A.C. was supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada and the Training Program in Regenerative Medicine (Canadian Institutes of Health Research). F.Y.Y.H. was supported by the Training Program in Regenerative Medicine. This work was funded by a grant from Astellas Pharma Canada. We also would like to thank Dr. Mark Menenghini (University of Toronto) for use of his Axon microarray scanner.

1. Sulandırma antijen ve Yaratma Antijen Mikroarray&#39;ler <ol><li> 0.2 mg / ml&#39;lik bir son konsantrasyona PBS içinde antijenleri seyreltilir. 9 iğneli bir mikro-dizici konfigürasyonu ile iki nüsha halinde 162 kadar benzersiz antijenleri yazdırın. antijen kitaplık olarak pozitif kontrol IgG ve IgM antijenleri ekleyin. Yalnızca bir negatif kontrol olarak PBS içerir. </li><li> 384 gözlü bir kaynak plaka her bir antijenin 20 ul ekle. Kafasının kurulumu ayna gruplar halinde kaynak plakasına antijenleri ekleyin (9 pimleri baskı için kullanıldığında, örneğin, 9 kişilik gruplar halinde antijenleri düzenlemek). </li><li> diziler yazdırmak için hazır olana kadar -80 ° C&#39;de folyo ve dondurma ile kapak kaynak plakası. </li><li> 3 x 1 dakika boyunca iyonu giderilmiş su ile, bir sonikasyon banyosu içinde onları kuluçka katı mikro-dizici işaretçilerini temizleyin. rafa yerleştirin pimleri kurutun ve daha sonra mikroarray yazıcı kafasındaki işaretçilerine düzenlemek için. 9 pin için, 3 x 3 yapılandırmayı kullanın. </li><li> kafasının sayı işaretçilerine ayarlayarak tarafından işletilen baskı için program mikro-dizici,slaytlar sayısı her slaytta, pedleri numarasını yazdırmak ve her bir antijen için çoğaltmak lekelerin sayısı için. Tipik olarak, 9 işaretçilerine, 70 kaydırak, 2 pedleri / slayt ve her bir antijen için 2 suret noktalar kullanın. Program mikro-dizici antijenlerin farklı gruplar arasında suda işaretçilerine ses dalgalarına maruz için. </li><li> Çözülme kaynak plakası 100 x g, 1 dakika daha sonra santrifüj plaka. mikro dizicinin belirlenen noktada kaynak plaka koyun. Basılacak antijenlerin ilk grubu kapsayan folyo bölümünü kaldırın. </li><li> arrayer yüzeyi ve çalıştırma baskı programı baskısız slaytlar düzenleyin. Dizi makinesinin nemlendirici inşa ile% 60 - nem oda sıcaklığında baskı slaytlar 55 de arrayer ayarlanır. </li><li> antijenlerin her grup her slaytlarda basıldıktan sonra, mikro-dizici duraklatmak. sadece folyo ile basıldı antijenleri Kapak (buharlaşmasını önlemek için) ve antijenlerin sonraki grup basılacak ortaya çıkarmak. Baskı programına devam edin. </li><li> Tüm antijenler yazdırıldıktan sonra, kapak kaynak pl-80 ° C folyo ve dondurma yeni bir parça ile yedi. slayt kutu ve vakum mühür basılmış slaytlar yerleştirin. Slaytlar ertesi gün kullanılan veya en fazla bir ay sonra olabilir. </li></ol> 2. Seyreltik Sera ile Antijen mikrodizileri Probing <ol><li> Kuluçka odaları kullanarak çerçevelerin içine slaytlar yerleştirin. Her bir dizi yüzey engelleme tamponu (% 2.5 [hacim / hacim] fetal buzağı serumu (FCS), PBS içinde Tween 20,% 0.1 [/ hacim: hacim]) 700 ul ekle. </li><li> Islak bir doku parçası ile kapalı bir kapta çerçeve ve yerde yapışkan film yerleştirin. bir rocker 4 ° C&#39;de O / N inkübe edin. </li><li> Serum örnekleri 1 sulandırmak: 100 tampon engelleme. Aspire dizilerden engelleme çözümü ve her dizi yüzeyine seyreltilmiş örnek 500 ul ekleyin. </li><li> yapışkan film ile örtün ve sallanan 4 ° C sıcaklıkta 1 saat boyunca inkübe edilir. </li><li> tampon engelleme ikincil antikorlar seyreltilir. İnsan deneyleri için, 0.33 mg / ml ve Cy5 bir Cy3 etiketli keçi anti-insan IgG antikoru seyreltilmiş keçi anti-Huma etiketli0.25 ug / ml IgM antikoru n. Fare çalışmaları için, bir Cy3 mi 0.38 ug / keçi anti-fare IgG antikoru etiketlenmiş ve bir Cy5 / ml 0.7 ug keçi anti-fare IgM antikoru etiketli seyreltin. </li><li> dizileri aspire örnekler ve dizi yüzeyleri durulama tamponu (PBS içerisinde% 0.1 Tween 