayınYine PBST ile e üç kez aşırı antikor temizlemek için. </li><li> 1 ile alkalin fosfataz konjuge edilmiş keçi anti-fare IgG&#39;si ile membran inkübe: 1,250, oda sıcaklığında 1 saat süre ile hafif bir çalkalama ile engelleyici çözelti içinde. </li><li> Yine zarın yıkanması ve 5-bromo-4-kloro-3-indolil fosfat / p-nitro mavi tetrazolyum klorit içinde inkübe (BCIP / NBT) fosfataz alt-tabaka karışımı 10 ila 20 dakika süreyle renk gelişimi kadar. </li><li> damıtılmış su çeşitli değişiklikler membranın yıkama ederek reaksiyonu durdurun. </li></ol> 4. nokta leke <ol><li> iyi sinyaller elde etmek için uygun bir oran ile PBST içinde% 5 sığır serum albumin (BSA), kas üst fazlar (protokol 1.1-1.4) seyreltilir: 1: evcil hayvanlar ve ton 5 ve 1:25 deniz memelileri ve conta için. Noktası zarı üzerine numune 5 ul. Çözelti yavaşça uygulayarak (genellikle 3-4 mm çapında) nüfuz bölgeyi en aza indirin. </li><li> Oda sıcaklığında (zar kurutun, örneğin &lt;30-60 dakika boyunca, bir laminar akış / em&gt;), oda sıcaklığında, 1 saat süre ile Petri kabındaki çözüm engelleme ile bloke eder ve PBST ile yıkayın. </li><li> primer antikor ile membran inkübe (mAb 1 ile hibridoma süpernatanından: 10,000 veya assit sıvısı ile 1: 5% engelleme çözeltisi içinde seyreltildi 100,000), oda sıcaklığında 1 saat karıştırıldı. </li><li> Oda sıcaklığında 1 saat boyunca (% 5 Bloklama çözeltisi, 1.250 1 alkalin fosfataz konjuge edilmiş keçi anti-fare IgG&#39;si) kullanımı PBST fazla antikor kaldırmak ve daha sonra sekonder antikor ile inkübe edilmesi her biri 5 dakika MEMBRAN üç kez yıkamak . </li><li> Yine membran yıkayın ve renk gelişimi kadar 10 ila 20 dakika içinde BCIP / NBT fosfataz substrat karışımı içinde kuluçkaya yatmaktadır. </li><li> damıtılmış su çeşitli değişiklikler membranın yıkama ederek reaksiyonu durdurun. </li></ol> 5. Dolaylı ELISA <ol><li> Yıkama tamponu (% 0.02 Tween 20 ile 0,002 M imidazol tamponlu tuz) hazırlayın. plaka yıkama ile 3 kere yıkayınHer bir sonraki aşamada (protokol 5.2-5.5) arasında tampon. </li><li> kaplama tamponu kas süpernatanları (protokol 1.1-1.4) ve 01:25 seyreltme hazırlayın. Ceket 96-çukurlu bir ELISA gece boyunca 4 ° C&#39;de 100 ul seyreltilmiş supernatantlar plaka ve oda sıcaklığında 1 saat süre ile tampon (PBS içinde% 1 BSA) ile bloke ile bloke eder. </li><li> İnceltilmiş tampon ile saflaştırılmış mAb 2.000 seyreltme ve her bir seyreltilmiş, mAb 100 ul 1 hazırlayın. Oda sıcaklığında 1 saat boyunca inkübasyona bırakılır. </li><li> daha inkübasyon için: (seyreltilmiş tampon maddesi içinde 200 seyreltme 1) keçi anti-fare IgG yaban turpu peroksidaz ile konjüge edilmiş ekleyin. </li><li> her bir (100 ul / çukur) peroksidaz substratı ilave edin ve 10-15 dakika inkübe edilir. </li><li> Renk gelişimi gözlemlenir peroksidaz durdurma çözeltisi (100 ul / oyuk) enzim reaksiyonu durdurun. </li><li> Bir mikrolevha spektrofotometre kullanılarak 450 nm&#39;de optik yoğunluk okuyun. </li></ol> Kolloidal Altın etiketli mAb 6. hazırlanması </p&gt; Not: koloidal altın çözüm ve karışımın rengi her zaman kırmızı olmalıdır. Siyah çökelti fark edildiğinde pH, mAb konsantrasyonu, santrifüj hızını ayarlayın. 