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 for å utføre IIF eksperiment og Carol Buchner for hennes ekspert teknisk gjennomgang.

En. Prøveforberedelse og Underlags Selection <ol><li> Fjern reagenser fra emballasjen og la hvert element for å komme til romtemperatur. Forbered reagenser og fortynn pasientsera i henhold til retningen innsatsen. Merk: NOVA Lite lysbilder er strekkode og kan enkelt integreres i automatiserte systemer i forbindelse med Laboratory Information System LIS. Denne prosedyren illustrerer manuell lysbilde behandlingen; høy gjennomstrømning laboratorier, kan imidlertid velge automatisert lysbilde prosessutstyr. </li></ol> 2. Tilsetting av kontroller og prøver <ol><li> Tilsett en dråpe positiv kontroll og en dråpe negativ kontroll til de riktige glide brønner. Pipetter 20 til 25 pl av fortynnede pasientserum til de gjenværende brønnene. Behandle ett lysbilde om gangen. </li><li> Plasser glideren (r) i en fargingsbeholder med en fuktig papirhåndkle på bunnen. Dekk beholderen og inkuber glideren (e) for 30 min. Merk: Den fuktige forhold vilhindre underlaget tørker ut. Tørking av brønnene kan resultere i kunstig farging. I løpet av denne inkubasjonsperiode blir den anti-nukleære antistoffer i pasientens serum bindes til antigen på cellene som er festet på hver brønn. </li><li> Etter inkubasjonsperioden, skyll av serum ved hjelp av en svak strøm av vaskebuffer. For å unngå skader på underlaget og hindre at cross-over av prøver mellom brønnene, vinkel glide litt for å unngå å rette strømmen direkte til brønnene. Tapp av overflødig vaskebuffer og plasser lysbilde i en Coplin krukke inneholder vaskebuffer i ca 5 min. </li></ol> Tre. Tilsetting av Fluorescent konjugat <ol><li> En etter en, fjerner du lysbildene fra vaskebuffer og banke forsiktig for å fjerne overflødig vaskebuffer. Påfør en dråpe av fluorescerende konjugat (FITC-merket anti-humant IgG) på hver brønn. Inkuber lysbilder for 30 min i befuktet container, og sørg for å erstatte flekkerbeholderlokket. Merk: For ANA testing, er bruken av en IgG Fc-konjugatet spesifikt anbefalt. Konjugatet er lysfølsom, og dekselet vil beskytte skinnene mot eksponering for lys i tillegg til å opprettholde den befuktet miljø. I løpet av denne inkubasjonsperiode, blir konjugatet bindes til pasientens anti-nukleære antistoffer som er bundet til celleantigener. Dette konjugat bindingsresultater i nærvær av fluorescens i brønnene. </li><li> Skyll og vaske lysbildet som beskrevet ovenfor. </li><li> Plasser et dekkglass på et papirhåndkle og gjelder monteringsmedium i en kontinuerlig linje til nedre kant av dekk. </li><li> Fjern hvert lysbilde fra vaskebuffer og trykk raset forsiktig for å fjerne overflødig vaskebuffer. Trykk på nedre kant av lysbildet til kanten av dekkglasset. </li><li> Senk lysbildet på dekkglass på en slik måte at monteringsmedium på dekkglass strømmer til den øvre kant av side uten luftbobledannelse. Merk: Coverslipping, inkludert bruk av den optimale mengden av montering medium, er en teknikk som krever øvelse for å perfeksjonere. </li></ol> 4. Identifikasjon av positive og negative resultater Manuell tolkning <ol><li> Vis lysbilder med et fluorescerende mikroskop, som ligger i et mørkt rom. Utfør skanning av hele godt med en 20X eller 25X mål å vurdere cellefordeling og ensartethet av fluorescens </li><li> Bytt til en 40X objektiv for å gjøre den endelige tolkningen om positivitet og mønster. </li><li> Se på de positive og negative kontroller. Grad positivitet ved hjelp av en reaktivitet karakterskala fra 1 + til 4 +. Merk: Den negative kontrollen vises kanskje ikke helt mørkt, men vil ofte vise lavt nivå spesifikk fluorescens. Den positive kontrollen viser lyse eple grønn fluorescens i kjernen (figur 1). </li></ol> Automatisert tolkning Merk: I tilvidere til manuell tolkning, last-og skanne lysbilder ved NOVA Vis Automated fluorescens mikroskop eller andre automatiserte digital bildebehandling instrument; ingen mørkerom er nødvendig. Når du har opprettet et prosjekt ved å velge den aktuelle lysbildet type, kjøper instrumentet og lagrer høyoppløselige digitale bilder av celler i hver brønn. I tillegg, NOVA Vis måler fluorescerende lys intensitet, og kategoriserer resultatene som positive eller negative, og gir mønstergjenkjenning for positive prøver. Bilder blir sett fra brukeren på en datamaskin med høy oppløsning på skjermen, slik at for endelige tolkningen, revisjon og bekreftelse av NOVA Vis resultat. Rapporter kan genereres på bekreftede resultater. Digital mikros skal brukes i forbindelse med en erfaren laboratorietekniker som det er beregnet på å hjelpe til med tolkning av resultater.

