1. Mikropartikül Hazırlık <ol><li> Biotin NHS Kimya kullanarak Etiket Antikorlar </li><li> Biyotinilasyon Derece belirlenmesi </li><li> Biotinlenmiş Antikor Konjugat Streptavidin Boncuk <ol><li> 1-2 dakika karıştırın orijinal flakon boncuk süspanse edin </li><li> Bir mikrosantrifüj tüp içine boncuk istenen birimde kısım </li><li> Boncuk PBST ile 3 kere yıkayın: <ol><li> 2 dakika boyunca boncuk mıknatıs içeren tüp Yeri </li><li> Mıknatıs tüp ile aspirasyon ile süpernatantı çıkarın. Boncuklar bozmak için dikkatli olun </li><li> Mıknatıs tüp çıkarın ve 1 ml PBST ekleyin </li><li> Boncuklar tekrar süspansiyon için 20 saniye boyunca Vortex </li><li> Boncuk kapağı kaldırmak için 5 saniye boyunca santrifüjleyin </li><li> Iki kez daha 2.3.1-2.3.5 tekrarlayın. </li></ol></li><li> Mıknatıs üçüncü yıkama yeri tüp sonra PBST aspirat </li><li> Boncuk biotinlenmiş antikor ekleyin ve tekrar süspansiyon </li><li> Gerekirse PBS ile 300 mcL biotinlenmiş antikor ses getirmek. Antikor hacmi ise 300 mcL daha herhangi bir PBS katmayan </li><li> Vorteks çalkalayıcı veya tüp rotator, RT 30 dakika boncuk / antikor süspansiyon inkübe </li><li> 3. adımda PBST ile 3 kere yıkayın. </li><li> Biotinlenmiş BSA ile boncuk Blok <ol><li> 300 mcL üçüncü yıkama 10 mikrogram / mg biotinlenmiş BSA ekledikten sonra, PBS gerekirse ile ses seviyesini ayarlamak ve ajitasyon ile 30 dakika inkübe </li></ol></li><li> 3. adımda PBST ile 3 kere yıkayın </li><li> PBS/0.05% BSA 100 mcL boncuk süspanse edin. Sodyum azid, uzun süreli depolama için eklenebilir </li></ol></li><li> Standart NHS Floresein protokolleri kullanarak, dedektör antikor fluoresceinate </li><li> Fluorescination, Derece belirleyin </li></ol> 2. OP - Geliştirme <ol><li> Çalışma çözümler hazırlayın: <ol><li> Floresein etiketli Antikor (Fl-Ab): En CHO-HCP testlerin son bir 400 ng / ml konsantrasyonda Fl-Ab kullanın. Fl-Ab nihai tahlil hacminin ¼ &#39;ü olarak eklenir, bu nedenle çalışma çözeltisi konsantrasyonu 1.6 mg / ml. Flab stok BioScale Boncuk / flab Dilüent ile seyreltilmesi ile bu çözüm hazırlayın. </li><li> Boncuk: En CHO-HCP Testler 2x10 5 boncuk / mL nihai konsantrasyonda boncuk kullanmayın. Boncuk reaktif son tahlil hacminin ¼ &#39;ünü oluşturmaktadır, bu nedenle çalışma çözeltisi konsantrasyonu 5 boncuk / ml 8x10 konumundadır . Boncuk stok BioScale Boncuk / flab Dilüent ile seyreltilmesi ile bu çözüm hazırlayın. Vorteks, tüm boncuk yapmak için boncuk hisse senedi, bir kısım çıkarmadan önce süspansiyon vardır. </li><li> Tampon Koşu: tampon kompozisyon ancak birkaç CHO HCP deneyleri 10 mM HEPES tamponu,% 1 Tween, pH 7.5 kullanılarak geliştirilmiş olan testin geliştirilmesi için kullanılabilir antikorlar için optimize edilmesi gerekebilir. BioScale için bir katkı maddesi kullanmadan önce% 1 &#39;lik bir konsantrasyon tampona eklenen çalışan tamponu (geliştirme kiti ile birlikte) geliştirmiştir. </li></ol></li><li> BioScale Sample Diluent kalibratörler ve örnekler sulandırınız. </li></ol> 3. ViBE Sistemi Kurma <ol><li> Üzerinde ViBE Workstation açın </li><li> Connect sistem kaynağı ve atık </li><li> Başbakan sistem </li><li> ViBE Kartuş Ekle </li><li> ROUTE tüpler </li><li> &quot;Örnek Setup&quot; etiketli sac doldurarak tahlil şablon hazırlayın. BioScale her ViBE İş İstasyonu tahlil için bir test şablonu vardır yeteneği sağlar. Bu kullanıcı, farklı deneyleri, deney tam bir kaydını tutmak için şablon &quot;Notlar&quot; sayfasında özel notlar düzenlemek ve kaydetmek için izin verir. </li><li> İlk tahlil şablonu kurdu: &quot;Örnek Setup&quot; sayfasında tayininde standartlarının konsantrasyon aralığı tablo (sol üst köşe) doldurarak başlayın. </li><li> Sonra, uzun örnekleri inkübe için ne