6.1 Adımları ve 6.2 optimizasyon adımlardır. <ol><li> 5-9 arasında değişen pH değerleri ile, koloidal altın çözeltisi 100 ul mAb (50 ug / ml, 3 ul) tespit saflaştınlmış ekleyin. İki saat süre ile kırmızı rengi tutan en az optimum pH değeri pH olarak kabul edilir. Not: Bu çalışmada, 0.1 M potasyum karbonat kolloid altın (40 mil), pH 8.0 (optimum pH) çözüm ayarlamak için kullanıldı. </li><li> pH 8.0&#39;da koloidal altın çözeltisi 100 ul mAb tespit saflaştırılmıştır çeşitli bir miktar (500 mg / ml, 1-20 ul) eklenir. Not: Bu çalışmada, optimum konsantrasyonu 6 ng / ml (siyah tortu) &#39;dir. </li><li> Yukarıdaki sonuçlara göre, arıtılmış, mAb damla damla kolloid altın çözeltisi 10 ml saptama 60 ug ekleyin. 10 dakika süre ile, oda sıcaklığında, karışımın hafifçe emülsifiye. ilankarışıma PBS içinde% 5 BSA çözeltisi (pH 7.4), 2 ml ve 15 dakika boyunca oda sıcaklığında yavaşça emülsifiye D interferansı azaltır. </li><li> 4 ° C&#39;de 30 dakika boyunca 10,000 x g&#39;de santrifüjleyin karışımı. </li><li> dikkatli bir şekilde konjüge edilmemiş antikor ile süpernatantı ve% 1 BSA ve% 0.1 Tween 20 içeren PBST ile 4 ml elde pelet askıya ve santrifüj ve süspansiyon birkaç kez tekrarlayın. </li><li> 1 ml PBST son çökeltiler askıya alma ve kullanılana kadar 4 ° C sıcaklıkta saklayın. </li></ol> Bağışıklık Strip 7. İnşaat Not: Şekil 1, bağışıklık şerit tasarımı gösterir. Hazırlayın ve &lt;(1 yıl) uzun süreli depolama ömrü için (% 20 Bağıl Nem düşük nem laboratuarı çevre durumda şeritler)&gt; monte edin. pedleri ve zarın boyutları: Birleşmiş Yastığı, 300 mm x 10 mm, emici ped, 300 mm x 24 mm, Örnek Yastığı, 300 mm x 24 mm, NC membranı 300 mm x 25 mm, 300 çalışma alanınamm x 80 mm. <ol><li> Birleşmiş Yastığı, doyurulması ve sonra monte edilmeden önce 1 saat boyunca 37 ° C&#39;de kurutmak için mikropipet ile adım 6.6 arasında kolloid altın etiketli mAB solüsyonu eklenir. </li><li> Bir pipet ya da İmmunoStrip yazıcı kullanılarak Kuzey Carolina zar üzerinde mAb saptanması için kontrol alanının spesifik antijen yakalama test bölgesi üzerinde mAb (500 ug / ml) ve tavşan anti-fare IgG (500 ug / ml) dağıtın. bir girişimi engellemek için iki bölge arasındaki mesafeyi (&gt; 5 mm) tutun. </li><li> çift ​​taraflı bant ile çalışma alanında konjuge pad, emici ped ve NC membran yapıştırın. Not: yaklaşık 2 mm NC zarın her iki tarafındaki pedleri üzerinden geçin. Uygunsuz şerit inşaat tamamlanmamış bir testte neden olur. </li><li> eşlenik pedi üzerinde numune pedi (2 mm) yerleştirin ve çalışma alanındaki yapıştırın. </li><li> Bir kağıt kesici ile 6 mm çapında şeritler oluşturun. kurutucu ile alüminyum folyo çanta içinde şeritler paketleyin ve kullanılana kadar 4 ° C sıcaklıkta saklayın. </li></ol> 8. Çapraz reaktivite Testi <ol><li> 1.5 ml santrifüj tüpüne 1 ml PBS içeren (% 0.1 BSA) bambu çubuk kullanarak ya da çubuğu taşlama ham kas numunesi 0.03 g homojenize edilir. </li><li> Test alanlara son tersi şeridi tutun ve 5-10 dakika süreyle numune içine numune pedi parçası daldırma ve doğrudan sonucu gözlemleyin. <ol><li> İsteğe bağlı: 500 ul süpernatant toplayın ve yeni bir santrifüj tüpüne aktarın. Not: şerit doğrudan süpernatant içine batırılmış zaman sinyal açık değilse, bu adım önerilir. </li></ol></li><li> üç nüsha olarak çeşitli kas örnekleri test edin. Not: Burada orkinos, tavuk, mühür, deniz memelileri 15 tür ve karasal memeli 5 tür test. </li><li> , Beş bağımsız müfettişler, yani beş kez 8.3 tekrarlayın sahip toplam 575 şeritler kullanın. </li></ol>

