sorbent assay. <— ala _ tet Have you ever gotten sick? How did you know you were sick? yo. bret bot What were your symptoms? hot Did you get better? esThe Immune Response: Why you are still alive oh | primary reponse secondayrespanse (ew Have you ever had chickenpox? Do you know anyone who had the chickenpox twice? ee Have you ever had the measles? Why do we get some diseases only Yo sparse once and some diseases not at all? . The Body’s Defense System we Immune Response Nonspecific ‘Specific wo Triggered by a specific Physical Nonspecific aiusen (disease - Barriers: response’ bacteria, protozoa, virus, cin inflammation etc) membranes _£¢v¢rSpecific Immune Response Lin tonsug) Gutaers molecules produced by pathogens (e.g. proteins) Antibody _Y-shaped proteins that bind to specific aviii'g «/'¢ White Blood Ci © B-cells - generate specific antibodies © T-gells -detect antigens, stimulate B cells otonty An A die wt net How can we tell what disease a patient has? Answer: By the gy fip oc ec present in the patient's blood. Aniiliedé+¢ produced by the body are Specie to each pathogen (disease), How can we tell what antibodies are present in a patient's blood? ‘Answer: Perform an ELISA test!What is ELISA? ELISA is enzyme-linked immunosorbent assay Enzyme - proteins that make reactions happen. Immuno- - having to do with the immune system (antibodies) -sorbent - substance that absorbs another Assay - test to determine the contents of a substance In other words, ELISA is a test that determines if a disease is. present in a given sample (e.g. a patient's blood). ELISA test Five groups of Patients: Zika virus. HIV Lyme disease Avian influenza West Nile virus ohoneHow ELISA works Label Figure 2.(d) on the next slide (page S-3 in Lab Packet): Bound antigen - protein molecule produced by the pathogen (disease) ‘Serum antibody - produced by the body's immune system, found in blood ‘Secondary antibody - from test kit, binds to antibodies produced by patient Conjugate enzyme - reacts with dye, so we can see the antibodies Chromogen - molecules (dye) that change color in presence of enzyme Label Figure 2(d) conjugated Word Bank conjugate enzyme ‘serum (blood) antibody ‘secondary antibody antigen cehromagenELISA Simulation Lab ‘We are going to test blood samples taken from patients who may be at risk for contracting one of five diseases. We are going to be adding antigens, positive control, negative control, and ‘samples from patients A, B, C, D, E, and F to a microtiter plate using a pipette. ELISA Lab: General Procedure 1. Add antigen (from pathogen, i.e. the disease) to each well of microtiter plate Add controls to specific wells (from testing kit) ‘Add blood sample to be tested (from patients) to specific wells ‘Add secondary antibodies (from testing kit) to each well ‘Add chromogen substrate (from testing kit, causes color change) to each well Incubate (wait) 5-10 minutes Observe color change and record results (table in lab packet) NOUNAdditional Lab Instructions 1, Follow all written lab instructions carefully (beginning on page S-3) 2. Follow microtiter plate layout - Figure 3: $060060O6000 : ©©000OOOOOO® ipo ice pasar 3. Do not touch liquid already in the wells with the pipette (avoid contamination!) 4. Use each pipette only once per sample! (Do not reuse pipettes)‘Student Guide 241248211249 ELISA Simulation Introduction ‘The Body's Defense System “The body possesses several ines of defense aginst infection by pathogenic organisms. Pathogens ar ty slisease-ausing agen including vinss, taser, poze, mold, and thet misroonzmnisms, Pathogens invade the body and mull; they ean cause sickness or even the death ofthe invaded individual. The body employstive line of defense to prevent ae fight off sich dangerous intrsins, The fist 0 defense modes ae nonspeeti. They include the body's phsial bares andthe nonspecific immune ‘systm. These defenses funtion witht regard to the typeof pathogenic intruder. The thind layer of slefense isthe body's peifc immune system, Speeiielmmune responses we toed tothe peat invading pathogen, Specific Immune Response ‘Specific immune responses are triggered by antigen molecules. Antigens include prin and other moleules produced by pathogens. The key pliers in the specifi immune defense are denditic eels ‘macrophages an small white blood ellsealled B Iymphocjes(B cell) and TIytnphocyes (Teel), Phagocytic macrophages and dendritic ells beak dow pathogens and display antigen fragment from the pathogens on the surface a ther cell membranes, Ban T lymphocytes circulate through the body in the blood and Iymph. When T-cell see displayed antigenic fragments, thoy stimulate specific Bells to reproduce an generate antibodies designed agaist he specifi vigen encountered ‘Thus, the word antigen is derived fom the ter "antibody generator” Anu ins (also refereed