2014’ ELISA Workshop - WOONGBEE

1 ELISA .. 1 ELISA . 2 ELISA . 3 ELISA . 4 ELISA . ELISA . ELISA (Enzyme-Linked ImmunoSorbent Assay).. - . target , . Enzyme-Linked Y YY ImmunoSorbent ELISA . Why ELISA ? Specific simple Sensitive Quantifiable Ag-Ab binding Microplate, Enzyme Color ( ). detection . Microplate reader High capacity sensitivity . (pg~ng/ml) Data . Rapid , . Cheap Kit . ELISA . , (cytokine, chemokine, growth factor) , . (virus, animal, human disease, food ). (toxicology, pharmacy ). (immunology, cancer, neuroscience ). Quality-control check (food, drug industry ). Antibody screening ELISA . ELISA . Direct ELISA . Indirect ELISA . Sandwich ELISA . Competitive ELISA . ELISA - Direct ELISA . Y Enzyme conjugated Antibody Antigen microplate Antibody 1 low sensitivity Antigen(sample) coating Sample antigen level.

2 ELISA - Indirect ELISA . Y Enzyme conjugated Antibody Y Y Ag-speicific Antibody Antigen microplate Antibody 2 . Sample antibody level . (virus , ). ELISA - Sandwich ELISA . Sample antigen level . (Ag level OD ). High specificity, Commercial ELISA kit ELISA - Competitive ELISA . Small molecule level . Small molecule level OD . High specificity ELISA - Sandwich ELISA . Capture Ab coating & Blocking WASH. STD, sample incubation WASH Tip! Biotinylated Detection Ab . incubation Reagent , incubation , .. WASH. warm up~. SA-HRP incubation pipette technique WASH Contamination . Washing !!! Substrate incubation Stop & Detection ELISA assay Pipetting tip , pipette calibration pipetting . Tip ( ). pipetting . Contamination (sample/reagent tip , reservoir ). Pipette volume.

3 Surface tension tip pre-rinse tip . , tip . Air bubble ( drawing tip ). X X X. ELISA assay Microplate Control Blank Microplate: ELISA (high binding). Coating microplate .. - Duplicate - STD, blank, control . sample : 40 sample 400. 400 400. Standard ELISA assay- Standard Standard !! Sample standard mass-value sample . OD standard . Control . Reconstitution buffer , . 15 rolling aliquot . Serial Dilution 7 point + blank Sample type diluent . vortexing tip . ELISA assay- Control Control !! Standard curve sample , . negative control = blank positive control = analyte = control set (high, medium, low). Control set Control set , high, medium, low recombinant protein ( , commercial lab, ). 1 Lot . ( degradation ) Sample analyte detection range .. ELISA assay- Antibody & Diluent Antibody.

4 High affinity High purity, High binding capacity Diluent . Diluent sample . binding interference .. Sample type, analyte . Specific diluent ELISA assay- Washing Washing !!! insufficient washing background non-specific binding . Wash buffer volume(400ul) . 30 sec soaking washing method squirt bottle > multipipette > autowasher washing paper towel . washing well . ELISA assay Detection method Colorimetric assay Chemiluminescent assay HRP + TMB, H2O2 HRP + Luminol Alkaline phosphatase + NADPH Stop solution X. Stop solution Luminometer . Microplate reader . ELISA assay Data analysis Microplate reader (OD , STD curve).. - wavelength: 450 nm - wavelength correction: 540 nm or 570 nm Standard curve - X: , Y: OD . - X,Y scale: log or linear - curve fit: 4-parameter Unknown sample OD value Sample.

5 - sample . - Sample dilution , dilution factor .. ELISA assay Data analysis Corrected = 450 nm 540/570 nm . - OD CV < 10%. - non-specific binding (blank) < - R2 value = ~ ELISA assay ELISA 5 Tip! Kit Insert Washing !!!! Reconstitution Squirt bottle, manifold Pre-warming Method insert washer, autowasher . buffer . /lot .. specific option Buffer . Aliquot !!! ( ).. Pipetting Data .. Scale: X/Y log, linear Contamination . Curve fit: 4-parameter ELISA .. ELISA . ELISA , check . 1. Kit . 2. Kit . 3. kit warm up assay (Insert ). 4. Calibrator diluent assay diluent warm up .. 5.. 6. Incubation , , shaker ( kit ) . 7. kit lot kit . 8. Standard . ELISA Technique ELISA Wash wash . 1. Wash strip numbering . 2. Buffer 10-15 soak time . 3. Wash buffer paper towel plate buffer.