ViBE İş İstasyonu talimat standartları (Basit Sütun Kurulum Oku, Bir Reaktif ekle ve İki Reaktif Ekle kurulumları) sağ tabloda ek bilgileri girin, ne kadar reaktif hacmi aktarılacak. Örnek birikimi gereken ne kadar uzun ve sensör klima ve probları ve kartuş dekontaminasyon gerekli olup olmadığını, vb. </li><li> Plaka düzeni doldurun. </li><li> Plakalar halinde numune / reaktifler yükleyin. ViBE reaktif plaka için gerekli hacim sağlayacaktır. Örnek hacmi değişir, ancak toplam 120 mcL tahlil hacminin yarısını iyi bir başlangıç ​​yeridir. </li><li> Plakası örtüleri uygulayın. Iş istasyonu güverte üzerine tabak yükleyin. </li><li> Testin başlangıç ​​sihirbazı çalıştırın, sihirbaz üzerinden gidin ve tahlil başlatmak. </li></ol> 4. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2493/2493fig1.jpg" alt="Şekil 1" /> Şekil 1: AMMP Sandviç İmmünoassay </p&gt; ViBE İş İstasyonu, bir AMMP test antijen konsantrasyonu ölçer. Sinyal boncuk-antijen-hapten antikor hapten / anti-hapten etkileşimi aracılığıyla sensör bağlanır ölçülür. Yenileme bağışıklık kompleksleri çıkarmadan, başlangıç ​​durumuna geri sensör getirir. <img src="/files/ftp_upload/2493/2493fig2.jpg" alt="Şekil 2" /> Şekil 2: CHO-OP AMMP Testi kalibrasyon eğrisi CHO OP AMMP Testi kalibrasyon eğrisi Şekil 2 yukarıdaki bir örnek. Bu örneklerin analizi de dahil olmak üzere, yüksek hassasiyet ve tekrarlanabilirlik ile reaktör arıtma akışı genelinde yapılabilir. <img src="/files/ftp_upload/2493/2493fig3.jpg" alt="Şekil 3" /> Şekil 3: Protein AMMP Testi kalibrasyon eğrisi Şekil 3 VIBE Artık Protein A Testi Kiti ile yapılan bir Kalıntı Protein AMMP Testi için bir kalibrasyon eğrisi. Aynı kiti bir 30 dakikalık inkübasyon dönemi ile bir hızlı tahlil, için ya da genellikle eşdeğer ELISA Kullanıcı kullanıcıları daha iyi olan, her ikisi de hassas ya da doğruluk kaybı olmadan bir daha uzun kuluçka dönemi, daha hassas sonuçlar ve geniş dinamik aralık için kullanılan olabilir. &#39; eller <img src="/files/ftp_upload/2493/2493fig4.jpg" alt="Şekil 4" /> Şekil 4: Ürün titresi AMMP Testi ViBE İş İstasyonu Ürün titresi tahlil, şekil 4&#39;deki sonuçlar için de kullanılabilir. Diğer AMMP Tahliller farklı seslendirilen bu testte basit seyreltme veya boncuk daha örnek tedavi gerektirmeyen, ve kantitatif canlılığında geniş bir yelpazede üzerinde 10 milyondan fazla hücre içeren &quot;spin&quot; örnekleri onaylandı. <img src="/files/ftp_upload/2493/2493fig5.jpg" alt="Şekil 5" /> Şekil 5: GST-Gadd34 biyomarker tahlil için AMMP Testi standart eğri Şekil 5 ViBE BioAnalyzer (Panel A) veya rekombinant GST-Gadd34 AlphaScreen Teknolojisi kullanılarak elde edilen sinyal standart bir eğri gösterilmiştir. ViBE platformu Sonuçlar aynı antikorları kullanarak daha duyarlı yaklaşık bir günlük. AMMP henüz az büyük parçacıkları hala çözüm faz yakalama ve algılama kompleks oluşum faydaları ölçümü yararlanır. <img src="/files/ftp_upload/2493/2493fig6.jpg" alt="Şekil 6" /> Şekil 6: tümör örnek GST-Gadd34 AMMP Testi standart eğri Şekil 6, CWR22 xenografts Gadd34 indüksiyonu ViBE BioAnalyzer (Panel A) veya AlphaScreen (B Paneli) veya analiz gibi bir boronate proteazom inhibitörü varlığı ya da yokluğu test edildi. ViBE platformu AlphaScreen sinyali ise in vivo tümör xenografts artan protein mikrogram ile söndürülür hassas ve tekrarlanabilir sonuçlar elde etmek mümkün oldu. Bu örnek, son derece karmaşık örnekler AMMP teknolojinin gücünü gösterir. Yongaları yakın mıknatıs hassas algılama yongası ve yüksek verimlilik nedeniyle, az sayıda manyetik mikro, verimli ve tekrarlanabilir parçalanmış hücresel (lizis reaktifler de dahil olmak üzere) ve kan enkaz karmaşıklığı ortasında düşük bolluk hedef için scavenge.