<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 ansatte 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.

Betegnelsen antinukleære antistoffer (ANA) beskriver en rekke av auto-antistoffer som reagerer med bestanddeler av cellekjerner, inkludert DNA, proteiner og ribonucleoproteins 1, 2. Hep-2 celle, en innfødt protein array med hundrevis av antigener, gir et ideelt underlag for påvisning av ANA en. Påvisning av ANA i humant serum er et viktig verktøy for screening bindevevssykdommer, og IIF er referansemetoden for testing av en ANA. Nylig, IIF på HEp-2-celler er blitt erstattet i noen laboratorier med antigen-spesifikke immunanalyser og multipleks-metoder. På grunn av bekymringer over falske negative resultater og mangel på åpenhet for klinikere, American College of Rheumatology dannet en arbeidsgruppe som konkluderte med at IIF bruker HEp-2 celler bør være &quot;gullstandarden&quot; for ANA screening en. På grunn av den subjektive natur ANA screening, er kvaliteten på HEp-2-celler itegral til nøyaktig og trygg rapportering av resultater. Tilstedeværelse av et høyt antall av mitotiske celler, optimal cellemorfologi, tilstrekkelig til konfluens, og ekspresjon av relevante antigener er spesielt viktige. IIF mønstergjenkjenning fungerer som et viktig verktøy for å hjelpe til pasientens diagnose. Forstå betydningen av ulike mønstre gjør at klinikere og laboratoriepersonell til å utføre den aktuelle oppfølging testing for å bekrefte diagnosen. For eksempel kan homogene ANA mønster forekommer i nærvær av anti-dsDNA-eller kromatin-antistoffer, og kan være assosiert med systemisk lupus erythematosus (SLE) 3. Fra den andre siden, har det nylig blitt beskrevet at den såkalte tette fin flekkete mønster (DFS) som vanligvis ses hos opptil 12% av rutineprøver, for det meste blitt assosiert med anti DFS70 antistoffer. Disse autoantistoffer, da funnet i isolasjon (uten andre sykdomsspesifikke ANA) er ikke assosiert med systemiske autoimmune revmatiske sykdommer &lt;sup&gt; 4-9. Dermed bekreftende testing for anti-DFS70 antistoffer kan bidra til å redusere unødvendig refleks testing, gir store kostnadsbesparelser, og lette pasientens angst. Gitt at IIF er den første linjen screening metode for å påvise antistoffer, er det viktig at brukeren forstår hvordan valg av reagenser og vev underlag kan påvirke resultatene. Siden IIF teknikken er i seg selv subjektivt, er det viktig at reagensene som benyttes gi den høyeste kvalitet. Dette målet av denne videoen protokollen er å sette brukeren med de riktige trinnene for å utføre IIF metoden, de vanlige mønstre knyttet til ANA, og å innføre nye fremskritt som kan effektivisere laboratoriet arbeidsflyt og standardisere resultatene.

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anti-nuclear antibody screening using hep-2 cells

