Guide to Lateral Flow Immunoassays · 2018-04-06 · A guide to lateral flow immunoassays | 5 3.1...

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Guide to Lateral Flow Immunoassays

Innova Biosciences Guide

Lateral flow immunoassays

Components, process, assay formats and nanoparticles

A guide to lateral flow immunoassays | 2

Contents

1. Evolutionoflateralflowimmunoassays

2. Immunoassays

3. Lateralflowimmunoassays

3.1 Thesampleapplicationpad

3.2 Theconjugatereleasepad

3.3 Thedetectionreagent

3.3.1 Theantibody

3.3.2 Thedetectionmoiety

3.4 Themembrane

3.5 Thewickingpad

3.6 Theplasticcassette

4. Processoptions

5. Assayformats

6. Advantagesanddisadvantagesoflateralflowimmunoassays

7. Nanoparticlesforlateralflowimmunoassays

8. CustomservicesfromInnovaBiosciences


1. Evolutionoflateralflowimmunoassays

Lateralflowimmunoassaysareawell-establishedandextremelyversatiletechnologythatcanbeappliedtoawidevarietyofdiagnosticapplications.Sincetheirinceptioninthelate1980sahugerangeoflateralflow immunoassayshavebeen launched,with theglobal lateralflow immunoassaymarket expectedtobeworthapproximately$6billionby2020.Lateralflowimmunoassaysarewidelyusedinhospitalsandclinicallaboratories,aswellasinveterinarymedicine,inenvironmentalassessment,andforsafetytestingduringfoodproduction.Duetothelowdevelopmentcostsandtherelativeeaseofproduction,thislistofapplicationscontinuestogrow.

The technologyonwhich lateralflow immunoassaysarebasedwasderived from theworkofSingerandPlotzwho, in1956,developedalatexagglutinationassaytodiagnoserheumatoidarthritis.Latexparticleswereusedascarriersforhumanantibodies;uponbindingoftheseantibodiestorheumatoidfactorfromtheserumofpatientswithrheumatoidarthritistheparticlesbecamecross-linkedtoformvisibleclumpsinaprocesstermedagglutination.

At around the same time as Singer and Plotz were developing their agglutination assay, the firstradioimmunoassay, designed tomeasure insulin levels in human serum,was invented byYalowandBerson.Guineapigswereimmunizedwithbovineinsulin,towhichtheyproducedanimmuneresponseandgeneratedantibodies.Theseguineapigantibodieswereincubatedwithhumaninsulinandiodine131-labeledbovineinsulin,bothofwhichcompetedforantibodybinding.Thepresenceofinsulininhumanserumwassubsequentlydetectedbypaperchromato-electrophoresis,andRosalynYalow receivedaNobelPrizeforherworkinthedevelopmentoftheradioimmunoassay.

Enzyme immunoassays evolved shortly after radioimmunoassays, providing a number of significantadvantagesincludingfasterreactiontimes,greaterspecificitiesand,mostimportantly,thereplacementofradioisotopeswithenzymes.Otherenablingtechnologieswhichprogressedduringthistimeincludedantibody production methodologies, nitrocellulose membrane manufacturing processes and liquiddispensingpractices.AlloftheseeffortsculminatedinthefilingofthreeUSpatentsin1987(fromBectonDickinson,UnileverandCarterWallace)fortheveryfirstlateralflowimmunoassays.

Themainapplicationtodrivethedevelopmentoftheseearlylateralflowimmunoassayswasthehumanpregnancytest,whichreliedonthedetectionofhumanChorionicGonadotropin(hCG)inurine.hCGisahormonewhichissecretedduringtheearlystagesofpregnancy,andplaysanessentialroleinearlypregnancysurvival;itspresenceinurinecanbeusedtodetermineapregnancy.Thesefirstlateralflowimmunoassaysclearlydemonstratedtheutilityofpoint-of-care(POC)testing,andpavedthewayfortheevolutionofawiderangeoftests.