Bu makalenin yazarları, bu yazıda kullanılan bazı araçlar ve reaktifler üretir Bioscale, Inc tarafından istihdam edilmektedir.

Yukarıdaki örnekler, karmaşık örneklerinde AMMP son derece duyarlı kantitatif sonuçlar göstermektedir. Bu performans, kolaylık ve kontrol sistemi içine reaktifler biçimlendirme ile teslim çeşitli uygulamalar için sağlıyor. Bu uygulamalar tümör Gadd34 gibi bir hedef için özel ya da konak hücre proteini tespit ve kantitatif arıtma akışı çapraz örneklerinde olduğu gibi hedeflerin bir spektrum için genel olabilir. Son derece esnek AMMP ile birlikte düşük yükü ViBE platformu kritik testleri sunmak için güçlü bir araç sağlar.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NHS-Chromogenic-biotin or NHS-Chromogenic Biotin Labeling kit</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>If NHS-Chromogenic-biotin is purchased as a reagent the following additional materials will be required</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS) or other amine free buffer (pH 7 &#x2013; 9)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting columns or dialysis units for buffer exchange</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Spectrophotometer able to measure 354 and 280 nm &#x2013; for measuring protein and biotin concentrations</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated antibody (2-5 biotins/Ab)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin magnetic beads</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>(10 mg/mL) ~108 beads/mg</td>
</tr>
<tr>
<td>Magnet for microcentrifuge tubes</td>
<td>&nbsp;</td>
<td>New England BioLabs</td>
<td>cat # S1510S</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated BSA for blocking</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS 0.1% Tween 20</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vortex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge holder for Vortex or tube rotator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Antibody or protein to be labeled</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NHS-Fluorescein or NHS-Fluorescein Labeling kit</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>If NHS-Fluorescein is purchased as a reagent the following additional materials will be required</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 mM borate, pH 8.5 or other amine free buffer (pH 7 &#x2013; 9)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting columns or dialysis units for buffer exchange</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Measurement of protein and fluorescein concentrations requires a spectrophotometer able to measure 493 and 280 nm.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Diluent</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>to dilute beads and FlAb</td>
</tr>
<tr>
<td>Sample Diluent</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>to dilute samples and calibrators</td>
</tr>
<tr>
<td>Running Buffer Additive</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

developing custom chinese hamster ovary-host cell protein assays using acoustic membrane microparticle technology