20) ile 4 kez yıkayın. slaytlar üzerine tampon ve çabuk fiske dökün. </li><li> sallanan oda sıcaklığında her dizi yüzeyini yıkamak ve 10 dakika inkübe etmek blokaj tamponu 700 ul ekle. Tekrarlayın yıkama adımı iki kez daha. </li><li> Her slayt yüzeyine seyreltilmiş ikincil antikor 500 ul ekleyin ve yapışkan film ile kaplayın. sallanan 4 ° C&#39;de 45 dakika inkübe edin. </li><li> Aspire ikincil antikor slaytlar gelen karışım ve yukarıdaki gibi 4 kez yıkayın. Yıkama yukarıdaki gibi engelleme / seyreltme tamponu 700 ul ile 3 kez kayar. </li><li> PBS batırılır metal slayt raf çerçeveleri ve yerden slaytları çıkarın. yörünge çalkalama ile oda sıcaklığında 20 dakika kuluçkalayın. PBS yeni kapta koyun ve başka bir 20 m inkübesallayarak ile. </li><li> deiyonize su 15 saniye bir kap içine koyun slayt raf. su, yeni bir kapta raf yerleştirin ve başka bir 15 saniye kuluçkaya yatmaktadır. </li><li> Kuru slaytlar, oda sıcaklığında 5 dakika boyunca 220 xg&#39;de santrifüj ve spin bir ELISA plakası adaptörü üzerindeki yeri slayt rafa. </li><li> tarama için hazır olana kadar ışık geçirmez kutu slaytlar yerleştirin. </li></ol> 3. Tarama Antijen Mikroarray&#39;ler ve İhracat Verileri <ol><li> Cy3 ve Cy5 floresan sinyalleri algılayabilen bir mikrodizi tarayıcı kullanarak tarama slaytlar. Tarayıcının photomultiplier tüp (PMT) seviyesini ayarlamak için, sadece sekonder antikor ile problanmıştır bir slayt önceden tarayın. Not: Bu slayt tam pozitif (IgG ve IgM) de dahil olmak üzere basılmış antijen kütüphanesi yanı sıra negatif (PBS) kontrolleri olmalıdır. ön tarama optimum PMT ayarlarını bulmak için hızlı bir şekilde kullanıcı sağlayan bir düşük çözünürlüklü tarama olduğunu. </li><li> PMT değerlerini ayarlayın, böylece (Cy3 kanalında) insan IgG özellikleri ve uğultu(Cy5 kanalında) bir IgM özellikleri benzer medyan floresan eksi arka plan (MFI-B) (tipik olarak 40.000) var. PMT düzeyleri &quot;donanım&quot; düğmesine basarak ayarlanır. ayarlandıktan sonra, sabit bu PMT ayarları korumak. </li><li> &quot;Tarama&quot; tuşuna basarak iki kanal deneysel slaytlar tarayın. Her taramadan sonra slayt görüntüleri (iki kanal) kaydedin. </li><li> slayt yükle &quot;Dosya&quot; düğmesini kullanarak mikroarray yazılımı içine analiz edilecek. </li><li> &quot;Dosya&quot; düğmesini kullanarak gen dizisi listesi (GAL) dosyasını yükleyin. GAL dosya dizisi özellikleri kimlikleri ile dizinin düzeni vardır. </li><li> diziler özellikleri mümkün olduğunca yakından şablonu eşleşecek şekilde taranan görüntünün üzerine dizi şablonu yerleştirin. </li><li> &quot;Hizalama&quot; düğmesine basarak şablonu ile tüm bloklarda özellikleri aynı hizaya getirin. Program genellikle dairesel özellikleri anahat bulacaksınız ama bazı manuel ayarlamalar gerekebilir. Bu ayarlamalar özellik moduna girerek yapılabilir. Yazılım daha sonra antijenlerin her biri için bir MFI-B hesaplar. </li><li> Bir metin dosyası olarak bu sonuçları ihracat &quot;dosya&quot; düğmesini kullanın. </li></ol> Mikroarray&#39;ler Önemi Analizi ile 4. Analiz Dizi Verileri (SAM) <ol><li> excel içine tek metin dosyaları yüklemek ve Cy3 ve Cy5 kanalları için MFI-B var sütunları belirlemek. </li><li> yinelenen özellikler için ortalama hesaplayın. </li><li> herhangi bir olumsuz MFI-B, 10 ile negatif sayı günlük tabanı 2 hesaplayarak sonra 100 ile ham MFI-B bölünmesi ve verileri Transform değiştirin. </li><li> Daha önce 7 açıklandığı gibi Mikroarray&#39;ler (SAM) Önemi Analizi ile günlük dönüştürülmüş verileri analiz edin. </li><li> Iki grup (örneğin, sağlıklı kontrol vs hasta), etiket bir grup &quot;1&quot; ve grup kullanarak bir çalışma için &quot;2.&quot; ler vardır antijenler reaksiyonlarını belirlemek için SAM iki sınıf, eşleşmemiş analizini kullanınHer iki grup arasında önemli ölçüde farklıdır (q &lt;0.05). </li><li> Sunum 8 görüntüleri yapmak için kümeleme ve ısı haritası üreten yazılımını kullanın. </li></ol>