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Technol. 39, 5703-5708 (2005).</li><li>Tryland, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pathogens+in+marine+mammal+meat.">Human pathogens in marine mammal meat.</a> Norwegian Scientific Committee for Food Safety (VKM). , (2011).</li><li>Matsunaga, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+simple+method+for+the+identification+of+meat+species+and+meat+products+by+PCR+assay.">A quick and simple method for the identification of meat species and meat products by PCR assay.</a> Meat Sci. 51, 143-148 (1999).</li><li>Dalebout, M. L., Helden, A. V., van Waerebeek, K., Baker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetic+identification+of+southern+hemisphere+beaked+whales+(Cetacea:+Ziphiidae).">Molecular genetic identification of southern hemisphere beaked whales (Cetacea: Ziphiidae).</a> Mol. Ecol. 7, 687-694 (1998).</li><li>Dalmasso, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+PCR+assay+for+the+identification+of+animal+species+in+feedstuffs.">A multiplex PCR assay for the identification of animal species in feedstuffs.</a> Mol. Cell. Probes. 18, 81-87 (2004).</li><li>Kesmen, Z., Sahin, F., Yetim, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+assay+for+the+identification+of+animal+species+in+cooked+sausages.">PCR assay for the identification of animal species in cooked sausages.</a> Meat Sci. 77, 649-653 (2007).</li><li>Liu, L., Chen, F. -. C., Dorsey, J. L., Hsieh, Y. -. H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+Monoclonal+Antibody-based+Sandwich+ELISA+for+the+Detection+of+Porcine+Skeletal+Muscle+in+Meat+and+Feed+Products.">Sensitive Monoclonal Antibody-based Sandwich ELISA for the Detection of Porcine Skeletal Muscle in Meat and Feed Products.</a> J. Food Sci. 71, 1-6 (2006).</li><li>Kotoura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Sandwich+ELISA+for+the+Determination+of+Beef+Meat+Content+in+Processed+Foods.">A Sandwich ELISA for the Determination of Beef Meat Content in Processed Foods.</a> Food Sci. Techno. Res. 15, 613-618 (2009).</li><li>Hsieh, Y. H., Chen, Y. T., Gajewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody-based+sandwich+ELISA+for+reliable+identification+of+imported+Pangasius+catfish.">Monoclonal antibody-based sandwich ELISA for reliable identification of imported Pangasius catfish.</a> J. Food Sci. 74, 602-607 (2009).</li><li>Ross, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+surveillance:+web-based+molecular+identification+of+whales,+dolphins,+and+porpoises.">DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises.</a> J.Hered. 94, 111-114 (2003).</li><li>Ngom, B., Guo, Y., Wang, X., Bi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+lateral+flow+test+strip+technology+for+detection+of+infectious+agents+and+chemical+contaminants:+a+review.">Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review.</a> Anal. Bioanal. Chem. 397, 1113-1135 (2010).</li><li>Muldoon, M. T., Onisk, D. V., Brown, M. C., Stave, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets+and+methods+for+the+detection+of+processed+animal+proteins+in+animal+feedstuffs.">Targets and methods for the detection of processed animal proteins in animal feedstuffs.</a> Int. J. Food Sci. Technol. 39, 851-861 (2004).</li><li>Rao, Q., Hsieh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+commercial+lateral+flow+feed+test+for+rapid+detection+of+beef+and+sheep+content+in+raw+and+cooked+meats.">Evaluation of a commercial lateral flow feed test for rapid detection of beef and sheep content in raw and cooked meats.</a> Meat Sci. 76, 489-494 (2007).</li><li>Tamura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEGA5:+molecular+evolutionary+genetics+analysis+using+maximum+likelihood,+evolutionary+distance,+and+maximum+parsimony+methods.">MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.</a> Mol. Biol. Evol. 28, 2731-2739 (2011).</li><li>Atassi, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+structure+of+myoglobin:+the+complete+immunochemical+anatomy+of+a+protein+and+conclusions+relating+to+antigenic+structures+of+proteins.">Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigenic structures of proteins.</a> Immunochemistry. 12, 423-438 (1975).</li><li>Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybridoma+technology+for+the+generation+of+monoclonal+antibodies.">Hybridoma technology for the generation of monoclonal antibodies.</a> Methods Mol. Biol. 901, 117-135 (2012).</li><li>Kao, D. J., Hodges, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+a+synthetic+peptide+immunogen+over+a+protein+immunogen+in+the+development+of+an+anti-pilus+vaccine+for+Pseudomonas+aeruginosa.">Advantages of a synthetic peptide immunogen over a protein immunogen in the development of an anti-pilus vaccine for Pseudomonas aeruginosa.</a> Chem. Biol. Drug Des. 74, 33-42 (2009).</li><li>Dolar, M. L., Suarez, P., Ponganis, P. J., Kooyman, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoglobin+in+pelagic+small+cetaceans.">Myoglobin in pelagic small cetaceans.</a> J. Exp. Biol. 202, 227-236 (1999).</li><li>Lo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+immune+colloidal+gold+strip+for+cetacean+meat+restraining+illegal+trade+and+consumption:+implications+for+conservation+and+public+health.">Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health.</a> PLoS ONE. 8, e60704 (2013).</li></ol>

The authors declare that they have no competing financial interests.

taşıyıcı proteine ​​konjüge edilmiş sentetik bir peptidin kullanımıyla kognat proteine ​​kıyasla önemli ölçüde daha etkilidir. mAb bilinen göreceli yerle epitopları kullanılarak geliştirilmiştir, çünkü sandviç temelli bir yöntem için, bu çalışmada iki mAb hedef antigen epitopu ile birbirlerinin etkileşime müdahale olası değildir. Ayrıca, doğal proteinin, sentetik peptit bağı ile bağışıklaştırılan farelerin antikor arasında tepkime nativ protein ve doğal proteinin ...

Historically, cetacean meat has been consumed in many parts of the world and this consumption continues today1. Due to the trophic level of cetaceans, high levels of mercury and other toxins are known to be present in their meat2. Therefore, the consumption of cetacean meat could lead to a health problem not only for high-risk groups such as pregnant women but also for the general population3. Furthermore, the contamination of cetacean meat with zoonotic or potentially zoonotic pathogens can also occur during its processing and storage4. It is difficult even for experienced agents to identify cetacean meats by their appearance alone. Therefore, a reliable scientific method of identification is required to differentiate cetacean meat from other meats. This would help to limit the consumption of cetacean meat.
Current methods of species identification include molecular techniques and immunological methods. Molecular techniques, such as polymerase chain reaction (PCR) and DNA sequencing, can be used to identify samples not only from raw meat5 and decomposed samples6 but also from processed foods such as cooked sausage and feedstuffs7,8. Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), are commonly applied in food production to detect the meat content of, for example, pork9, beef10 and catfish11. PCR-based DNA analysis for the identification of cetacean meat is available12, and has helped prevent the illegal international trade of cetacean meat in Japan, South Korea, the Philippines, Taiwan, Hong Kong, Russia, Norway, and the United States1. These methods are effective and reliable, but they can take hours or days to complete and involve laborious steps. The identification of cetacean meats is usually based on molecular techniques and there is currently no immunological method available. For regulatory agencies, it is highly desirable to develop a dependable and rapid technique that can be used in the field to identify cetacean meats. 
Immunochromatographic strips are used as detection tools with the advantage of producing rapid result via a simple protocol that is suitable for use in the field. The principles of the immunochromatographic strip and ELISA are very similar, and includes antibodies, antigens and labels. Many different labels such as colloidal gold, carbon and latex have been used in the development of immunochromatographic strips. At present, this method is commonly used for detecting antibiotics, toxin, bacteria and viruses13, but it is rarely used for identifying proteins in meat14,15. Here we propose a lateral-flow chromatographic enzyme immunoassay for rapid detection of cetacean myoglobin (Mb).