to us immunoglobulins) tht are found in the Dlooseam or bound to ell membranes. These proteins all ave te same hase Y-shped stuctur, but have different antigen binding sites a her ends. Antigen binding sits are designed to fitthe shape of specific antigens. Antibodies bind t antigens likea lok and key forming antigen-antibody complexes (GeeFigue 1). Figure 1 Antgen-antbody compiax ‘When a anibody forms an antigen-antibody complex, generally it marks the invading orzanismantgen for destruction or for lerance from the bloadstream by phagoeyte eel, This removal i designed to prevent the organis/ntigen from infecting the cel. Antger-atibody complexes also stimulate addtional immune responses to ad the body in clearing an lfection, (©2008 Carolin Bitoni! Supply Company s4ELISA ‘Scientists have applied the basic principles of antibody-mediated immunity to an assay for {detecting infection by specific organisms. Ths assay is called an ELISA (gnzyme-Jinked Jimmunogorbent assay) and isbased on the principle that antibodies produced in response to pathogens their antigen targets with great to form antigen-antibody complexes, ‘There ae two types of ELISA tests-direct ELISA and indieet ELISA. Indirect ELISA is used to detect infection by testi | blood for the presence or absence of against a particular pathogen. The presence of such antibodies indieates that the individual thas been infected and that their body has launched an immune response against the disease- ‘causing agent ‘STEP ONE - ANTIGENS In the first step of an indirect ELISA, antigen proteins purified from the infectious agent, or ‘genetically engineered versions ofthe antigens, are the wells of plastic microtiter plates, These antigen proteins bind to the bottom of the well by fanning hydrophobic associations with the plastic surhlee (see Figure 2a). The wells of the plate are then washed with a buffer to remove any unbound material, STEP TWO ~ PATIENT BLOOD "Next, blood serum ftom the patent(s) being tested is added to the treated_wells If these serum samples coniain antibodies against the bound antigen, the antibodies willatach to the antigens, forming tight complexes (see Figure 2b), Such antigen-antibody complexes are not visible by eye, so detection steps (described in the next paragraph) must be employed to visualize them. The wells are again washed to remove any unbound proteins ‘STEP THREE ~ SECONDARY ANITBODY Detection of antigen-antibody complexes scarred out through the following steps. A dary antibody that recognizes antibodies prod anti-human antibod isadded to the wells IFantigen-antibody complexes formed in the wells, his secondary antibody’ recognizes and hinds to the primary antibodies from the patients’ serum (see Figure 2c) The secondary antibody is attached to an enzyme that will aciltate the final detection. This antibodvieazyme combination js.called » conjugate. The wells ae rinsed ‘one last time to remove unbound molecules. STEP FOUR - CHROMOGEN COLOR CHANGE In the final step, a chromogen substrate jsadded to the wells of the plate If present, the enzyme that is linked to the secondary antibody facilitates achemical reaction that changes the color of the chromogen (see Figure 2d). A color change indicates that the patient possesses antibodies to the antigen and has been infected. No change in color indicates that the patient has not been infected, or that their body has not yet launched an immune response to produce antibodies against the invading antigen. Positive results determined by ELISA undergo a different test to confirm the findings. (©2008 Carona Stelegie! Supply Company s2{ahbound angen (Dl anionserum bound (alensymesrauees miboaycompice secondary anody ‘slorenon secondary entbody (pe = conga neve SK _He ervomesen eotr change Figure EUSA earatc ofa polve aut |v thi simulated ss (es), each Sample wl be tstod in trpict (tre ines) to ensure reproducibility. Known sive samples are inh Forease of performance the well washing steps ofa time ELISA have een eliminate inthis simulation. ris imporant to note that the washesare «necessary sep of an actual assy. Likes, is erica to use acean ppe for each new sample or reagent o prevent css: ‘contamination ofthe well Instructions 1. Using one plastic pipet, caefilyaiminises 2 doops ofsimulated angen (ANT) in each well of, rows and 8 ofthe microtiter plate. Discard the pipet after use, Ina tue ELISA asa, the antigen ‘would bind othe tion ofthe microtiter plate. The wels would then be washed witha buffer to Temove any unbound moleules, In his simulated lab activity, the washing step has been eliminated, 2. Using a clean pipet, ald 3 dros of posi 16020 wells ofthe microtiter plate see Figue3). Do not allow the pipet to Touch te Tui aleady inthe well. Discard the pipet afer tse 43. Using clean pipet, ald 3 drops of =C)t0 wes Ad, AS, and