Name: antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae
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Denne undersøgelse blev finansieret af det tunesiske sundhedsministerium og det tunesiske ministerium for videregående uddannelse og videnskabelig forskning.

Denne undersøgelse er udført i overensstemmelse med etiske aspekter fastlagt af det etiske udvalg ved Pasteur-instituttet i Tunis.
1. Trin før ELISA: Forudsætninger og forbehandling
<ol><li>Bakteriestammer og vækstmedier BEMÆRK: Mollicutes-arter og de murede bakterier, der anvendes i denne undersøgelse, og deres vækstmedier er anført i tabel 1.<ol><li>Vækst af Mollicutes arter<ol><li>Der podes 200 μL fra glycerolstammen af hver art i 1 800 μL ...

<ol>
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Dette dokument indeholder en generel beskrivelse af en intern ELISA, der hovedsagelig er udviklet for at sikre specifik screening af M. pneumoniae Infektion. Detaljerne om protokollen for selve ELISA-analysen samt nogle for- og efterbehandlingstrin er angivet. Specificiteten af dette assay sikres ved adsorptionsteknikken. Denne procedure er tidligere beskrevet i ELISA-test udviklet til diagnosticering af mennesker og fugle Mycoplasmas17,24. Det ...

Mycoplasmer er blandt de mindste og enkleste kendte prokaryoter. De adskiller sig hovedsageligt fra andre bakterier ved manglen på en cellevægsstruktur. Således blev mycoplasmer klassificeret i en separat klasse ved navn Mollicutes1. Cellevægsmanglen giver iboende resistens over for disse mikroorganismer mod nogle antimikrobielle midler og er i høj grad ansvarlig for deres polymorfisme. Mycoplasmer har lille genom og reduceret størrelse, hvilket begrænser deres metaboliske og biosyntetiske evner og forklarer deres parasitære og saprofytiske natur1.
Mycoplasma pneumoniae er en af de Mycoplasmer, der inficerer mennesket og menes at være den mest virulente2. M. pneumoniae koloniserer de øvre luftveje, hvilket fører til atypisk lungebetændelse hos børn og unge voksne. De kliniske tegn fremkaldt af M. pneumoniae infektion er influenzalignende, med hovedpine, feber og hoste3. Cytadherensen af M. pneumoniae til værtsceller medieres af en vedhæftningsorganel, herunder P1 større adhæsion og flere tilbehørsproteiner 4,5. Flere kliniske manifestationer kan forekomme på grund af lokal inflammation og stimulering af værtsimmunsystemet som følge af den intime vedhæftning af M. pneumoniae til luftvejsslimhinden6. Selvom lungebetændelse er et kendetegn ved M. pneumoniae-infektion, er det blevet afsløret, at infektion med denne bakterie også kan være ansvarlig for et bredt spektrum af ikke-pulmonale manifestationer på forskellige anatomiske steder såsom centralnervesystemet, hjerte, hud og led7.
Som for alle Mycoplasma-arter er diagnosen M. pneumoniae udfordrende. De kliniske tegn, der fremkalder mycoplasmosis, er for det meste usynlige og ikke-karakteristiske8. Da det er meget svært at diagnosticere M. pneumoniae-infektion ved kun at stole på kliniske manifestationer og symptomer, er laboratoriescreening af særlig interesse9. Påvisning af M. pneumoniae-kolonier ved kultur er guldstandardmetoden til en korrekt diagnose. Imidlertid komplicerer de kræsne vækstkrav og den lange tid, der er nødvendig for levering af endelige resultater (1-2 uger), kulturen og betyder således, at den sjældent bruges til rutinemæssig diagnose10. Nukleinsyreamplifikationsteknologier blev valideret med hensyn til hastighed og effektivitet, selvom disse molekylære teknikker på grund af deres relativt høje omkostninger og utilgængelighed i nogle sundhedsfaciliteter ikke betragtes som diagnostiske førstelinjetest. Det er rigtigt, at kommercielle PCR-tests i vid udstrækning anvendes til at diagnosticere M. pneumoniae-infektioner, men de kan stadig ikke erstatte serologi. Også den hyppige forekomst af både falsk negative og falsk positive resultater har begrænset brugen af PCR9. Rutinemæssigt forbliver serologi den mest praktiserede i laboratorier til diagnosticering af M. pneumoniae-infektion. Flere serologiske tilgange er blevet rapporteret i årtier, såsom kolde hæmagglutininer, komplementfikseringstest11, indirekte hæmagglutinationstest 12, immunofluorescens 13 og ELISA-teknologien, som først blev anvendt til mycoplasma-serologi i begyndelsen af 1980'erne14,15,16. Et af de største problemer, der opstår ved udførelse af ELISA-serodiagnose af M. pneumoniae-infektion, er krydsreaktioner, hvilket reducerer teknikkens specificitet betydeligt. Uspecifik adsorption af humane sera med M. pneumoniae-antigener er tidligere rapporteret; Faktisk er mange af de antistoffer, der påvises af ELISA i humane sera, muligvis ikke altid bundet til mycoplasmalantigener 17 på grund af M. pneumoniaes fællestræk med nogle bakterier18,19 og nogle animalske og humane væv20.
På grund af de høje baggrundsaflæsninger, der blev observeret i den konventionelle ELISA-test, der blev praktiseret i laboratoriet, var fortolkningen af resultaterne ofte kompliceret, og derfor var leveringen af en ordentlig M. pneumoniae-diagnose en hård opgave. Mens vi stod over for dette problem, valgte vi at forbedre M. pneumoniae ELISA ved at fjerne uspecifikke reaktioner af M. pneumoniae-antigener med antistoffer, der skal testes. Til dette formål arbejdede vi på selektiv udtømning af de uspecifikke M. pneumoniae-antigener ved hjælp af adsorptionsteknikken. Faktisk er hovedmålet med antigen-capture ELISA specifikt at detektere M. pneumoniae immunoglobulin (Ig) G i humane serumprøver. Konceptet med denne ELISA består hovedsagelig af selektiv indfangning af M. pneumoniae-specifikke antigener, før de humane serumprøver tilsættes. Denne selektivitet sikres ved at inkubere M. pneumoniae råantigen med et M. pneumoniae polyklonalt antiserum, der produceres hos kaniner i laboratoriet, og gøre det artsspecifikt ved adsorption mod et panel af heterologe bakterier, der tilhører eller ikke tilhører Mollicutes-klassen, og som deler antigener med M . pneumoniae arter og / eller kendt for at kolonisere luftvejene. Adsorptionsproceduren blev gentaget tre gange, og dens effektivitet til eliminering af krydsreaktivitet blev testet ved immunoblotting. Det udviklede ELISA-assay er en kombination af sandwich og indirekte ELISA. Kort fortalt er brøndene i ELISA-pladen først belagt med et polyklonalt antiserum, der er specifikt for M. pneumoniae. Derefter tilsættes M. pneumoniae-antigen og fanges mellem antiserumet og antistofferne i serumprøven, der skal testes. Det dannede immunologiske kompleks detekteres af et sekundært enzymkonjugeret antistof (peroxidasekonjugeret IgG). Reaktionerne visualiseres ved tilsætning af kromogent substrat, og absorbansen måles spektrophotometrisk. Denne interne ELISA er skematisk præsenteret i figur 1. Den hjemmelavede ELISA viste sig at være effektiv til specifikt at detektere M. pneumoniae-infektion og er i øjeblikket en af de mest praktiserede tests i rutinemæssig diagnostisk aktivitet.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-chloro-1-naphtol</td>
			<td>Sigma-Aldrich</td>
			<td>C6788-50 TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-50 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate bicarbonate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>C3041-50 Cap</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL1066&#160;</td>
			<td>VWR</td>
			<td>P0058-N1L</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco PPLO Broth</td>
			<td>BD</td>
			<td>255420</td>
			<td>Frey media</td>
		</tr>
		<tr>
			<td>ELISA plate-Reader</td>
			<td>MULTISKAN GO, Thermo Scientific</td>
			<td>Ref: 51119200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Capricorn Scientific</td>
			<td>FBS-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-human IgG</td>
			<td>Abcam</td>
			<td>ab6759</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>656120</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid 37%</td>
			<td>Prolab</td>
			<td>2025.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2), solution 30%</td>
			<td>Scharlau</td>
			<td>HI01361000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-arginin</td>
			<td>Sigma-Aldrich</td>
			<td>A5006-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Prepared in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Provided by the laboratory of bacteriology of the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Mycoplasma pneumoniae polyclonal antiserum produced in rabbit</td>
			<td>Produced in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Serum was produced by rabbit immunization at the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Nicotinamide adenine dinucleotide</td>
			<td>Sigma-Aldrich</td>
			<td>N7004-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Maxisorp flat-bottom 96-well microtiter plate&#160;</td>
			<td>Invitrogen</td>
			<td>44-2404-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium (1 MIU)</td>
			<td>PANPHARMA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>fluka chemika</td>
			<td>77660</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>MP Biomedicals</td>
			<td>902887</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Novachim</td>
			<td>PS02805</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid (H2SO4) 95-97%</td>
			<td>Merck</td>
			<td>1007311011</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>USB</td>
			<td>22215</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB substrate (3,3&#8217;, 5,5&#8217; TetraMethyl-Benzidine solution)</td>
			<td>Abcam</td>
			<td>ab142042</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T6791-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>1379-500 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract, powder, Ultrapure</td>
			<td>Thermo Scientific&#160;</td>
			<td>J23547&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae

Mycoplasma pneumoniae er en prokaryot med cellevægsmangel, der hovedsageligt er kendt for at kolonisere det menneskelige luftveje og være endemisk, med epidemiske toppe hvert 6. år hos ældre børn og unge voksne. Diagnose af M. pneumoniae er udfordrende på grund af patogenens kræsne natur og muligheden for asymptomatisk transport. Laboratoriediagnose af M. pneumoniae-infektion baseret på antistoftitrering i serumprøver af patienter forbliver den mest praktiserede metode. På grund af det potentielle problem med immunologisk krydsreaktivitet ved anvendelse af polyklonalt serum til M. pneumoniae er der udviklet et antigenindfangningsenzymbundet immunosorbentassay (ELISA) for at forbedre specificiteten af serologisk diagnose. ELISA-plader er belagt med M. pneumoniae polyklonale antistoffer, hævet hos kaniner og gjort specifikke efter adsorption mod et panel af heterologe bakterier, der deler antigener med M. pneumoniae-arter og / eller vides at kolonisere luftvejene. De reagerede M. pneumoniae homologe antigener genkendes derefter specifikt af deres tilsvarende antistoffer i serumprøverne. Yderligere optimering af de fysisk-kemiske parametre, som antigenindfangnings-ELISA'en udsættes for, førte til en meget specifik, følsom og reproducerbar ELISA.

Immunoblottingaktiviteten af det ikke-adsorberede polyklonale Mycoplasma pneumoniae-antiserum mod heterologe bakterier Krydsreaktivitet eksisterer faktisk som vist i immunoblotting-resultaterne (figur 2), og sammenlignet med den positive kontrol (bane 1) deles nogle af M. pneumoniae-antigenerne med de screenede bakterier. Intensiteten af disse krydsreaktioner varierede. For eksempel viste M. gallisepticum og M....

Watch this Scientific Journal Video about Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae at JoVE.com

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

I Mycoplasma pneumoniae infektion, serologi test kan generere gode resultater, men med lav specificitet på grund af immunologisk krydsreaktion. Den interne antigen-capture ELISA, beskrevet i dette papir, garanterer høj artsspecificitet og har vist sig at være en pålidelig screeningstest til nøjagtig diagnose af M. pneumoniae.

Antigen-capture enzym-linked immunosorbent assay til specifik påvisning af mycoplasma pneumoniae

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks ...