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The authors have nothing to disclose.

Burada açıklanan protokol antijen mikrodizi tekniği kullanılarak otoantikorlann ölçümü sağlar. Antijen mikrodizileri otoantikorlar için eleme testi esnasında klasik ELISA göre birçok avantajı vardır. Her şeyden önce, nükleik asitler, proteinler, peptidler ve hücre lizatları dahil çeşitli antijenlere ve böylece oto antikorların çoğaltılmış çok katlı taranması için izin nitroselüloz kaplı slaydlar üzerine dizilmiş olabilir. antijen nanolitre Her bir slayt üzerine fark vardır Ek olara...

Autoantibodies are present in many disease states and can often have direct pathogenic activity1. Identification of autoantibodies is important for diagnosis of certain diseases, for prognosis of disease outcome, and for the classification of patients who may benefit from specific therapies2. Autoantibodies are typically identified in patient serum using the ELISA technique; however, screening for multiple antigens with this technique is laborious and consumes a large quantity of patient sample. New technologies are therefore needed to profile autoantibodies on a larger scale.
The antigen microarray technique is a proteomic technology that allows autoantibodies to be profiled in a multiplex fashion3. In the first step of this process, an antigen library is arrayed onto a slide surface using a robotic microarrayer. The slides are probed with diluted serum and then fluorescent-labeled secondary antibodies are added. Antibody reactivities are visualized by scanning the slides with a microarray scanner and quantified by fluorescent intensities. Antigen microarrays offer multiple advantages over the ELISA technique in screening for autoantibodies: 1) they require only microliters of serum to profile autoantibodies to multiple antigens simultaneously, 2) they use antigen sparingly, as only nanoliters of antigen are spotted onto the arrays, 3) they have enhanced sensitivity3 compared to ELISA and 4) they allow for the simultaneous yet, separate detection of more than one antibody isotype. Antigen microarrays have been used to profile autoantibodies in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus4-6. In all three of these diseases, new insight into disease pathogenesis was obtained from profiling autoantibodies on a large scale with the arrays. 
Here we describe a protocol to generate antigen microarrays using nitrocellulose-coated slides. A variety of antigens including proteins, peptides, and cell lysates can be arrayed onto the slides using this technique. The arrays can be easily customized by including antigens of interest in the source plate that holds the antigen library. In addition, we show how a pair of secondary antibodies can be used to separate IgG and IgM reactivities on the same slide surface. We have now optimized this technique to measure autoantibodies in both humans and mice.