<table><tbody><tr><td>Phosphate buffered saline</td><td>AMRESCO</td><td>J373</td><td></td></tr><tr><td>Protein G HP SpinTrap&amp;nbsp;</td><td>GE&amp;nbsp;Healthcare</td><td>28-9031-34</td><td>spin column containing Protein G Sepharose</td></tr><tr><td>IsoStrip Mouse Monoclonal Antibody Isotyping Kit</td><td>Roche</td><td>11493027001</td><td>Isotyping Strips, precoated with subclass- and light-chain-specific anti-mouse-Ig antibodies</td></tr><tr><td>Mini Trans-Blot</td><td>Bio-Rad</td><td>170-3935</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Whatman</td><td>Z613630</td><td></td></tr><tr><td>Antibody blocker solution&amp;nbsp;</td><td>LTK BioLaboratories</td><td></td><td>To minimize nonspecific binding interactions of nonspecific IgG in the samples</td></tr><tr><td>BCIP/NBT phosphatase substrate&amp;nbsp;</td><td>KPL</td><td>50-81-00</td><td></td></tr><tr><td>Protein Detector HRP Microwell Kit, Anti-Mouse</td><td>KPL</td><td>54-62-18</td><td></td></tr><tr><td>Nunc Immunoplate MaxiSorp ELISA plate</td><td>Thermo&amp;nbsp;Fisher Scientific</td><td>EW-01928-08</td><td></td></tr><tr><td>Multiskan&amp;nbsp;EX ELISA reader&amp;nbsp;</td><td>Thermo&amp;nbsp;Electron&amp;nbsp;Corporation</td><td>51118170</td><td></td></tr><tr><td>Colloid gold (40 nm) solution&amp;nbsp;</td><td>REGA biotechnology Inc.</td><td></td><td>40-50 nm is appropriate for immunostrip</td></tr><tr><td>Bovine serum albumin</td><td>Gibco</td><td>15561-020</td><td></td></tr><tr><td>Rapid test immno-strip printer</td><td>REGA biotechnology Inc.</td><td>AGISMART RP-1000&amp;nbsp;</td><td>Only suited for small scale production of immunostrips for research and development purposes</td></tr><tr><td>Strip components (NC membranes, sample pads (#33 glass, S&amp;amp;S), conjugate pads (#16S, S&amp;amp;S) and absorbent pads (CF6, Whatman))</td><td>REGA biotechnology Inc.</td><td></td><td></td></tr><tr><td>Freund&amp;rsquo;s adjuvant and incomplete Freund&amp;rsquo;s adjuvant&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F5881, F5506</td><td>Used to produce water-in-oil emulsions of immunogens</td></tr><tr><td>Acrylamide, gel buffer, ammonium persulfate (APS), tetramethylethylenediamine (TEMED)&amp;nbsp;</td><td>Protech</td><td></td><td>Gel preparation for SDS-PAGE</td></tr><tr><td>Coomassie brilliant blue R-250</td><td>Bio-Rad</td><td>1610436</td><td>Protein staining in SDS-PAGE gels</td></tr><tr><td>Laemmli sample buffer and &amp;beta;-mercaptoethanol</td><td>Bio-Rad</td><td>1610737, 1610710</td><td>Dilute protein samples before loading on SDS-PAGE gels</td></tr></tbody></table>

development of a colloidal gold-based immunochromatographic test strip for detection of cetacean myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