2. Immunoassays

An immunoassay is defined as a bioanalytical method which relies on the interaction between anantibodyandanantigen(theanalyte).Awidevarietyofimmunoassaytechniqueshasbeendeveloped,withWestern blotting, immunocytochemistry (ICC), immunohistochemistry (IHC), flow cytometry andELISAbeingamongstthemostwell-known.Allofthesemethodsshareanumberofadvantages:theyarehighlyselective,oftenhavelowlimitsofdetection,areapplicabletothedeterminationofarangeofanalytesandarerelativelyinexpensivetoperform.


Thereadoutofanimmunoassaycantakeanumberofdifferentforms.Colorimetricdetectionreliesonthegenerationofacoloredproduct,fluorometricdetectionrequirestheexcitationandsubsequentemissionoflightbyafluorophore,chemiluminescenceoccurswhenasubstrateiscatalyzedbyanenzymeandproduceslightasaby-productofthereaction,andanother(lesspopular)optionistheuseofradiolabels.Sincelateralflowimmunoassaystendtobequalitativeratherthanquantitative,acolorimetricmethodis usually the preferred readout. The demand for quantitative lateral flow immunoassays is growingsteadily,howeverthedevelopmentofthesepresentsachallenginggoalsincequantitativeassaysrequireconsiderablymorefromthefinishedproductintermsofreproducibility,stability,sensitivityanddynamicrangethanisneededfromaqualitativeassay.

3. Lateralflowimmunoassays

Lateral flow immunoassays, also known as immunochromatographic assays or strip tests, areimmunoassayswhichhavebeendesignedtooperatealongasingleaxis.Althoughthereareanumberofdifferentvariationsofthetechnology,theyalloperateusingthesamebasicconcept.

Whenalateralflowimmunoassayisrun,thetestsampleisaddedtoasampleapplicationpadattheendofthestrip.Thesamplethenmigratestotheconjugatereleasepad,whereadetectionparticle(typicallygoldorlatex)thathasbeenconjugatedtoabiologicalcomponentoftheassayisheld.Nextthesampleandthedetectionreagentmigratetothereactionmembrane;asecondbiologicalcomponentoftheassaywillhavebeenimmobilizedheretofunctionasacapturereagent.Thecapturereagentusuallyexistsasatestlinewhichspansthewidthofthemembrane;acontrolreagentwillbeimmobilizedinasecondlinefurtheralongthemembrane.Theanalyteiseithercapturedatthetestline,orcontinuestomigrateuntilreachingtheabsorbentwickingpadattheotherendofthestrip.Thedetectionreagentbindsatthecontrollinetoindicatethattheassayhasrunsuccessfully.Thisprocessisillustratedinfigure1.

Figure 1. Schematic representation of a typical lateral flow assay. The antigen is illustrated in yellow.


3.1 Thesampleapplicationpad

Thisisanabsorbentpadontowhichthesampleisapplied,andistypicallycomposedofawovenmeshorcellulosefiber.Wovenmesheshavegoodtensilestrengthandalowbedvolume,meaningthattheyretainvery littlesamplevolume; theycan thoughbe ratherexpensive.Cellulosefiberpadshave lowtensilestrengthandamuchgreaterbedvolume;theyarealsorelativelycheap.Irrespectiveofwhichmaterialischosen,thesampleapplicationpadshouldexhibitconsistentabsorbency,thicknessanddensitysothatuniformwickingratesensureassayreproducibility.Thesampleapplicationpadshouldalsodemonstratelowproteinbindingtoavoidlossofanalyte.

Themainfunctionofthesampleapplicationpadistopromoteevenandcontrolleddistributionofthesample,howeverthesampleapplicationpadcanbemodifiedtoenableconditioningofthesample.Bypre-treatingthesampleapplicationpadwithcomponentssuchasproteins,detergentsorsaltsitispossibletoreducenon-specificbinding,increasethesampleviscosity,oralterthepH.Sampleconditioningisanimportantconsiderationduetotheverydifferentnatureofthesampleswhichmaybetestedinalateralflowimmunoassay;thesecanbeasdiverseaswater,urine,serum,plasma,blood,saliva,cerebrospinalfluid,milk,amplifiednucleicacids,andsolubilizedsolidmaterialssuchasfeces,food,plantsandsoil.Duetothelowbedvolumeitisimpracticaltopre-treatwovenmeshes;cellulosefibersaremuchmoreamenabletothesemodifications.

