Name: anti-nuclear antibody screening using hep-2 cells
Uploaded: 2014-06-23T18:15:00
Duration: 13 min 1 s
Description: Watch this Scientific Journal Video about Anti-Nuclear Antibody Screening Using HEp-2 Cells at JoVE.com

Vi takker Cassandra Bryant til at udføre IIF eksperimentet og Carol Buchner for hendes ekspert teknisk gennemgang.

1.. Prøve Selection Forberedelse og substrat <ol><li> Fjern reagenser fra emballage og give hvert element til at komme til stuetemperatur. Forbered reagenser og fortyndes patientsera overensstemmelse med retningen indsatsen. Bemærk: NOVA Lite dias Barcoded og kan nemt integreres i automatiserede systemer i forbindelse med Laboratory Information System LIS. Denne procedure illustrerer manuel slide behandling; høje throughput laboratorier kan dog vælge automatiseret slide udstyr. </li></ol> 2.. Tilsætning af kontroller og prøver <ol><li> Doser 1 dråbe af den positive kontrol og 1 dråbe af den negative kontrol til de relevante slide brønde. Pipetter 20-25 pi fortyndet patientserum til de resterende brønde. Proces ét dias ad gangen. </li><li> Anbring slide (r) i en farvning beholder med en fugtig papirserviet på bunden. Dæk beholderen og inkubere objektglasset (r) i 30 minutter. Bemærk: De fugtige forhold vilforhindre substratet i at tørre ud. Tørring af brøndene kan resultere i kunstig farvning. I løbet af denne inkubationsperiode vil anti-nukleare antistoffer i patientens serum binder til antigener af de celler, der er fastgjort på hver brønd. </li><li> Efter inkubationsperioden skylle serum under anvendelse af en mild strøm af vaskepuffer. For at undgå at beskadige underlaget og forhindre cross-over af prøver mellem brønde, vinkel slide lidt for at undgå at lede strømmen direkte på brøndene. Bank overskydende vaskebuffer og placere dias i en Coplin krukke indeholdende vaskebuffer i ca 5 min. </li></ol> 3.. Tilsætning af fluorescerende konjugat <ol><li> Én efter én, skal du fjerne dias fra vaskebuffer og forsigtigt trykke for at fjerne den overskydende vaskebuffer. Anvend 1 dråbe fluorescerende konjugat (FITC-mærket anti-humant IgG) på hver brønd. Glassene inkuberes i 30 minutter i det befugtet beholder, og sørg for at udskifte farvningenbeholderdækslet. Bemærk: ANA test, anbefales brug af en IgG Fc specifik konjugat. Konjugatet er lysfølsomt, og dækslet vil beskytte objektglassene mod lys ud over at opretholde den befugtede miljø. I løbet af denne inkubationsperiode vil konjugatet binde til patientens anti-nukleare antistoffer, som har bundet til celle-antigener. Dette konjugat bindende resultater i nærvær af fluorescens i brøndene. </li><li> Skyl og vask det dias, som beskrevet ovenfor. </li><li> Placer et dækglas på et stykke køkkenrulle og anvende montering medium i en ubrudt linje til den nederste kant af dækglasset. </li><li> Fjern hver dias fra vaskebuffer og trykke på Skub forsigtigt at fjerne den overskydende vaskebuffer. Tryk på den nederste kant af dias til kanten af ​​dækglasset. </li><li> Sænk forsigtigt slæden på dækglasset på en sådan måde, at deres montering medium på dækglasset strømme til den øverste kant af objektglasset uden luft bobledannelse. Bemærk: Coverslipping, herunder ved hjælp af den optimale mængde af montering medium, er en teknik, der kræver øvelse at perfektionere. </li></ol> 4.. Identifikation af positive og negative resultater Manuel Fortolkning <ol><li> Se objektglas med et fluorescensmikroskop, der ligger i et mørkt rum. Udføre en scanning af hele godt med en 20X eller 25X formål at vurdere cellefordeling og ensartethed af fluorescens </li><li> Skift til en 40X målsætning at foretage den endelige fortolkning med hensyn til positivitet og mønster. </li><li> Ser på de positive og negative kontroller. Grade positivitet ved hjælp af en reaktivitet karakterskala fra 1 + til 4 +. Bemærk: Den negative kontrol vises muligvis ikke helt mørkt, men vil ofte vise lavt niveau uspecifik fluorescens. Den positive kontrol vil vise lyse æble grøn fluorescens i kernen (figur 1). </li></ol> Automatisk tolkning Bemærk: I additipå manuel fortolkning, belastning og scan dias fra NOVA View Automatiseret Fluorescens mikroskop eller andre automatiserede digital billedbehandling instrument; ingen mørkekammer er nødvendig. Efter oprettelse af et projekt ved at vælge den relevante slide type instrumentet erhverver og gemmer højopløselige digitale billeder af celler i hver brønd. Derudover NOVA View måler fluorescerende lys intensitet, og kategoriserer resultater som positiv eller negativ, og giver mønstergenkendelse for positive prøver. Billeder er set af operatøren på en høj opløsning computerskærm, der giver mulighed for den endelige fortolkning, revision og bekræftelse af NOVA View resultat. Rapporter kan genereres bekræftede resultater. Digital mikroskopi bør anvendes sammen med en erfaren laborant, som det er beregnet til at hjælpe i fortolkning af resultaterne.