Som for alle mycoplasms patienter er deres diagnose af Mycoplasma pneumoniae udfordrende på grund af krydsreaktioner. Vores foreslåede ELISA er baseret på selektiv udtømning af ikke-specifikke Mycoplasma pneumoniae-antigener ved hjælp af en adsorptionsteknik. Den interne antigenoptagelses-ELISA, der er beskrevet i denne video, garanterer høj rummelig specificitet.Og faktisk har det vist sig at være en pålidelig screenet test for en nøjagtig diagnose af Mycoplasma pneumoniae infektion. Demonstrere præ-ELISA trin vil være Imen Chniba, en ph.d.-studerende. Og ELISA-trinene vil være Nadine Khadraoui, en tekniker fra vores laboratorium.For at indlede antigenadsorptionsproceduren trækkes de 12 heterologe bakterieantigener ind i et mikrocentrifugerør, og proteinkoncentrationen svarende til halvdelen af det antiserum, der skal adsorberes, justeres. Derefter centrifugeres antigenblandingen ved 14.000 G i 10 minutter ved fire grader Celsius for at opsamle pelleten. Derefter suspenderes pelleten igen med den rensede polyklonale anti-Mycoplasma pneumoniae IgG og inkuberes i to timer ved 37 grader Celsius med langsom omrøring.Ved inkubationens afslutning centrifugeres suspensionen igen ved 14.000 G i 10 minutter ved fire grader Celsius og genvinder supernatanten svarende til det specifikke polyklonale Mycoplasma pneumoniae antiserum. For at belægge en ELISA-plade med 96 brønde med et indfangningsantistof fortyndes antistoffet til 10 mikrogram pr. Milliliter og 0,1 molær carbonatbicarbonatbuffer ved pH 9,6. Derefter tilsættes 100 mikroliter af det fortyndede antistof til hver brønd på mikropladen og inkuberes natten over ved fire grader Celsius.Den følgende dag fjernes belægningsopløsningen og vaskes pladen fem gange med vaskebufferen. Bloker derefter de ubundne overflader af de belagte brønde ved at tilsætte 100 mikroliter af blokeringsopløsningen til hver brønd. Efter inkubation af pladen fra en time ved stuetemperatur fjernes blokeringsopløsningen med en pipette, og pladen vaskes som tidligere vist.Tilsæt en lige stor mængde Mycoplasma pneumoniae hele proteiner i en koncentration på 10 nanogram pr. Brønd, og lad antigen-antistofreaktionen finde sted i to timer ved stuetemperatur. Efter inkubation fjernes det overskydende antigen, og pladen vaskes som vist. Derefter fortyndes de humane serumtestprøver og henvises til positive og negative serumkontroller.Derefter tilsættes 100 mikroliter af de fortyndede testprøver og kontroller i de relevante brønde og duplikater. Hullerne, der hverken indeholder antigen eller sera, markeres som blinde. Efter inkubation af pladen i 90 minutter ved stuetemperatur fjernes serumopløsningen, og pladen vaskes fem gange med vaskebufferen.Der pipetteres 100 mikroliter af de passende konjugerede detektionsantistoffer for fortyndet enzym til hvert hul og inkuberes i en time ved 37 grader Celsius i mørke. Efter fjernelse af de ubundne detektionslegemer og vask af pladen tilsættes 100 mikroliter TMB Chromogen-opløsning for at visualisere antigen-antistofbindingen. Lad pladen udvikle sig i 30 minutter ved stuetemperatur.Stop derefter reaktionen ved at tilsætte 100 mikroliter 7,5% svovlsyre. Endelig aflæses absorbansen ved 450 nanometer ved hjælp af en mikropladelæser og sorteres resultaterne baseret på beregningen af positivitetsindekset. Immunoblot-analyse mellem Mycoplasma pneumoniae ikke-adsorberet polyklonalt antiserum og heterologe bakterielle antigener afslørede krydsreaktivitet, men med varierende intensitet.For eksempel viste antigenerne Mycoplasma gallisepticum og Mycoplasma imitans den stærkeste reaktivitet på trods af at de var aviære mycoplasmer. I modsætning hertil viste ingen af de to humane kliniske isolater af Ureaplasma urealyticum nogen reaktion. Imidlertid gav antigener fra de resterende humane genitale mycoplasmaarter og andre bakterielle antigener betydelige krydsreaktioner med antiserumet.Immunoblot-analyse bekræftede effektiviteten af Mycoplasma pneumoniae polyklonal antiserumadsorption mod de heterologe bakterielle antigener. Adsorptionsproceduren eliminerede alle krydsreaktioner, hvilket gjorde Mycoplasma pneumoniae-antiserummet specifikt for alle efterfølgende serologiske og immunblotting-test. Endvidere viste sættet af humane sera testet ved hjælp af antigenfangst-ELISA, der blev udviklet i denne undersøgelse, positivt for Mycoplasma pneumoniae IgG med god specificitet.Specificiteten af denne ELISA er hovedsageligt forsikret af adsorptionsteknikken, da det er primordial at udføre dette kritiske trin omhyggeligt og gentage det mindst to eller tre gange. Princippet om denne teknik kan anvendes til at diagnosticere andre mycoplasmaarter eller andre bakterier generelt. Udvælgelsen af bakterielle antigener til adsorption bør imidlertid være irrelevant og tilstrækkelig.

antigen-capture-enzyme-linked-immunosorbent-assay-for-specific

Research

JoVE Journal

Biology

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

En hurtig og konkret Microplate Assay til bestemmelse af intra-og ekstracellulære Ascorbat i dyrkede celler

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

En Multi-påvisningsassay for Malaria Fremsendende Myg

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Påvisning af Intracellulær genekspression i levende celler af murine, Human og porcin oprindelse anvendelse af fluorescens-mærkede Nanopartikler

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Parallel høj gennemløb enkelt molekyle kinetisk analyse for site-specifikke DNA spaltning

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.