3.2 Theconjugatereleasepad

The conjugate release pad is typically composed of non-woven glass fiber, intowhich the detectionreagenthasbeendried.Oncethesampleapplicationpadhasbeensaturated, thesampleflows in totheconjugatereleasepadwhereitreleasesthedetectionreagent;thedetectionreagentthenleavestheconjugatereleasepadandmoveswiththesampleintothemembrane.

The roleof theconjugate releasepad is to ensureuniform transfer of the sampleand thedetectionreagent,andtopreservetheconjugateupondryingandre-wetting.Itplaysapivotalroleincontrollingtheperformanceof the lateralflow immunoassay.Theconjugatereleasepadshouldexhibit lownon-specificbindingsothatthedetectionreagentdoesnotremaintrappedinthepad.Apre-treatmentmaybenecessarytoincreasewettability,decreasenon-specificinteractionsandtocontrolthepH.Itisimportantthat theconjugate releasepadhasaconsistentbedvolume toensure that theamountofdetectionreagentineachteststripremainsconstant.

3.3 Thedetectionreagent

Thedetectionreagentemployedinalateralflowimmunoassaytypicallyconsistsofantibodieswhichhavebeenconjugatedtocoloredorfluorescentparticles.Theoveralleffectivenessofthefinishedlateralflow immunoassaywillbedependenton thequalityofboth theantibodiesand thedetectionmoiety,amongstotherfactors.


3.3.1 Theantibody

Whenselectingsuitableantibodiesforuseinalateralflowimmunoassaythereareanumberoffactorswhichmustbetakenintoconsideration.Firstly,itiscriticaltoensureaconsistentsupplyofgoodqualityantibodies.Secondly,theantigenspecificityshouldbeassessed.Theantigenspecificityisdefinedbytheantibody’svariableregion;ifthisregionrecognizesacommonmolecularmotif,theantibodymaybindtothesameepitopeonmultipletargets.Sincenon-specificbindingcouldleadtothemisinterpretationofresultsitispreferabletoselectanantibodywhichrecognizesasingletarget.

Thebindingaffinityoftheantibodyisalsoimportant;unlessthelateralflowimmunoassayisintendedforthedetectionofananalytewhichispresentatrelativelyhighconcentrationsinthesample,anantibodywith high binding affinity is required. The binding affinity is defined by the equilibrium dissociationconstant,KD,andisgovernedbythesameprincipleswhichdefineanyreversiblebiomolecularinteraction:

Ab+Ag⇌ Ab-Ag KD=[Ab][Ag] [Ab-Ag]

Inthisequation,[Ab]isthemolarconcentrationofunboundantibody,[Ag]isthemolarconcentrationofunboundantigen,and[Ab-Ag]isthemolarconcentrationoftheAb-Agcomplex.ThelowertheKDvalue,thehighertheaffinityoftheantibodyforitstarget,andthereforethelessantibodythatisrequiredinthelateralflowimmunoassayfordetectionoftheanalyte.AntibodieswithhighbindingaffinitytypicallyhaveaKD≤10

-7M.

Aswellasbeingdesirabletouseanantibodywithhighbindingaffinity,inalateralflowimmunoassayformatitisvitalthattheforwardreactionisdrivenbyafaston-rate.Theon-ratedefineshowquicklytheantibodybindstotheantigentoformacomplex.Thisisofparticularimportanceforthereactionwhichoccursatthetestline,sincethetimewhichtheanalytespendsincontactwiththecaptureantibodythathasbeenimmobilizedherewillbejustafewseconds.Althoughtheflowrateandthewidthofthetestlinecanbothbemanipulatedtoaffectthiscontacttime,afaston-rateisessentialtoarobustlateralflowimmunoassay.Afaston-rateisalsoimportantduringthebindingoftheanalytetothedetectionantibody,althoughtheanalytewillspendslightlylongerinthepresenceofthedetectionantibodysincethedetectionantibodyislocatednearertothebeginningoftheteststrip.AcommonapproachtofindingsuitableantibodypairsistoperformanELISA,howevertheassayconditionsofanELISAareverydifferenttothoseofalateralflowimmunoassay,withincubationtimesintheregionofhoursratherthanseconds.Becauseofthis,itisimportanttotesttheantibodiesassoonaspossibleinaformatthatcanpredicttheirperformanceinalateralflowimmunoassay.