Monoklonal antikor özelliklerinin Bu memeli Mb sırasıyla iki sentetik peptidler (MKASEDLKKHGNTVLC ve AIIHVLHSRHPAEFGC) tanıyan, iki IgG 1 mAb (CGF5H9 ve CSF1H13) geliştirildi ve bu memeli Mb hızlı tespiti için yapılan bir sandviç tipi koloidal altın immünokromatografik test şeridi oluşturmak için kullanıldı. Şekil 2, CGF5H9 yaklaşık 17 kDa&#39;lık tahmini bir moleküler ağırlığında tek bir boyanmış bandın olarak, deniz...

Watch this Scientific Journal Video about Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin at JoVE.com

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

Deniz Memelileri Miyoglobin Tespiti için bir Kolloidal Altın merkezli İmmunokromatografik Testi Strip Gelişimi

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

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Research

JoVE Journal

Biology

14.1K Görüntüleme.  National Chiayi University. This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals. 

Video: Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Akustik Membran mikropartikül Teknoloji Geliştirme Özel Çin Hamsteri Yumurtalık-host Hücre Proteini Tahliller

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Biyomühendislik

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Bir UV-Vis ve Raman Spektroskopisi İmmunoassay Platformu imalatı

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biyokimya

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Saptamak için deneysel protokol<em&gt; Siyanobakteri</em&gt; Bir Antikor Mikroarray Chip ile Sıvı ve Katı Örneklerinde

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Çevre

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Yerelleştirmek için Autometallography ve yarı ölçmek gümüş deniz doku kullanımı

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Proteinler için Poliakrilamid Jeller Hızlı ve Hassas Kolloidal Coomassie G-250 Boyama

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Biyoloji

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Küçük Molekül Bileşiklerinin Hızlı ve Nicel Tespiti için Yanal Akış İmmünokromatografik Şeridinin Geliştirilmesi

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has gained prominence in recent decades. Each microsphere that is read by the equipment represents an analysis event of the interaction capacity between the molecules under test. Thousands of these events are read in a single assay, thus ensuring high assay accuracy and reproducibility. This methodology can also be used in the validation of new inputs, such as IgY antibodies, for the diagnosis of diseases. These antibodies are obtained through immunizing chickens with the antigen of interest and then extracting the immunoglobulin from the yolk of the animals' eggs; therefore, this is a painless and highly productive method for obtaining the antibodies. In addition to a methodology for the high-precision validation of the antibody recognition capacity of this assay, this paper also presents a method for extracting these antibodies, determining the best coupling conditions for the antibodies and latex beads, and determining the sensitivity of the test.

This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

Yumurta sarısı antikorlarına dayalı sitometrik boncuk testinin standardizasyonu

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Elektrokemiluminesan immunoassay Serum Kardiyak Miyozin Bağlayıcı Protein-C belirlenmesi için hassas ve özel miktar tayini yöntemi

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

In-alan uygulamaları için tasarım ve Aptamer-Altın Nanopartikül Tabanlı Kolorimetrik Tahliller Gelişimi

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

A Paper-Based Immunoassay for the Detection of Immunoglobulin G

Source: Peng, Y. et al., <a href="https://app.jove.com/v/54111">Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.</a>&#160;J. Vis. Exp. (2016)
The video demonstrates a paper-based immunoassay, involving amine-functionalized cellulose discs cross-linked with glutaraldehyde. Covalent immobilization of IgG-Fc-specific capture antibodies, followed by the introduction of immunoglobulin G and horseradish peroxidase-conjugated detection antibodies, results in a color change confirming the presence of immunoglobulin G.

İmmünoglobulin G'nin Tespiti için Kağıt Tabanlı Bir İmmünoassay

1. Grafting Amine Functional Groups on Cellulose Paper Discs

	Prepare one piece of square paper with a dimension of 1 cm &#215; 1 cm, and 100 paper discs ...