<ol>
	<li>Meroni, P. L., Schur, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANA+screening:+an+old+test+with+new+recommendations.">ANA screening: an old test with new recommendations.</a> Ann Rheum Dis. 69 (8), 1420-1422 (2010).</li><li>Tan, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+to+nuclear+antigens+(ANA):+Their+immunobiology+and+medicine.">Autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine.</a> Advances in immunology. 33, 167-240 (1982).</li><li>Sack, U., 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=Autoantibody+detection+using+indirect+immunofluorescence+on+HEp-2+cells.">Autoantibody detection using indirect immunofluorescence on HEp-2 cells.</a> Ann NY Acad Sci. 1173, 166-173 (2009).</li><li>Watanabe, 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=Anti-DFS70+antibodies+in+597+healthy+hospital+workers.">Anti-DFS70 antibodies in 597 healthy hospital workers.</a> Arthritis Rheum. 50 (3), 892-900 (2004).</li><li>Mahler, 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=Importance+of+the+dense+fine+speckled+pattern+on+HEp-2+cells+and+anti-DFS70+antibodies+for+the+diagnosis+of+systemic+autoimmune+diseases.">Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases.</a> Autoimmun Revi. 11 (9), 642-645 (2012).</li><li>Mahler, M., Fritzler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+significance+of+the+dense+fine+speckled+immunofluorescence+pattern+on+HEp-2+cells+for+the+diagnosis+of+systemic+autoimmune+diseases.">The clinical significance of the dense fine speckled immunofluorescence pattern on HEp-2 cells for the diagnosis of systemic autoimmune diseases.</a> Clin Dev Immunol. , (2012).</li><li>Miyara, 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=Clinical+phenotypes+of+patients+with+anti-DFS70/LEDGF+antibodies+in+a+routine+ANA+referral+cohort.">Clinical phenotypes of patients with anti-DFS70/LEDGF antibodies in a routine ANA referral cohort.</a> Clin Dev Immunol. , (2013).</li><li>Mariz, H., 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=Pattern+on+the+antinuclear+antibody-HEp-2+test+is+a+critical+parameter+for+discriminating+antinuclear+antibody+positive+healthy+individuals+and+patients+with+autoimmune+rheumatic+diseases.">Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody positive healthy individuals and patients with autoimmune rheumatic diseases.</a> Arthritis Rheum. 63 (1), 191-200 (2011).</li><li>Muro, Y., 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=High+concomitance+of+disease+marker+autoantibodies+in+anti-DFS70/LEDGF+autoantibody-positive+patients+with+autoimmune+rheumatic+disease.">High concomitance of disease marker autoantibodies in anti-DFS70/LEDGF autoantibody-positive patients with autoimmune rheumatic disease.</a> Lupus. 17 (3), 171-176 (2008).</li><li>Agmon-Levin, N., 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=International+recommendations+for+the+assessment+of+autoantibodies+to+cellular+antigens+referred+to+as+anti-nuclear+antibodies.">International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies.</a> Ann Rheum Dis. 73 (1), 17-23 (2014).</li><li>Craft, J., 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=The+U2+small+nuclear+ribonucleoprotein+particle+as+an+autoantigen.">The U2 small nuclear ribonucleoprotein particle as an autoantigen.</a> J Clin Invest. 81 (6), 1716-1724 (1998).</li><li>Salomonsson, 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+serologic+marker+for+fetal+risk+of+congenital+heart+block.">A serologic marker for fetal risk of congenital heart block.</a> Arthritis Rheum. 46 (5), 1233-1241 (2002).</li><li>Miyawaki, 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=Clinical+and+serological+heterogeneity+in+patients+with+anticentromere+antibodies.">Clinical and serological heterogeneity in patients with anticentromere antibodies.</a> J Rheumatology. 32 (8), 1488-1494 (2005).</li><li>Raijamkers, R., 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=PM-Scl-75+is+the+main+autoantigen+in+patients+with+the+polymyositis/scleroderma+overlap+syndrome.">PM-Scl-75 is the main autoantigen in patients with the polymyositis/scleroderma overlap syndrome.</a> Arthritis Rheum. 50 (2), 565-569 (2004).</li><li>Yang, J. 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+scleroderma+sera+contain+autoantibodies+to+protein+components+specific+to+the+UC+small+nucleolar+RNP+complex.">Human scleroderma sera contain autoantibodies to protein components specific to the UC small nucleolar RNP complex.</a> Arthritis Rheum. 48 (1), 210-217 (2003).</li><li>Granito, 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=Antinuclear+antibodies+as+ancillary+markers+in+primary+biliary+cirrhosis.">Antinuclear antibodies as ancillary markers in primary biliary cirrhosis.</a> Expert Rev Mol Diagn. 12 (1), 65-74 (2012).</li></ol>

Forfatterne Gabriella Lakos, Cassandra Bryant, Carol Buchner, og Anna Eslami er medarbejdere i Inova Diagnostics.

Screening by HEp-2 cells is a critical first step in the diagnosis of patients with SARD. However, IIF methods lack harmonization. Sources of variability include preparation of slides, conjugate specificity, and efficiency of the fluorescent microscope and experience of the reader. Despite these concerns, IIF remains the &#8220;gold standard&#8221; for ANA testing. HEp-2 cells contain a large variety of autoantigens, and provide the ideal substrate for the detection of these autoantibodies. In some laboratories, IIF has been replaced with solid phase screening methods such as multiplex assay or ELISA. The shortcoming of such tests is that they do not display the full range of antigens to be sufficiently sensitive. As a consequence, true positive patients may be missed which can have deleterious consequences In addition to the issues of treatment delay and wrong diagnosis, additional healthcare costs may occur due to the repetition of confirmatory tests or unnecessary diagnostic investigations. Given the inherent challenges to IIF, it is paramount to perform this technique properly to avoid subjectivity in results.
To ensure accurate interpretation and reporting of results for ANA screening, it is vital to use the highest quality substrate. When selecting a HEp-2 substrate, it is critical that the cells be optimized to express clinically relevant epitopes in their native protein state. Transfected or otherwise modified HEp-2 cell lines may not allow proper antibody-antigen binding. In addition, when several different autoantibodies are present simultaneously, recombinant cells may mask one or more patterns. High number of mitotic cells should also be present in the substrate. Adequate number of mitotic cells allows quick and accurate identification of patterns. 
In addition to the attributes of the substrate, the objective of this video protocol is to describe the IIF technique by showing critical steps such as the addition of sample, slide washing, addition of conjugate, cover slipping, and determination of positive and negative results. Results can be compromised if proper technique is not used for each of these steps. Proper washing is important to remove all unbound antibody. Some patients display very high amounts of autoantibodies and in these cases it is important to wash the serum from the wells such that it does not contaminate other wells. 
Although IIF has traditionally been a very labor intensive and subjective laboratory method, new technologies such as slide processors, barcoded slides and automated digital microscopy can greatly simplify the workflow, increase the consistency and reduce the sources of variability of interpretation.

Udtrykket antinuclear antistof (ANA) beskriver en række autoantistoffer, som reagerer med bestanddele af cellekerner inklusive DNA, proteiner og ribonucleoproteiner 1, 2. Den HEp-2 celle, et nativt protein array med hundreder af antigener, tilvejebringer et ideelt substrat til påvisning af ANA 1. Påvisning af ANA i humant serum er et vigtigt screeningsværktøj til bindevævssygdomme, og IIF er referencemetoden for ANA test af 1.. For nylig har IIF om HEP-2 celler blevet erstattet i nogle laboratorier med antigen-specifikke immunanalyser og multiplex metoder. Grund af bekymring over falske negative resultater og den manglende gennemsigtighed for klinikere, American College of Rheumatology dannet en taskforce, der konkluderede, at IIF hjælp HEP-2 celler skal være &quot;gold standard&quot; for ANA screening 1. På grund af den subjektive natur af ANA screening, kvaliteten af ​​HEp-2-celler iTegral til nøjagtige og selvsikker rapportering af resultater. Tilstedeværelsen af ​​et stort antal mitotiske celler, optimal cellemorfologi tilstrækkelig konfluens og ekspression af relevante antigener er særligt vigtige. IIF mønstergenkendelse fungerer som et vigtigt redskab til at støtte patientens diagnose. Forstå betydningen af ​​forskellige mønstre gør det muligt for klinikere og laboratorie personale til at udføre den relevante opfølgning test for at bekræfte diagnosen. For eksempel kan homogen ANA mønster forekommer i tilstedeværelsen af anti-dsDNA eller kromatin antistoffer og kan være forbundet med systemisk lupus erythematosus (SLE) 3. Fra den anden side, er det for nylig blevet beskrevet, at den såkaldte tætte fine spættede mønster (DFS), der ofte ses i op til 12% af rutinemæssige prøver, for det meste har været forbundet med anti-DFS70 antistoffer. Disse autoantistoffer, da de blev fundet i isolation (uden anden sygdom-specifikke ANA) er ikke forbundet med systemiske autoimmune gigtsygdomme &lt;sup&gt; 4-9. Derved bekræftende test for anti-DFS70 antistoffer kan hjælpe med at reducere unødvendige refleks test, tilbyder betydelige omkostningsbesparelser, og lette patientens angst. Da IIF er den første linje screening metode til at opdage autoantistoffer, er det altafgørende, at brugeren forstår, hvordan udvælgelsen af ​​reagenser og væv substrater kan påvirke resultaterne. Da IIF teknik er i sagens natur subjektive, er det vigtigt, at de anvendte reagenser levere den højeste kvalitet. Dette mål af denne video protokol er at gøre brugeren med de rigtige skridt, der er nødvendige for at udføre IIF-metoden, de fælles mønstre forbundet med ANA, og indføre nye fremskridt, som kan effektivisere arbejdsgangen laboratorium og standardisere resultater.

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:252px;">NOVA Lite HEp-2 IgG (DAPI conjugate)</td>
			<td style="width:189px;">INOVA Diagnostics</td>
			<td style="width:135px;">708102</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NOVA View Instrument</td>
			<td>INOVA Diagnostics</td>
			<td>NV2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QUANTA Link Workstation</td>
			<td>INOVA Diagnostics</td>
			<td>LINK010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QUANTA Link Workstation License</td>
			<td>INOVA Diagnostics</td>
			<td>LINK001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barcode Scanner</td>
			<td>INOVA Diagnostics</td>
			<td>LINK019</td>
			<td></td>
		</tr>
	</tbody>
</table>

anti-nuclear antibody screening using hep-2 cells

American College of Rheumatology position erklæring om ANA test fastsætter anvendelse af IIF som guldstandarden metode til ANA screening 1. Selvom IIF er en fremragende screening test i ekspert hænder, de tekniske vanskeligheder for forarbejdning og læsning IIF slides - såsom arbejdskrævende slide behandling, manuel aflæsning, behovet for erfarne, uddannede teknologer og brugen af ​​mørkt rum - gør IIF-metoden svært at passe i arbejdsgangen af ​​moderne, automatiserede laboratorier. Det første og afgørende skridt i retning af høj kvalitet ANA screening er omhyggelig slide forarbejdning. Denne procedure er arbejdskrævende, og kræver fuld forståelse af processen, samt opmærksomhed på detaljer og erfaring. Slide læsning er udført af fluorescerende mikroskopi i mørke rum, og er udført af uddannede teknologer, der er bekendt med de forskellige mønstre i forbindelse med cellecyklusog morfologi interfase og delende celler. Forudsat at IIF er den første linje screeningsværktøj til SARD forstå de skridt til korrekt udføre denne teknik er kritisk. For nylig er der blevet udviklet digital billedbehandling systemer til automatiseret læsning af IIF dias. Disse systemer, såsom NOVA View Automated fluorescerende mikroskop, er designet til at strømline den rutinemæssige IIF workflow. NOVA View erhverver og gemmer højopløselige digitale billeder af brøndene, og dermed adskille billede erhvervelse fra fortolkning; billederne ses en fortolket på høj opløsning computerskærme. Den gemmer billeder til fremtidig reference og støtter operatørens fortolkning ved at give fluorescerende lys intensitet data om billederne. Det er også foreløbigt kategoriserer resultater som positiv eller negativ, og giver mønstergenkendelse for positive prøver. Sammenfattende det eliminerer behovet for mørkekammer, og automatiserer og strømliner IIF om enhedenng / tolkning workflow. Vigtigst er det, det øger overensstemmelsen mellem læsere og aflæsninger. Desuden med brug af stregkoder dias, er transskriptionsfejl elimineres ved at prøve sporbarhed og positiv patient identifikation. Dette resulterer i øget patientsikkerhed dataintegritet og sikkerhed. Det overordnede mål med denne video er at demonstrere IIF proceduren, herunder slide behandling, identifikation af fælles IIF mønstre, og indførelsen af ​​nye fremskridt for at forenkle og harmonisere denne teknik.

