Methods in Molecular Biology 2612 Robert S. Matson Editor ELISA Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. ELISA Methods and Protocols Edited by Robert S. Matson QuantiScientifics LLC, Orange, CA, USA Editor RobertS.Matson QuantiScientificsLLC Orange,CA,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2902-4 ISBN978-1-0716-2903-1 (eBook) https://doi.org/10.1007/978-1-0716-2903-1 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. 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Dedication for LielleandAyla Preface This edition of ELISA: Methods and Protocols was written during the challenging times associatedwiththeonslaughtoftheCovid-19worldwidepandemic.Itistrulyremarkableto havewitnessedtherapidemergenceoftherecombinant(mRNA/DNA)vaccinestrategyto combat this formidable enemy as well as others we have yet to know. We must also acknowledge the tremendous effort of the diagnostic industry to provide reliable testing on such a scale and with very short notice. In the backdrop, there was an army of skilled scientists supporting these efforts.It is very likelythat manyused theELISA in support of theirimportantwork. Whiletheenzyme-linkedimmunosorbentassay(ELISA)isoneofthemostwidelyused techniques, its nuances are not always fully understood. Case in point, the revelation that excessivebiotinconsumptionmayledtoassayinterferencewhenusingthewell-established streptavidin-biotinreportersystem.Youwillfindthattheauthorsoffergreatinsightintothe theory and practice of the ELISA with useful notes to help you through the step-by-step protocolswhilesteeringyouclearofcommonpitfalls. In this compendium, with the introductory chapter we start with a review of the basic formatsusedintheELISA,theirapplication,strengths,andweaknesses.Thisisfollowedby examining the essential components and features comprising the technique-enzyme, anti- body, and sorbent. Approaches to the immobilization of antibodies are provided for those who may find a need to prepare their own immunosorbent. Since the antibody specificity andsensitivityaremostcriticaltothesuccessoftheassay,achapterisdedicatedtoscreening andselectionofantibodies.Next,wediscussmeanstoeffectivelyblockthesorbentsurface toreducenon-specificbinding.Inanotherchapter,theadvancesinsurfacemodificationof the plastics commonly used as the sorbent are described. A chapter on interference com- pletesthefirstpartofthebook. In the second half of the book, we highlight applications of the ELISA. These include the determination of cytokines in tissues and cerebral spinal fluid by sandwich ELISA, a microarray-based method for carbohydrate characterization, and a chapter on mycotoxin extractionwithdeterminationbycompetitiveimmunoassay.AutomationofELISAforhigh- throughputdiagnosticapplications,multiplexallergentesting,andadaptationofELISAfor lateral flow are described in other chapters. There is exciting work on a homogeneous immunoassay and a final chapter examining the role of ELISA in biosensors. It is the goal ofthisbooktoprovidethereaderwithabasicunderstandingofthetechniquewithmethods andprotocolsthatenablesuccessatperformingyourenzyme-linkedimmunosorbentassay. Orange,CA,USA RobertS.Matson vii Contents Dedication.................................................................. v Preface ..................................................................... vii Contributors................................................................. xi 1 Enzyme-LinkedImmunosorbentAssay:TypesandApplications...... ........ 1 HovhannesHayrapetyan,ThaoTran,EglisTellez-Corrales, andCharithaMadiraju 2 ELISAEssentials:Surfaces,Antibodies,Enzymes,andSubstrates..... ........ 19 RobertS.Matson 3 AntibodyImmobilization........... ....... ....... ........ ....... ........ 33 RobertS.Matson 4 ScreeningforAntibodySpecificityandSensitivitywithELISA........ ........ 45 MichaelJ.HalatandEmilyGravens 5 EffectiveBlockingandStabilizingMethodsUsingSynthetic PolymeronELISA......... ........ ...... ....... ....... .. ....... ........ 59 TomozumiNoda,MasaruMatsuda,HirotakaSuzuki, YusukeOkawa,andMotohiro Mitani 6 GasPlasmaSurfaceModificationforBiologicalAssays........ ....... ........ 73 KhorenSahagian 7 InterferenceinELISA...... ........ ....... ....... ........ ....... ........ 91 RobertS.Matson 8 DeterminationofProinflammatoryandAntiinflammatoryCytokines byELISATechnique ....... ...... .. ...... ........ ........ ....... ........ 101 OsmanyBlancoMun˜oz 9 GyrolabImmunoassays:Miniaturization,Automation,andIntegration intoaRapidWorkflow...... ........ ....... ....... ........ ....... ........ 109 JohanEngstr¨om 10 AssessmentofImmunologicallyPotentCarbohydrates ....... ....... ........ 129 DenongWang 11 LateralFlowMicroarray-BasedELISAforCytokines....... .. ....... ........ 141 RobertS.Matson 12 AUniqueMultiplexELISAtoProfileGrowthFactorsandCytokines inCerebrospinalFluid...... ........ ....... ....... ........ ....... ........ 157 CharithaMadiraju,AmaniSastry,MarthaOppong,JacobKarp, MarylaKrajewska,StanKrajewski,BarbaraTomik,AndrzejSzczudlik, andRobertS.Matson 13 Well-BasedMultiplexFoodAllergenColorimetricELISA..... ....... ........ 169 RobertS.Matson 14 MycotoxinQuantificationbyCompetitiveELISA.... ........ ....... .... .... 183 BrynE.MerrillandRobertS.Matson ix x Contents 15 Lumit:AHomogeneousBioluminescentImmunoassayforDetecting DiverseAnalytesandIntracellularProteinTargets ........... ....... ........ 195 Byounghoon(Brian)Hwang,JulianoAlves,DanLazar,NidhiNath, LaurieEngel,MarthaO’Brien,KevinHsiao,KevinKupcho, BeckyGodat,RodFlemming,SaidGoueli,andHichamZegzouti 16 ELISA-BasedBiosensors.... ........ ....... ....... ........ ....... ........ 225 RobertS.Matson Index .......... ........ ....... ........ ....... ....... ........ ....... ........ 239 Contributors JULIANOALVES • R&DDepartment,PromegaCorporation,Madison,WI,USA BYOUNGHOON (BRIAN)HWANG • R&DDepartment,PromegaCorporation,Madison,WI, USA;Abbvie,NorthChicago, IL,USA LAURIEENGEL • R&DDepartment,PromegaCorporation,Madison,WI,USA JOHANENGSTRO¨M • GyrosProteinTechnologiesAB,Uppsala,Sweden RODFLEMMING • R&DDepartment,PromegaCorporation,Madison,WI,USA BECKYGODAT • R&DDepartment,PromegaCorporation,Madison,WI,USA SAIDGOUELI • R&DDepartment,PromegaCorporation,Madison,WI,USA EMILYGRAVENS • OmegaBiologicals,Inc.,Bozeman,MT,USA MICHAELJ.HALAT • OmegaBiologicals,Inc.,Bozeman,MT,USA HOVHANNESHAYRAPETYAN • MarshallB.KetchumUniversity,Fullerton,CA,USA;Wayne StateUniversity,SchoolofMedicine,Detroit,MI,USA KEVIN HSIAO • R&DDepartment,PromegaCorporation,Madison,WI,USA JACOBKARP • MarshallB.KetchumUniversity,Fullerton,CA,USA MARYLAKRAJEWSKA • CellestanImmunoquant(CIQ),Inc,Oceanside,CA,USA STANKRAJEWSKI • CellestanImmunoquant(CIQ),Inc,Oceanside,CA,USA KEVIN KUPCHO • R&DDepartment,PromegaCorporation,Madison,WI,USA DANLAZAR • R&DDepartment,PromegaCorporation,Madison,WI,USA CHARITHAMADIRAJU • MarshallB.KetchumUniversity,Fullerton,CA,USA ROBERTS.MATSON • QuantiScientificsLLC,Orange,CA,USA MASARU MATSUDA • NOFCORPORATION,LifeScienceProductsDivision,Tokyo,Japan BRYNE.MERRILL • ChapmanUniversity,Orange,CA,USA MOTOHIROMITANI • NOFCORPORATION,LifeScienceProductsDivision,Tokyo,Japan OSMANYBLANCOMUN˜OZ • MedicinayEnfermerı´a,FacultadeCienciasdelaSalud, UniversidaddelMagdalena,SantaMarta,Magdalena,Colombia NIDHINATH • R&DDepartment,PromegaCorporation,Madison,WI,USA;Bio-Techne, Minneapolis, MN,USA TOMOZUMINODA • NOFCORPORATION,LifeScienceProductsDivision,Tokyo,Japan MARTHAO’BRIEN • R&DDepartment,PromegaCorporation,Madison,WI,USA YUSUKEOKAWA • NOFCORPORATION,LifeScienceProductsDivision,Tokyo,Japan MARTHAOPPONG • MarshallB.KetchumUniversity,Fullerton,CA,USA KHORENSAHAGIAN • PlasmatreatUSAInc.,Hayward,CA,USA AMANISASTRY • NoordaCollegeofOsteopathicMedicine,Provo,UT,USA HIROTAKASUZUKI • NOFCORPORATION,LifeScienceProductsDivision,Tokyo,Japan ANDRZEJSZCZUDLIK • DepartmentofNeurology,JagiellonianUniversityMedicalCollege, Krakow,Poland EGLIS TELLEZ-CORRALES • MarshallB.KetchumUniversity,Fullerton,CA,USA BARBARATOMIK • DepartmentofNeurology,JagiellonianUniversityMedicalCollege, Krakow,Poland THAOTRAN • MarshallB.KetchumUniversity,Fullerton,CA,USA DENONG WANG • TumorGlycomicsLaboratory,SRIInternationalBiosciencesDivision, MenloPark,CA,USA HICHAMZEGZOUTI • R&DDepartment,PromegaCorporation,Madison,WI,USA xi