Anotherconsiderationwhenselectingantibodiesforalateralflowimmunoassayiswhethertochoosemonoclonals or polyclonals.Monoclonals are preferred since large quantities of a specific antibody,recognizingasingleantigenicepitope,canbeproduced.Althoughpolyclonalscanproduceadequateresults,theyaresubjecttovariabilitybetweenanimalsandovertime,andasupplycannotbeguaranteedforthelifetimeofthelateralflowimmunoassay.


Whetheramonoclonalorapolyclonalantibodyisselected,itshouldminimallybeaffinitypurifiedsinceanycontaminatingproteinsmaycompete forbindingduring theassay.Theantibodymustalsohavesufficient stability towithstand the variousextremes that itwill be subjected toduringmanufactureandstorageofthelateralflowimmunoassaydevice;itneedstobeabletomaintainitsreactivityafterbeingadsorbedtotheconjugatereleasepad,itshouldtoleratebeingcompletelydriedforadefinedtimeperiod(theexpirationdateofthelateralflowimmunoassay),anditshouldinstantlybereactivefollowingrehydrationbythesample.

3.3.2 Thedetectionmoiety

Themostcommonlyuseddetectionmoieties in lateralflow immunoassaysaregoldnanoparticlesorlatex beads. These particles produce a colored readout which requires no development process forvisualization.Fluorescent labels,enzymes,othercolloidalmetalsandmagneticparticlescanalsobeemployed,andinterestintheseisgrowingsteadily.

Whenworkingwithgoldnanoparticlesorlatexbeads,itisessentialthattheseparticlesareofauniformsizeandhavearegularsphericalshape.Thisensuresaconsistentrateoftransferthroughthemembrane;smallparticleswillmovemorerapidlythanlargeparticles,soanydiscrepanciesinsizecanmanifestasdifferentapparentsensitivities.Theparticlesshouldalsodemonstratereproducibleconjugationtotheantibody,theconjugationprocedureshouldbeeasyandscalable,andtheparticlesshouldbereasonablypriced.

Traditionally,passiveadsorptionmethodshavebeenused toattachantibodies todetectionmoietieshowever passive adsorption can be time-consuming and requires specialist knowledge. Covalentconjugationallowstheproductionofhighlystableconjugates,andusesconsiderablylessantibodythanpassiveadsorptionmethods.ThedirectconjugationofantibodiesorproteinstogoldnanoparticlesorlatexmicrospherescanbecarriedoutquicklyandeasilyusingInnovaCoat®GOLDandLATEXconjugationkitsfromInnovaBiosciences.

3.4 Themembrane

Themembraneisconsideredtobethemostimportantelementinalateralflowimmunoassay.Nitrocelluloseisthemostcommonly-usedmaterial,althoughcelluloseacetate,polyvinylidenefluoride(PVDF),charge-modifiednylonandpolyethersulfone(PES)mayalsobeemployed.Nitrocellulosemembranesexhibitingarangeofporesizes(0.05to12μm)arecommerciallyavailable,butsincetheporesdonothaveanevendistribution it ismoreappropriate toconsidercapillaryflowtimewhenselectingamembranefor thelateralflowimmunoassay.Capillaryflowtimeistypicallyexpressedasseconds/cmandisdefinedasthetimetakenforthesampleliquidtotraveltoandcompletelyfillthestripofmembrane.Thecapillaryflowtimecanaffectthesensitivityandspecificityofthelateralflowimmunoassay,aswellastheconsistencyofthetestline.

Captureantibodiesareimmobilizedacrossthemembrane,typicallyintwolines.Thetestlineisusedtobindthesampleprotein,whilethecontrollineconsistsofspecies-specificantibodiesforthedetectionantibodies(e.g.anti-mouseantibodies)andisusedtodemonstratethatthelateralflowimmunoassayisperformingasitshouldbe.Instrumentationisrequiredforpreciseapplicationofthecaptureantibodies;


positivedisplacementsystemsandairjettingsystemsaremostcommonlyused.Inpositivedisplacementsystemsaliquidstreamisdispenseddirectlyontothemembrane,eitherusingnon-contactorcontacttips.Non-contacttipsaresuspendedatafixeddistanceabovethemembrane,whilecontactdispensingrequiresaflexibletubetobedraggedacrossthesurfaceofthemembraneatthesametimeastheliquidisdispensed.Inbothcasesitiscriticalthatthemembraneisofaconsistentthicknesstoavoidcausinganymechanicaldamage.Inair-jettingsystemstheliquidstreamisinjectedwithpressurizedairtoformanaerosolandisthendispensedontothemembrane.Onceagainaconstantmembranethicknessisvital, sincedispersionof theaerosolwillbegreatlyaffectedby thedistancebetween the tipand themembrane.

To avoid assay variability it is imperative that themembrane remains perfectly flatwhile it is beingstripedwithantibodies,andforthisreasonitmaybepreferabletoselectanitrocellulosemembranewithanon-porousbackingfilm.Thesearereadilyavailable,andaremucheasier tohandlethanunbackedmembranes. If the test strip is to be placed in a plastic housing it is important to ensure that thethicknessoftheplasticbackingisconsistentotherwisetheteststripcanbesubjectedtovariablelevelsofcompression.

Inadditiontothechoiceofmembrane,anumberofothervariablesshouldbetakenintoconsiderationwhenstripingthemembrane.Theseincludetheconcentrationofthecaptureantibodies,thechoiceofantibodydiluent,thereagentdispensingrateandthedryingmethod.Completedryingiscriticaltoensurethatthecaptureantibodiesbecomefixedtothemembrane,howeveritisimportanttonotethatbackedmembranestakeconsiderablylongertodrythanunbackedmembranes.

3.5 Thewickingpad

Thisisfoundattheendofthelateralflowimmunoassay,andfunctionstoincreasethevolumeofsamplewhichenterstheteststrip.Theincreasedvolumecanbeusedtowashawayunbounddetectionantibodiestherebyreducingbackgroundandincreasingassaysensitivity.Wickingpadsalsopreventbackflow.Thewickingpadistypicallycomposedofacellulosefilter;bychangingthedimensionsofthewickingpaditispossibletooptimizethetotalvolumethatistakenupbytheteststrip.

3.6 Theplasticcassette

Foreaseofuse,manylateralflowimmunoassaysareplacedintoaplasticcassette.Theaimofthisisprimarilytoensurethattheenduserappliesthesampleonlytothesampleapplicationpad,andnottoanyotherregionoftheteststrip.Theplastichousingalsoprotectstheteststrip.Duringthemanufacturingprocesstheplastichousingcanbelabeled,forexampletoclearlyindicatethepositionofthetestlineandthecontrolline.Plasticcassettesareavailableasoff-the-shelfproducts,andthechoiceofcassetteforthefinishedtestshouldbematchedtothedimensionsoftheteststrip.

4. Processoptions

Therearetwogeneralapproachestolateralflowimmunoassayassembly,namelybatchprocessingandin-lineorreel-to-reelassembly.Thebatchprocessingapproachallowsfortheuseoflow-costequipment


andissuitableforrelatively lowproductionvolumes,however itsdisadvantagesarethat it involvesahighdegreeofmanuallaborandcanbepronetoproductvariability.Duringin-linemanufacturing,lateralflowimmunoassaystripsaremachine-processedincontinuousrolls;thisresultsinhigherthroughputandconsiderablylowerproductvariability.

Whethercarryingoutbatchprocessingorin-lineassembly,itisvitaltocontroltheambientconditions(temperatureandrelativehumidity)duringpaddrying,dispensingprocessesandstripassembly.Proteindispensing is usually performed at 18-25oC with 40-60% relative humidity, while strip assembly andstorage is generally carriedout at 18-25oCwith <20%humidity. Fluctuations in these conditions canintroduceanadditionalsourceofvariabilitytothelateralflowimmunoassay.