Özel testlerin benzersiz proteinler için genellikle zaman alıcı manuel algılama ve ELISA veya alternatif platformları üzerinde geliştirme karmaşıklığı nedeniyle Western blot gibi kantitatif teknikleri ile sınırlıdır. BioScale tescilli Akustik Membran mikropartikül (AMMP) teknolojisi daha iyi bir hassasiyet, tekrarlanabilirlik ve otomatik kullanım sağlayan, sandviç immunoassay kolay ViBE platform üzerinde kullanım için geliştirilmiş olmasını sağlar. Bir örnek olarak verilmiştir, bu protokol, özel bir Çin Hamsteri Yumurtalık-Host Hücre Proteini (CHO-OP) tahlil geliştirmek için prosedür özetlenmektedir. Burada özetlenen immun geniş bir yelpazede gelişmesi için genel ilkeler takip edilebilir. Hesaplamak için sensör yüzeye mikro bağlama kaynaklanan salınım frekans değişiklikleri ölçerek bir AMMP tahlil önlemleri antijen konsantrasyonu. Bu dört ana bileşenden oluşur: (1) Fonksiyonlu sekiz sensör çipi içeren bir kartuş, (2) antikor etiketli manyetik mikro Fonksiyonlu yonganın yüzey antijeni içeren örnekler (4) bağlanır (3) hapten etiketli antikor ilgi. BioScale Kullanıcı biyosensör, sekiz ayrı akışkan yolları ile bireysel membranlar içeren bir rezonans bir cihazdır. Membranlar kütlesi yüzeyde biriken ve bu frekans değişikliği eklendi kütle miktarını ölçmek için kullanılır yanıt salınım frekansını değiştirmek. Sensörü anti-hapten antikor ile işlevselleştirildiyse immunoassay geniş bir yelpazede kullanım kolaylaştırmak için. Testi spesifik antikorların tek bir antikor ve diğer biotin hapten etiketi kovalent konjugasyon ile modifiye edilmiştir. Biotin etiketi hapten etiketli antikor ile birlikte, bir sandviç analit yakalamak için kullanılan, çiftli manyetik boncuk streptavidin antikor bağlamak için kullanılır. Kompleks anti-hapten/hapten etkileşim yoluyla çip bağlanır. Her testin sonunda sensörleri 96-plaka sıralı sütun analizi sağlayan bir seyreltik asit ile temizlenir çalıştırın. Burada, biyoproses geliştirme için özel bir CHO-OP AMMP testi geliştirmek için yöntemi mevcut. AMMP testlerin geliştirilmesi veya AMMP testleri mevcut testlerin değiştirerek karmaşık örnekleri ve azaltılmış operatörü saati (tekrarlanabilirlik, hassasiyet) daha iyi performans sağlayabilir. Protokol, kalkınma için adımlar gösterir ve tartışma bölümüne temsilcisi sonuçlarının gözden geçirilmesi. BioScale tahlil optimizasyon ve özelleştirme parametreleri hakkında daha ayrıntılı bir açıklama için başvurun. Bu kit Artık Protein A, Ürün titre ve CHO-OP gibi genel biyoproses geliştirme testleri sunuyor.

Watch this Scientific Journal Video about Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology at JoVE.com

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

ViBE karmaşık matrisler daha hassas, tekrarlanabilir, otomatik sonuçlar için platform üzerinde özel testlerinin geliştirilmesi açıklanmıştır. Evrensel kartuşu testleri özel testlerin kullanım için kolayca adapte edilmelidir sağlar. Bu çok yönlülük, roman deneyleri ya da bu video örneklediği mevcut manuel testleri otomatik sürümleri, hızlı geliştirme ve doğrulama sağlar.

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 ...

developing-custom-chinese-hamster-ovary-host-cell-protein-assays

Research

JoVE Journal

Bioengineering

13.1K Görüntüleme.  BioScale, Inc.. ViBE karmaşık matrisler daha hassas, tekrarlanabilir, otomatik sonuçlar için platform üzerinde özel testlerinin geliştirilmesi açıklanmıştır. Evrensel kartuşu testleri özel testlerin kullanım için kolayca adapte edilmelidir sağlar. Bu çok yönlülük, roman deneyleri ya da bu video örneklediği mevcut manuel testleri otomatik sürümleri, hızlı geliştirme ve doğrulama sağlar.

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

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

Akustik Microstreaming kullanarak Standart Laboratuvar Reverse Transcriptase reaksiyonlarda mRNA Tek hücreli Miktarları cDNA Verim artırılması

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Biyomühendislik

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Protein A Membran Adsorberleri ve Kolaylaştırılmış bir İş Akışı Kullanarak Kimerik İnsan Fc Ekspresyon Proteinlerinin İzolasyonu ve Karakterizasyonu

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

Otomatik Microbioreactor teknolojisini kullanarak Contegra Protein üretim duruma getirilmesi için genel iletişim kuralı

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Otomatik bir Mikrobioreactor sistemi kullanılarak Çin hamster yumurtlama hücrelerinden bir monoklonal antikor arıtma ve analitik

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Çin Hamster Yumurtalık Hücre Yetiştiriciliğinde Yüksek İş-İş BulaMetli Otomatik Mikro-Biyoreaktörler Kullanılarak Proses Optimizasyonu

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

Hücre Serbest Bakteriyel Özler, Lipozomlar ve Emülsiyon Transferi Kullanarak Protein Üreten Sentetik Hücreler Hazırlama

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Akustik Ayırıcı Kullanarak CHO Hasat Edilen Hücre Kültürü Sıvısının Birincil Netleştirilmesi

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Optik Olarak Buharlaştırılmış Perflorokarbon Nanodamlacıkların Formülasyonu ve Akustik Modülasyonu

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Çin Hamster Yumurtalık Hücrelerinde Hızlı Antikor Glikomühendisliği

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Here we present a protocol to express and purify recombinant Drosophila caspases Dronc and Drice, and their use in in vitro cleavage assays.

İn Vitro Substrat Taraması için Saflaştırılmış Rekombinant Drosophila Caspases Kullanarak Bölünme Testleri

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis ...