2 ELISA Immuno Explorer™ Kit Instructors: Stan HitomiCoordinator – Math and ScienceSan Ramon Valley Unified School DistrictDanville, CAKirk BrownLead Instructor, Edward Teller Education CenterScience Chair, Tracy High Schooland Delta College, Tracy, CASherri Andrews, Ph.D.North Carolina School of the ArtsWinston-Salem, NC
3 ELISA Enzyme-Linked Immunosorbant Assay Mammalian immune systemAntibody specificityBiology’s “magic bullet”Evolved over millions of yearsHarness nature’s tool kitImagine the applications!
4 Links to the Real World HIV, Mad Cow, SARS Biodefense Cancer treatment Drug and steroid testingPregnancy / ReproductionGMO
5 ELISA Immuno Explorer Kit Features Lab completed in a 45 min periodSupplies for 48 students (12 workstations)Comprehensive and flexible curriculumCompelling real-world linksCost effectiveClassroom SafeStriking results
6 What Can You Teach With The ELISA Kit? Hands-on ImmunologyOne lesson integrates multiple standardsHealth sciencesImmunologyImmune response – antibody/antigen interactionsDisease – infection, detection, transmissionBiowarfare – defense against acts of aggressionLinks to research, medicine, and consumer productsModern way to teach classic and contemporary scienceTeach more with less!
7 Immune Response C. Macrophage A. Pathogen D. Macrophage B. B cells F. T cellE. MacrophageG. B cellJ. Antibodies attach to pathogenH. Memory B cellsI. Plasma cells
11 ELISA Kit Workstation Inventory Reagents:Yellow tubes Test samples 2Violet tube (+) Positive control 1Blue tube (-) Negative control 1Green tube (PA) Primary antibody 1Orange tube (SA) Secondary antibody 1Lab Equipment and Supplies:Microplate strips, pipettor, pipette tips,transfer pipette, wash buffer, paper towels,marking penDiscuss materials at each workstation
12 ELISA Kit Quick GuideInstruct participants to follow Quick Guide procedure at their own pace.
13 ELISA Kit ResultsRepeat FLASH animation to solidify what they just did.
14 What Are The Reagents? And What Function Do They Perform? PBS: Phosphate buffered saline – provides stable buffered environment to maintain antibody structureTween 20: Nonionic detergent – removes non-specifically bound proteins to reduce background and blocks protein binding sites on the polystyreneMicroplates: Polystyrene – proteins absorb (bind) by hydrophobic bonds to the polystyrene
15 What Are The Reagents? And What Function Do They Perform? Antigen: Chicken gamma globulinPrimary antibody: Polyclonal anti-chicken antibody made by rabbitsSecondary antibody: Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish peroxidase (HRP)Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless solution that when oxidized by HRP turns blue
16 ELISA Kit Protocol Issues Controls:Protocols I and IIPositive control = antigenNegative control = PBSProtocol IIIPositive control = primary antibodyNegative control = wash bufferTriplicate samples: controls against cross contamination (2 out of 3 ain’t bad)Washing: eliminates non-specific binding and reduces background
17 Ways The ELISA Kit Can Be Used ProtocolType of ELISAReal-World ApplicationITracking outbreaks of diseaseHIV, SARS, smallpox & anthraxIIDetecting antigensPregnancy, drugs, GMO, BSE (and all the above)IIIDetecting antibodies in serumHIV, Lyme disease, smallpox and West Nile virus
18 Protocol I: Tracking Disease Outbreaks Real-World Application - HIV Transmission & DetectionStep 1 - Share ‘body fluid’ samples to simulate disease transmissionStep 2 - ELISA to detect infectionTube DescriptionActual Tube ContentsSimulated Tube ContentsStudent samplesAntigen or PBSSample derived from patient’s bloodPrimary antibodyAntibody against HIV from miceSecondary antibodyHRP-linked antibody against mouse antibodiesPositive controlAntigenHeat-inactivated HIV virusNegative controlPBSHIV negative human serumResults – Infection of about 50% of students
19 Protocol II: Antigen Detection ELISA Real-World Application – Pregnancy TestProtocol - ELISA on simulated urine samplesTube DescriptionActual Tube ContentsSimulated Tube ContentsStudent samplesAntigen or PBSPatient’s urine samplePrimary antibodyAntibody against hCG hormone from miceSecondary antibodyHRP-linked antibody against mouse antibodiesPositive controlAntigenPurified hCG hormoneNegative controlPBSUrine from woman that is not pregnant
20 Protocol III: ELISA Antibody Test Real-World Application – Lyme DiseaseProtocol - ELISA to detect exposure to a diseaseDo you have antibodies to the disease?Tube DescriptionActual Tube ContentsSimulated Tube ContentsPurified antigenAntigenB. Bergoferi purified proteinsStudent samplesPrimary antibody or wash bufferPatient’s serum samplesSecondary antibodyHRP-linked antibody against B. bergoferiPositive controlPrimary antibodySerum from patients with Lyme diseaseNegative controlWash bufferSerum negative for Lyme disease
21 Real-world Applications of Antibodies Uses– Disease diagnosis– Basic Research– ImmunotherapyApplications– Dipstick tests/ELISA– Immunostaining– Western blottingReal-world Applications of AntibodiesBio-Rad’s HIV-2ELISA KitBio-Rad’s HIVWestern Blot Kit