5. Assayformats

Thetwomainapproacheswhichareusedinalateralflowimmunoassayareadirect(sandwich)assayformatandacompetitiveassay.

In a sandwich assay the analyte is captured between two complementary antibodies. One of theseantibodies isconjugated to thedetection reagentand isheldat theconjugate releasepad,while theotherantibody is immobilizedat the test lineon themembrane.Anyexcess labeledantibodywillbecapturedatthecontrolline.Theintensityofthecolorwhichisseenatthetestlinecanbeindicativeoftheamountofanalytepresentinthesample.Thistypeoftestisgenerallyusedforlargeranalyteswithmultipleantigenicsites.

Figure 2. Sandwich assay format. A positive result is seen when the analyte becomes bound to the detection antibody and to the capture antibody which has been immobilized at the test line. The antibody which has been immobilized at the control line will recognize the detection antibody, producing a colored line to indicate that the lateral flow assay has performed as expected.


Thecomplexityofthelateralflowimmunoassaycanbeincreasedthroughtheuseofmultipletestlines,allowingthedetectionofmultipleantigenswithinthesamesample.Arecenttrendistousedotsinsteadof lines formultiplexing analysis. Dots can also be used to produce alpha-numeric symbols on themembrane,allowingintuitiveresultgeneration.

Competitive assays are used for smaller analytes which have a single antigenic determinant andthereforecannotbindtwoantibodiessimultaneously.Inthistypeofassayformattheantigenistypicallyimmobilizedatthetest line. Iftheanalyte ispresent inthesample, itbindstothedetectionantibodywhichissubsequentlyunabletobindatthetestline.Alackofsignalatthetestlineisthereforeindicativeofapositiveresult.

Figure 3. Competitive assay format. A positive result is seen when the analyte becomes bound to the detection antibody and therefore cannot bind to the antigen which has been immobilized at the test line. The antibody which has been immobilized at the control line will recognize the detection antibody, producing a colored line to indicate that the lateral flow assay has performed as expected.

Althoughmanydifferentpermutationsarepossible,alllateralflowimmunoassaysinvolvetheformationofacomplexbetweenadetectionreagentthatisfreeinthesamplestream,andacapturereagentwhichisboundtothemembraneatthetestline.

6. Advantagesanddisadvantagesoflateralflowimmunoassays

Theadvantagesofthelateralflowimmunoassaysystemarewell-known.Mostimportantamongthesearethepoint-of-care(POC)natureofthetechnology,thebroadrangeofapplications,andthespeedandrelativelyminimalcostatwhichanewly-developedlateralflowimmunoassaycanbebroughttomarket.


Thedisadvantagesare lessobvious,howeversensitivityand reproducibilityare themostchallengingobstacleswhichmustbeovercomewhendevelopingalateralflowimmunoassay.

Advantages Disadvantages

Low cost Qualitative or semi-quantitative readoutWide range of applications Restriction on total test volume can impose a

limit on sensitivityWell-established technology, ease of manufacture Inaccurate sample volume may reduce precision

Long shelf-life, no need for refrigeration Test-to-test reproducibility may be problematicSimple, user-friendly operation Difficult to miniaturize sample volumeHigh sensitivity and specificity Multiplexing can be challengingLow sample volume required Unclear patent situation in some instances

One-step assay, no washing steps necessary, short time to result

Relatively short timeline for development, time to market is reduced

High potential for commercializationEasily scalable

Can be integrated with reader systemsPossibility of multiplexing

Figure 4. Advantages and disadvantages of lateral flow immunoassays.

7. Nanoparticlesforlateralflowimmunoassays

Gold nanoparticles and latex beads are the most widely used labels in commercial lateral flowimmunoassays.Goldnanoparticlesarewell-suitedtothispurposebecauseoftheirintenserubyredcolor,andInnovaBiosciencesofferscolloidalgoldnanoparticlesthatareidealforlateralflowimmunoassays.Our colloidal gold has been developed using specialized techniqueswhich enable the production ofextremelyuniformsphericalparticleswithanarrowsizedistribution,therebyminimizingassayvariability.Theycanbeproducedinlargequantitieswithoutcompromisingonquality.Figure5showsaTEMimageofour40nmcolloidalgold.