Ved vurdering cellefarvning resultater for HEP-2, fem større nukleare mønstre er hyppigst rapporterede: homogen, spættede centromere, nucleolar og nukleare prikker. Disse mønstre er resultatet af autoantistof binde sig til specifikke bestanddele af kernen. Selvom ANA test er specifik for nukleare strukturer kan cytoplasmatiske mønstre forbundet med autoantistoffer mod cytoplasmatiske strukturer også overholdes. I nogle tilfælde kan flere autoantistoffer være til stede i en prøve, og billedet vises som en blandet mønster. Der er andre, mindre hyppigt observeret, og ofte cellecyklus specifikke mønstre, der kan registreres af IIF-metoden; Senest har en såkaldt tæt fint spættede mønster med særlig klinisk betydning blevet beskrevet i nogle ANA positive sera. Homogen Mønster At identificere en homogen mønster, scanne godt og identificere mitotiske eller dividereceller. Den kondenserede kromatin af mitotiske celler (figur 2, orange pil) udviser fast, ensartet fluorescens, som ofte er mere udtalt end i den hvilende celle nuclei.In det homogene mønster og hvilende celler udviser ensartet diffus fluorescens af hele kernen (figur 2, hvid pil). Denne karakteristisk mønster er ofte et resultat af anti-dsDNA-antistoffer 10. Spættet mønster I grove spættede mønster, de mitotiske celler viser ingen farvning af kondenserede kromosomale regioner (figur 3, orange pil). De hvilende celler udviser kornet fluorescens hele kernen. Det nukleare speckling kan defineres som groft eller fint. Den Grov speckling mønster er ofte et resultat af anti-Sm og anti-RNP 11. I det fine spættede mønster, de hvilende celler vise fine eller diffuse speckling hele kerner, typisk i uniform dFordeling (figur 4, hvid pil). Den fine plettet mønster er ofte et resultat af anti-SSAand anti-SSB 12. Dense Fin Speckled Mønster Det er nu anerkendt, at den tætte fine spættede mønster (DFS) er almindeligt i rutinemæssig afprøvning, og så meget som 12% af prøverne er positive for anti-DFS70 autoantistoffer. Men disse autoantistoffer, når der findes i isolation, er ikke forbundet med systemiske autoimmune gigtsygdomme 4 -6. Disse antistoffer er fremherskende hos raske personer og patienter, der bærer sygdomme relateret til SARD 6. Bekræftende test for anti-DFS70 antistoffer kan hjælpe med at reducere unødvendige refleks test, tilbyder betydelige omkostningsbesparelser, og lette patientens angst. Da DFS mønster er vanskeligt at identificere og ANA positivitet kan forårsage angst for både patienter og læger, er det stærkt anbefales at udføre en bekræftende test efter identifi<html> velse af et mønster, der tyder på anti-DFS70 antistoffer til at klarlægge den kliniske betydning af ANA positivty 5,6. I dette mønster mitotiske celler (figur 5, orange pil) viser positiv plettet farvning af metafase chromatin mens hvilende cellekerner (fig. 5, hvid pil) udviser ensartet fordelte fine prikker hele kernen. Centromer mønster At identificere centromeren mønster, scan brøndene og identificere mitotiske eller delende celler. I mitotiske celler (figur 6, orange pil), er disse diskrete prikker blevet tæt knyttet i det, der ofte beskrives som en &quot;metafase bar&quot;. I centromeren mønster, hvilende celler (figur 6, hvid pil) viser ca 40-60 diskrete prikker fordelt over hele kerner. Centromer mønster er et resultat af anti-CENP A, B, C antistoffer 13. skridt &quot;&gt; nucleolar Mønster Nogle nucleolar mønstre er forbundet med diffus cytoplasmatisk farvning i mitotiske celler (fig. 7, orange pil) og negative kromosomale område, medens andre nucleolear mønstre viser positiv farvning af det kromosomale region. Den nucleolar mønster er forbundet med homogene eller plettet farvning af nucleoli (fig. 7, hvid pil), sammen med svag plettet eller homogen farvning af nucleoplasm. Disse mønstre er forbundet med anti-RNA-polymerase III, anti-fibrillarin, anti-Th/To og anti-PM/Scl antistoffer 14,15. Nuklear prikmønster Det nukleare prik mønster er forbundet med negative metafase mitotiske celler (fig. 8, orange pil) og med få diskrete prikker i hvilende cellekerner (fig. 8, hvid pil). Denne karakteristisk mønster er ofte et resultat af sp-100, PML eller p80 Colin autoantistoffer. Disse antistoffer er som<html> som knyttes med primær biliær cirrose og autoimmun hepatitis 16.. <img alt="Figur 1" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig1highres.jpg" width="500" /> . Figur 1. Negativ kontrol (venstre): Celler vist lavt niveau af ikke-specifik fluorescens, men ingen specifik nuklear farvning Positiv kontrol (højre):. Celler vise æblegrøn nuklear fluorescens. <img alt="Figur 2" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig2highres.jpg" width="500" /> Figur 2. Homogen mønster. Mitotiske celler (orangearrow) viser solid fluorescens. Hvilende celler viser endda, diffus farvning (whitearrow). &quot;Width =&quot; 500 &quot;/&gt; Figur 3.. Grov plettet mønster. Mitotiske celler (orange pil) viser negativ farvning. Hvilende celler udviser et klart plettet farvning (hvid pil). <img alt="Figur 4" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig4highres.jpg" width="500" /> Figur 4.. Fin plettet mønster. Mitotiske celler (orange pil) viser negativ farvning. Hvilende celler udviser et klart fin plettet farvning (hvid pil). <img alt="Figur 5" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig5highres.jpg" width="500" /> Figur 5.. Tætte fint spættede mønster. Mitotiske celler (orange pil) viser et fint granuleret solid farvning. Hvilende celler viser en meget fin, diffus plettet farvning (whitearrow). Figur 6.. Centromer mønster. Mitotiske celler (orangearrow) viser uniform, diskrete prikker. Hvilende celler (whitearrow) viser 40-60 diskrete prikker pr cellekernen. <img alt="Figur 7" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig7highres.jpg" width="500" /> Figur 7. Nucleolar mønster. Mitotiske celler (orangearrow) vises som store klynger af granulær farvning. Resting celle nucleishows fluorescens i nucleoli (whitearrow). <img alt="Figur 8" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig8highres.jpg" width="500" /> Figur 8. Nuklear punktmønster. Mitotiske celler (orangearrow) fremgår negativ. Hvilende celler viser et par Discrete prikkerne i kernen (whitearrow). Derudover kan nucelar punktmønster sameksistere med cytoplasmatisk farvning er forbundet med autoantistoffer til mitokondrielle antigener (rød pil).