A6 ofthe microtiter late {sce Figure), Do not allow the pipet to toch the Hgud already inthe wells Discard the pipet after se 4. Using a ctean pet, ald 3 drops of Patent_A sample towels A 9 of the microtiter plate (See Figure). Do not allow the pipet to toh the ligud aleady in the wells: Discard the Pipet afer use 122006 Caran Blolegcal Supely Company 835. Using clean pipet, add 3 drops of Patent Bsample to wells AIO. All. an AI2 ofthe ‘microsite pate (ee Figure 3), Do not allow the pipet to touch the iui already inthe wells Discard the pipet afer wee, 6 Using clean pipe, add drops of Patent C sample to wells, B2.ond B3 ofthe microtiter plate (eeFigue 3), De not alow te pipet o touch the liguid already in the well. Discard the pipet afer se 7, Using clean pipet, add 3 dons of Patient D sample tn wells 4,95, and 26 ofthe microter plate (Gee Figue3). Do not allow te pipet to touch the liquid already in the wel. Diseard the pipet fer 8. Using clean pipe, ad 3 drops of Patent E:sample to wells 7.8. and B9 ofthe mertter plate (Gee Figure3}. De not alow the pipet o touch the lig already inthe els. Discard the pipet ter se. 9 Usinga clea pipet ad 3 drops of Patent fr 11 BIZ ofthe microtiter plate (s2 Figure 3. Do not allow the pipet to touch the liquid already inte wel, Discard the Pipe! afer use. true ELISA ny, a this pont, the wells would be washed witha ur to Femove any moleules that have not bound tothe adhered antigen. In this simulated lab stv, the washing step has been eliminated, 9OOOOOO06HOO : ©O©OOOOOQOOOOO +L paltive conto, -€ = negative contol AF = Paints AF igre 3. Mireter plat sample wets 10, Usinga clean pipet, ited secondary antibody (2° A rt and Bon the microtiter plate. Keep tack ofesch wel asthe reagent is added and do nat allow te Pipet to touch the liquid already inthe wells, Discard the pipet after use Ina tue ELISA, the wells ‘would again be washed fo remove unbound molecules, This step has been eliminated fom this Simulation 1. Using the remaining clean pipet, 3 drops of simulated chromogen (chrom) to cach wellof rs ‘Acai B onthe microtiter plata. Keep ack of each wellas the reagent isadlécd an! do at allow ‘ta pipet to touch the Fiquid already in the well Discard the pipet flr use 12, All the reaction ells willturn light geon when the chromogen is added. A. change fom iht treet purple indicates a postive rev. Incubate the miroir lat at room temperate for a ‘minimum of minutes end a maximum of 10 minutes to allow fr eolor development. 13, After $-10 minutes of incubation, record your results in the dat table on page 5. Compare the ‘olor ofeach patent sample to that ofthe positive and nopative control. Colored results thi are in between the postive and negative contol should be seared weak positives {©2008 Carona Biolegieal Supply Compary s4Student Guide Name 211248, 211249 Date ELISA Simulation i “Data Table Sample Color ‘Test Result pastve cont peste negative conta veztve Patent A » waive Pate B ” ative Paint © _ ae Pais | : ee Patient . becebive Pao F ea ratitve Questions ‘A. What basi pines fantboy-medited munity or wiz nan EL inoauton DED) glee ye assay? (refer to the WOOD 5 Mi bebier mn bya disease-causing agent? What she seconary antibody (om the testing kit actually etetng? =p Lebeeg trePreS 3. What isthe function ofthe secondary antibody and chromogen ia an ELISA? (ht: erom-maans “eoloe") 122005 Carona Sisages! Supply Company s5“A. Why did you perform three identical tests for each conto and patient sample? Ve Hi i4 WE bine 40 came ure it Sure ‘What sone posible soure of inthe lab? em ineiteton |S. What might cause some postive rests tobe lighter in color than others? mort Serr anitbedy oh | primaryresponte secondary responce [en > Time (oays) “The chart above shows the number of antibodies present ina patents blood over 180 days ‘Which ofthe patent's positive test results would you expett be darker: a sample taken st 15 days ora sump akan a 90 days? ee l v In Ge days the Seaple woul have bea deliver, 46. Describe the disease-causing pathogen for the disease you assayed (std) fo, including its mnt to people ‘Which dsease were you testing fr? Hiv +) What kind of pathogen vs, tera fing is sss with hisses? flu-like synebens ‘What re the symptoms ofthe disease? freien views J. How isthe dbase tani fom one pant another? Woody pregency, te reage mite, aad body Foluicht ssE bat ba Cre 4 ite tion tote dngosia te pies yo te "Slr yous by the cucone ote et on he scenarios provided? Pati cot A ec rmaned pros ver er beri! wos ab'agns he t 7 ert used protectin wun ho ow do you think ifeton cul re hen reveniad n the patents nh ete pote? fatient A ys Fereuy acrive, gant Shae weedic Pad AED MEME Ce Lovie nd ¥, han A “ n STR AUIP ect ue ho 8. Tebeapicur of your ELISA turns (rtp) with your onecame andy clin the eu onFlme 3 (age 8) ofthe lab packet, st