Figure 5. TEM image of 40nm colloidal gold, demonstrating the uniform spherical shape and narrow size distribution.


ThebenefitsofInnovaBiosciences’colloidalgoldinclude:

9 Differentnanoparticlesizesandconcentrationsavailable

9 Fullyscalable-availableathighconcentration(>20OD)andlargevolume(liters)

9 StringentlyQCtested

9 Uniformsphericalshapeandnarrowsizedistribution

9 Competitivelypriced

Theuseofcolloidalgoldforlateralflowimmunoassaysrequirespassiveadsorptionoftheantibodyontothesurfaceofthenanoparticle,aprocesswhichcanbeextremelychallengingandnecessitatesspecialistknowledge.Toovercomethis,wehavedevelopedInnovaCoat®GOLDconjugationkits.Thesecontain‘conjugationfriendly’nanoparticlesthathaveaproprietarysurfacecoatingwhichgreatlyenhancesgoldstabilityandpermitseasycovalentattachmentofavarietyofmolecules,includingantibodies,analytesandotherbiomolecules.InnovaCoat®GOLDconjugatescanbeusedinarangeofassayformats,includinglateralflowimmunoassays,anddemonstrateenhancedsensitivitywhencomparedtotraditionalpassiveconjugationmethods,asillustratedinfigure6.

Figure 6. Competitive lateral flow immunoassay comparing antibodies conjugated to 40nm gold particles by a traditional passive method against those conjugated covalently using InnovaCoat® GOLD.


ThebenefitsofInnovaCoat®GOLDinclude:

9 Quickandeasytouse

9 Proprietarysurfacecoatingpreventsmetal-proteininteractions

9 Differentnanoparticlesizesandamountsavailable

9 Freeze-dried

9 Fullyscalable

9 StringentlyQCtested:uniformsphericalshapeandnarrowsizedistribution

9 Covalentconjugationusessignificantlylessantibodythanpassiveconjugationandprovidesahighlystableconjugate

Latexbeadsareanotherpopularchoiceforlateralflowimmunoassayssincetheyareavailableinarangeofvibrantcolors,making themeasilyvisibleandallowing for thesimultaneousdetectionofdifferentanalytes.

Figure 7. Multiplexed lateral flow immunoassay using Innova Biosciences’ latex beads. Latex beads of three colors have each been conjugated to a different antibody or protein.

Foruseinalateralflowimmunoassay,latexbeadsrequireconjugationtothedetectionantibody.Traditionalmethodsoflatexconjugationaretechnicallycomplexandcanwastealotofpreciousmaterial;passiveconjugationrequiresextensiveoptimizationtoidentifytheappropriatepH,whilecovalentconjugationispronetoaggregationofthelatex.InnovaBiosciences’latexconjugationkitshavebeendesignedtogreatly simplify theprocess. These kits allowantibody conjugation to blue, redor black latexbeadswithouttheneedforextensiveoptimization.Highyieldsoffunctionalconjugatescanbemadewithouttheneedforharshresuspensionmethodssuchassonicationandvortexing.


ThebenefitsofInnovaBiosciences’latexconjugationkitsinclude:

9 Quickandeasytouse

9 Differentcolorsavailable

9 Freezedried

9 Fullyscalable

9 StringentlyQCtested

9 NoextensivepHoptimizationrequired

9 Resistanttoaggregation

9 Covalentconjugationprovidesahighlystableconjugate

Forfurtherinformationonourfullrangeofcolloidalgold,InnovaCoat®GOLDandlatexproducts,pleaseseeourwebsite.

8. CustomservicesfromInnovaBiosciences

Althoughlateralflowassaydevelopmentcanberelativelystraightforward,werecognizethatsometimesyou might need some assistance. You may, for example, be struggling to optimize your antibodyconjugation,timerestraintscouldmeanthatyouwishtooutsourceyourbulkconjugations,oryoumightbearelativenewcomertolateralflow.Tohelpyoustreamlineyourworkload,InnovaBiosciencesoffersawiderangeofcustomservices,includingourlateralflowassaydevelopmentservice.