Watch this Scientific Journal Video about Anti-Nuclear Antibody Screening Using HEp-2 Cells at JoVE.com

Anti-Nuclear Antibody Screening Using HEp-2 Cells

Indirekte immunofluorescens (IIF) assays er traditionelt blevet anvendt til påvisning af antinukleare antistoffer (ANA) i humant serum. Tilstedeværelsen af ​​disse antistoffer kan støtte i diagnosticering af systemiske autoimmune reumatiske sygdomme (SARD). Denne protokol viser, hvordan man effektivt udfører IIF teknik til præcist at opdage disse autoantistoffer.

Anti-nukleare Antibody Screening Brug HEp-2-celler

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

anti-nuclear-antibody-screening-using-hep-2-cells

Research

JoVE Journal

Biology

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

MISSION LentiPlex Pooled shRNA Bibliotek Screening i pattedyrceller

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

Omdannelse af en ELISA til en Luminex xMAP assay under anvendelse af en multiplex antistof screeningsmetode

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

High-throughput Funktionel Screening ved hjælp af en hjemmelavet Dual-glød Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induktion og analyse af Epithelial til Mesenchymale Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Antistoffarvning i<em&gt; C. Elegans</em&gt; Brug af &quot;Freeze-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

High-throughput screening for kemiske modulatorer af post-transkriptionelt regulerede gener

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identifikation af kinase-substrat Pairs Brug højkapacitetsscreening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Multi-enzym Screening Ved hjælp af en High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...