The American College of Rheumatology standpunkt på ANA testing fastsetter bruk av IIF som gullstandarden metode for ANA screening en. Selv IIF er en utmerket screening test på ekspert hender, de tekniske vanskeligheter for behandling og lese IIF glir - slik som arbeidskrevende glide behandling, manuell avlesning, behovet for erfarne, trent teknologer og bruk av mørkt rom - å gjøre det IIF metoden vanskelig å få plass i arbeidsflyten av moderne, automatiserte laboratorier. Den første og avgjørende skritt mot høy kvalitet ANA screening er forsiktig lysbilde behandling. Denne fremgangsmåte er arbeidskrevende, og krever full forståelse av prosessen, så vel som hensyn til detaljer og erfaring. Bilde lesing er utført av fluoriserende mikros i mørke rom, og er gjort av kvalifiserte teknologer som er kjent med de ulike mønstre, i sammenheng med cellesyklusog morfologi av interfase og delende celler. Forutsatt at IIF er den første linjen screening verktøy for SARD, forstå fremgangsmåten for å riktig utføre denne teknikken er kritisk. Nylig, har digitale bildebehandlingssystemer blitt utviklet for automatisk avlesning av IIF lysbilder. Disse systemene, som for eksempel NOVA Vis Automated fluorescerende mikroskop, er utviklet for å effektivisere rutine IIF arbeidsflyt. NOVA Vis kjøper og lagrer høyoppløselige digitale bilder av brønnene, og dermed skille image oppkjøpet fra tolkning; Bildene viste en tolket på høyoppløselige dataskjermer. Den lagrer bilder for fremtidig referanse og støtter operatørens tolkning ved å gi fluorescerende lys intensitet data på bildene. Det er også foreløpig kategoriserer resultatene som positive eller negative, og gir mønstergjenkjenning for positive prøver. I sammendraget, eliminerer behovet for mørkerom, og automatiserer og effektiviserer IIF reading / tolkning arbeidsflyt. Viktigst, øker det konsistens mellom lesere og opplesninger. Dessuten, med bruk av strekkode slides, blir transkripsjon feil elimineres ved å gi prøve sporbarhet og positiv pasientidentifikasjon. Dette resulterer i økt pasientdataintegritet og sikkerhet. Det overordnede målet med denne videoen er å vise IIF prosedyren, inkludert lysbilde behandling, identifisering av felles IIF mønstre, og innføring av nye fremskritt for å forenkle og harmonisere denne teknikken.