Ourlateralflowassaydevelopmentserviceisdesignedtotakeyourlateralflowimmunoassayfromaninitialidea,throughtoR&D,andthenontotrustedpartnersforbulkmanufacturing.Thefullserviceisamulti-stepprojectprovidingyouwithdifferentoptions,fromaproof-of-principlelateralflowimmunoassayusingadipstick,tofullstripswithsampleapplicationpad,conjugatereleasepadandwickingpad,andsmallscalemanufacture.

Module1–conjugationservices

Module1consistsofourconjugatemicro-optimizationservices,andourcustomconjugateformulationservices.Ourconjugatemicro-optimizationservicesenableyoutofindthebestperformingconjugateforyourassay,usingverysmallquantitiesofyourantibodyorprotein;ourexperiencedconjugationscientistswillproducearangeofdifferentantibody-goldnanoparticleconjugatesforyoutotestinyourapplication.Ourcustomconjugateformulationservicecanbeusedasafollow-upservicefromourconjugatemicro-optimizationservice,orasanindependentoption;ourteamofexpertconjugationscientistswillquicklyprovideyouwithbulkquantitiesofyourbespokeantibody-goldnanoparticleconjugate,inyourchosenbufferandatyourpreferredconcentration.


Module2–assaydevelopment

Ourlateralflowassaydevelopmentserviceisprovidedasadipstickassayformat,orasafulllateralflowteststrip.Thedipstickformatconsistsofawickingpadandanitrocellulosemembrane,andisdesignedtoprovethattheconjugationhasbeensuccessful.Theantibody-goldnanoparticleconjugateismixedwiththesamplecontainingtheantigen,andisthenappliedtothedipstick;acaptureantibodyonthenitrocellulosemembraneisusedtoshowthattheassaysetupiscorrect.Thefulllateralflowteststripformatprovidesthesamematerialsasthedipstickassay,withtheadditionofaconjugatereleasepadcontainingthedriedantibody-goldnanoparticleconjugate,andasampleapplicationpad.

Whicheverformatischosen,thecaptureantibodyisdispensedonthenitrocellulosemembraneaseitheraspotorasatestline;greateramountsofantibodyarerequiredforthelattertoallowforprimingofthedispenser.Ifspotsaredispensedonthenitrocellulosewewillprovideimages(qualitativemethod),whereasiflinesaredispensedthesignalwillbereadandthesensitivitycalculated(quantitativemethod).

Module3–lateralflowassayoptimization

Withinmodule3,ourexperiencedin-houseteamwilldeterminetheoptimalamountofcaptureantibodyandwilltestdifferentpadsandmembranetreatmentstoprovideyouwithyouroptimizedlateralflowassay.Assayoptimizationwillprovideyouwiththebestpossibleperformanceatthelowestmanufacturingcost.

Module4–stripmanufacturingforLFA

Thisserviceallowsyoutopurchaseassembledstripsforyourlateralflowassay,eitherasdipsticksorasfulllateralflowteststrips.

Forfurtherinformationonourcustomservices,pleasecontactus.

+44 (0)1223 661000 [email protected]

Innova Biosciences Ltd

Babraham Research Campus

Cambridge

UK, CB22 3AT

Phone: +44 (0)1223 661000

Email: [email protected]

Innova Biosciences products are sold for research purposes only, and our terms and conditions of sale include a limited use license to our Intellectual Property for internal research applications.

Commercial use, such as use within manufacturing, re-sale to third parties, or incorporation into kits, requires a separate written agreement, conferring relevant additional rights, with Innova Biosciences.Lightning-Link®, InnovaCoat® and Thunder-Link® are registered trademarks of Innova Biosciences. Cy and CyDye are trademarks of GE Healthcare. DyLight® is a registered trademark of Thermo FisherScientific Inc. and its subsidiaries. FluoProbes® is a registered trademark of interchim. Texas Red® is a registered trademark of Life Technologies Corporation

Guide to Lateral Flow Immunoassays · 2018-04-06 · A guide to lateral flow immunoassays | 5 3.1...

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