<table border="0" cellpadding="0" cellspacing="0" width="1054">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Ribosomal P0</td>
			<td style="width:225px;">Diarect</td>
			<td style="width:304px;">14100</td>
			<td style="width:240px;">dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human IgG</td>
			<td>Jackson Immuno</td>
			<td>009-000-003</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human IgM</td>
			<td>Jackson Immuno</td>
			<td>009-000-012</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse IgG</td>
			<td>Sigma-Aldrich</td>
			<td>I5381</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse IgM</td>
			<td>Biolegend</td>
			<td>401601</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double-stranded DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D1626</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">single-stranded DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D8899</td>
			<td>dilute to 0.2mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microarrayer</td>
			<td>Virtek</td>
			<td>VersArray Chipwriter Pro</td>
			<td>many types of arrayers are suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">solid printing pins</td>
			<td>Arrayit Corporation</td>
			<td>SSP015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">software for robotic microarrayer</td>
			<td>Virtek</td>
			<td>Chipwriter Pro&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST slides (2 Pad)</td>
			<td>GVS Northa America</td>
			<td>10485317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST frame</td>
			<td>GVS Northa America</td>
			<td>10486001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FAST incubation chambers (2 Pad)</td>
			<td>GVS Northa America</td>
			<td>10486242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384 well plates</td>
			<td>Whatman</td>
			<td>7701-5101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plate sealers</td>
			<td>VWR</td>
			<td>60941-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">foil plate covers</td>
			<td>VWR</td>
			<td>60941-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum</td>
			<td>Invitrogen</td>
			<td>12483020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3 goat anti-human IgG</td>
			<td>Jackson Immuno</td>
			<td>109-165-096</td>
			<td>use working stock in 50% glyercol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5 goat anti-human IgM</td>
			<td>Jackson Immuno</td>
			<td>109-175-129</td>
			<td>use working stock in 50% glyercol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3 goat anti-mouse IgG</td>
			<td>Jackson Immuno</td>
			<td>115-165-071</td>
			<td>use working stock in 50% glyercol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5 goat anti-mouse IgM</td>
			<td>Jackson Immuno</td>
			<td>115-175-075</td>
			<td>use working stock in 50% glyercol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microarray Scanner</td>
			<td>Molecular Devices</td>
			<td>Axon 4200A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microarray software</td>
			<td>Molecular Devices</td>
			<td>Genepix 6.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clustering software</td>
			<td>eisenlab.org</td>
			<td>Cluster 3.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heatmap software</td>
			<td>eisenlab.org</td>
			<td>Treeview 1.60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microarray statistical software</td>
			<td>Stanford University</td>
			<td colspan="2">SAM 4.0 (Significance Analysis of Microarrays)</td>
		</tr>
	</tbody>
</table>

generation of two-color antigen microarrays for the simultaneous detection of igg and igm autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

Şekil 1&#39;de gösterildiği gibi antijenler bir robot mikro dizicinin tarafından Slaytlarınıza 384 plaka olarak düzenlenmiş ve yazdırılır. 2 inkübasyon odaları ve işlenmesinden sonra taranmış bir slayt ile bir çerçeve içinde yerleştirildi slaytlar göstermektedir. 3 pozitif ve negatif kontrol slaytlar göstermektedir. Negatif bir slayt, sadece sekonder antikor ile incelendi ve pozitif...

Watch this Scientific Journal Video about Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies at JoVE.com

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

IgG ve IgM Antikorlarının Eşzamanlı Algılama için İki Renkli Antijen Mikroarray&#39;ler Üretimi

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

generation-two-color-antigen-microarrays-for-simultaneous-detection

Research

JoVE Journal

Immunology and Infection

12.7K Görüntüleme.  University Health Network. We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest. 

Video: Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Yapışkan bant tabanlı Örnekleme ve Floresans kombinasyonu In situ</emHızlı Algılama> Hibridizasyon Salmonella Taze üretin

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

İmmünoloji ve Enfeksiyon

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

Etiket-ücretsiz Biyosensör Teknolojisi kullanılması Meme Kanserinde İnsan Lökosit Antijen BİYOMARKÖRLERİN Algılama

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Ölümsüzleştirilmiştir Sıralama B hücrelerinden Rekombinant İnsan IgG Monoklonal Antikorlarının Üretimi

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Fenotip ve Fonksiyon Analizi için algılama ve Nadir zenginleştirilmesi antijene özgü B hücreleri

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Grip virüsü monoklonal antikorları nötralize kaçış türevleri üretimi

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo Tahlil antijen spesifik T-hücre Cytolytic işlevi bir aşı modeli kullanılarak tespiti için

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Electrochemiluminescence deneyleri için insan adacık otoantikorlar

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Salmonella spp. testi ile taranması için yüksek üretilen iş platformu /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

Formalin-Sabit Dokulardan Lyssavirüs Antijen Tespiti için İmmünohistokiniti Testi

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...