Når man skal vurdere cellefarging resultater for HEp-2, fem store atom mønstre er hyppigst rapportert: homogen, flekkete centro, nukleolært, og kjernefysiske prikker. Disse mønstrene er et resultat av autoantistoff binding til spesifikke bestanddeler av kjernen. Selv om ANA-testing er spesifikke for kjernefysiske strukturer, kan cytoplasmatiske mønstre forbundet med autoantistoffer mot cytoplasmatiske strukturer også observeres. I noen tilfeller kan flere autoantistoffer være til stede i en prøve, og bildet kan vises som et blandingsmønster. Det er andre, mindre hyppig observert, og ofte cellesyklus bestemte mønstre som kan oppdages av IIF-metoden; nylig, har en såkalt tett fin flekkete mønster med spesiell klinisk betydning blitt beskrevet i noen ANA positive sera. Homogen Mønster For å identifisere en homogen mønster, skanne godt og identifisere mitotic eller delelinjeceller. Den kondenserte kromatin av mitotiske celler (figur 2, orange pil) oppviser solid, ensartet fluorescens, som ofte er mer markert enn i hvilecelle nuclei.In den homogene mønster, den hvilende celler utstillings uniform, diffus fluorescens av hele kjernen (figur 2, hvite pilen). Dette karakteristiske mønster er ofte et resultat av anti-dsDNA-antistoffer 10. Flekkete mønster I grovspettet mønster, de mitotiske celler viser ingen farging av de kondenserte kromosomale regioner (figur 3, orange pil). De hvilende celler oppviser granulær fluorescens gjennom hele kjernen. Atom speckling kan defineres som grovt eller fint. Den grove speckling mønsteret er ofte et resultat av anti-Sm og anti-RNP 11.. I den fine flekkete mønster, hviler cellene vise fine eller diffuse speckling hele kjerner, typisk i uniform distribution (Figur 4, hvit pil). Den fine flekkete mønsteret er ofte et resultat av anti-SSAand anti-SSB 12.. Tett Fin Speckled Mønster Det er nå anerkjent som den tette fin flekkete mønster (DFS) er vanlig i rutinemessig testing, og så mye som 12% av prøvene er positive for anti-DFS70 autoantistoffer. Men disse autoantistoffer, da funnet isolert, ikke er forbundet med systemiske autoimmune revmatiske sykdommer 4. -6. Disse antistoffene er utbredt hos friske individer og pasienter som bærer sykdommer relatert til SARD seks. Bekreftende testing for anti-DFS70 antistoffer kan bidra til å redusere unødvendig refleks testing, gir store kostnadsbesparelser, og lette pasientens angst. Siden DFS mønsteret er vanskelig å identifisere og ANA positivitet kan føre til angst for både pasienter og leger, er det sterkt anbefalt å utføre en bekreftende test etter identi<html> fying et mønster som tyder på anti-DFS70 antistoffer for å avklare den kliniske betydningen av ANA positivty 5,6. I dette mønsteret, mitotiske celler (figur 5, oransje pil) viser positiv flekkete farging av meta kromatin, mens du hviler cellekjerner (figur 5, hvit pil) utstillingen jevnt fordelt fin flekker i hele kjernen. Centro Mønster Å identifisere cent mønster, skanne brønnene og identifisere mitotic eller dele celler. I mitotiske celler (figur 6, oransje pil), disse diskrete flekker bli nært forbundet i det som ofte beskrives som en &quot;meta bar&quot;. I cent mønster, hvilende celler (figur 6, hvit pil) viser ca 40-60 diskrete flekker fordelt i atomkjerner. Den cent mønsteret er et resultat av anti-CENP A, B, antistoffer C 13. trinn &quot;&gt; nucleolar Pattern Noen nucleolar mønstre er assosiert med diffus cytoplasmisk farging i mitotiske celler (figur 7, orange pil) og negative kromosomale region, mens andre nucleolear mønstre viser positiv farging av det kromosomale regionen. Den nukleolært mønster er forbundet med homogen eller flekkete farging av nucleoli (figur 7, hvite pilen), sammen med svak spettet eller homogen farging av nukleoplasma. Disse mønstrene er assosiert med anti-RNA polymerase III, anti-fibrillarin, anti-Th/To og anti-PM/Scl antistoffer 14,15. Nuclear Dot Mønster Atompunktmønster er forbundet med negativ metafase mitotiske celler (figur 8, orange pil), og med noen få adskilte flekker i hvilecellekjerner (figur 8, hvit pil). Denne karakteristiske mønsteret er ofte et resultat av sp-100, PML, eller P80 Colin autoantistoffer. Disse antistoffene er som<html> assosieres med primær biliær cirrhose og autoimmun hepatitt 16. <img alt="Figur 1" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig1highres.jpg" width="500" /> . Figur 1. Negativ kontroll (venstre): Celler vise lav ikke-spesifikk fluorescens, men ingen spesifikk nukleær farging Positiv kontroll (til høyre). Celler viser eplegrønn atom fluorescens. <img alt="Fig. 2" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig2highres.jpg" width="500" /> Figur 2. Homogenous mønster. Mitotiske celler (orangearrow) viser solid fluorescens. Resting cellene vise enda, diffuse flekker (whitearrow). &quot;Width =&quot; 500 &quot;/&gt; Figur 3. Grovspettet mønster. Mitotiske celler (oransje pil) viser negativ farging. Hvilende cellene vise en tydelig flekkete flekk (hvit pil). <img alt="Figur 4" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig4highres.jpg" width="500" /> Figur 4 Fin flekkete mønster. Mitotiske celler (oransje pil). Viser negativ farging. Hvilende cellene vise en tydelig fin flekkete farging (hvit pil). <img alt="Figur 5" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig5highres.jpg" width="500" /> Figur 5. Tett fin flekkete mønster. Mitotiske celler (oransje pil) viser en fin kornet solid farging. Hvilende cellene vise en veldig fin, diffus flekkete flekken (whitearrow). Figur 6. Centro mønster. Mitotiske celler (orangearrow) viser uniform, diskrete flekker. Hvile celler (whitearrow) viser 40-60 diskrete flekker per cellekjernen. <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 av kornet farging. Hvile 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. Nuclear prikkmønster. Mitotiske celler (orangearrow) vises negativ. Hvilende cellene vise noen discrete flekker i kjernen (whitearrow). I tillegg er nucelar prikkmønster kan sameksistere med farging i cytoplasma forbundet med autoantistoffer mot mitokondrie 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 immunfluorescens (IIF) assays har tradisjonelt blitt brukt for påvisning av antinukleære antistoffer (ANA) i humant serum. Tilstedeværelsen av disse antistoffer kan hjelpe til ved diagnostisering av systemiske autoimmune revmatiske sykdommer (SArd). Denne protokollen demonstrerer hvordan du effektivt utføre IIF teknikk for å oppdage nøyaktig disse autoantistoffer.

Anti-Nuclear antistoff screening med 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 Samlede shRNA Library 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.

Konvertering av en Capture ELISA til en Luminex xMAP Assay bruker en 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 ...

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.

Høy gjennomstrømming Funksjonell Screening ved hjelp av en hjemmelaget 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.

Induksjon og analyse av Epiteliale 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.

Antistoffarging i<em&gt; C. Elegans</em&gt; Ved hjelp av &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.

Høy gjennomstrømming Screening for kjemi modulatorer av Post-transcriptionally Regulerte 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.

Identifisering av kinase-substrat Pairs Bruke Høy throughput screening


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-enzymet Screening Ved hjelp av en High-throughput Genetisk 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 ...