6 4. Well . 5. Buffer well bubble . 6. aspiration buffer (plate buffer .). ELISA Technique ELISA Pipetting pipetting . 1. pipette calibration . 2. well volume . 3. Sample, reagent tip . 4. Tip pre- rinse pipetting . 5. bubble , tip . 6. reagent . ELISA Technique ELISA Pipetting Pipette Calibration . Pipette volume volume (10 ). CV 2% . Pipette volume volume (10 ). CV 2% . CV 2-3% test . CV 2-3% .. Q1. ? Q2. Standard ? Q3. Reference ? Q4. 4-parameter ? Q5. Capture antibody vial ! Q6. Standard blank ? Q7. ELISA microplate ? Q8. Assay procedure ? Q9. Insert sample type ? Q10. sample ? Q11. Wash buffer well ? & Question Answer Q1. ? A1. , . 18-23 .. & Question Answer Q2. Standard ? A2. ELISA detection range sensitivity , range standard curve .. & Question Answer Q3.

7 Reference ? A3. Reference plate .. plate . , . Plate reader reference insert . Reference . reader .. & Question Answer Q4. 4-parameter ? A4. Lot QC test suitable specific . 4- parameter . ELISA . reader software . & Question Answer Q5. Capture antibody vial ! A5. Antibody trehalose .. antibody vial , QC buffer .. & Question Answer Q6. Standard blank ? A6. standard , blank . 10% . & Question Answer Q7. ELISA microplate ? A7. , . Immunoassay microplate . Cell culture microplate cell . capture antibody . & Question Answer Q8. Assay procedure ? A8. procedure .. guarantee . & Question Answer Q9. Insert sample type . ? A9. ELISA kit insert sample type . validation . sample type test . spike/recovery test (validation test) validation .. protocol . & Question Answer Q10.

8 Sample ? A10. Standard curve . ( ) .. & Question Answer Q11. Wash buffer well ? A11. Well well washing . wash buffer .. Troubleshooting .. Troubleshooting Troubleshooting , Check point Substrate Stop solution Standard : . Blank: 10% . Troubleshooting ELISA Wash Technique wash . 1. Wash strip numbering . 2. Buffer 10-15 soak time . 3. Wash buffer paper towel plate buffer . 4. Well . 5. Buffer well bubble . 6. aspiration buffer (plate buffer .). Troubleshooting ELISA Wash Technique R&D wash device . 1. Squirt bottle . buffer . Bubble . 2. Multi-channel pipette . Tip volume . pipette . 3. Auto washer . Protocol buffer volume . wash paper towel buffer .. Case 1. Stop solution , . Case 2. Standard . Case 3. Blank (only diluent) . (High background). Case 4.. Case 5.

9 Duplicate . Case 6. Standard , sample . Case 7. Standard . (Standard curve flat non-linear). Troubleshooting Case 1. Stop solution , . Substrate . Substrate .. 15 . Substrate activity test: conjugate . substrate .. Standard reconstitution protocol . buffer volume . 15 . 1 aliquot Shaker . RPM orbit range . Substrate wash . Substrate stop solution . Troubleshooting Case 2. Standard . Wash . Wash buffer . volume well . Well wash buffer . Wash .. Contamination . , diluent, substrate, pipette, tip assay .. Standard serial dilution . Dilution tip . Incubation . incubation . (18-23 ) .. Troubleshooting Case 3. Blank (only diluent) . Wash . : . volume well . Blank : 10% Well wash buffer . Blank Contamination . , diluent, substrate, pipette, tip assay . ! . Pipette tip.

10 Plate sealer . Incubation , . Troubleshooting Case 4.. Kit . Substrate . Substrate .. 15 . Substrate activity test: conjugate . substrate .. Standard reconstitution . buffer volume . 15 . : Shaker . RPM orbit range .. signal . Troubleshooting Case 5. Duplicate . warm up . Well wash buffer volume . Wash buffer volume well variation . well bubble . bubble reaction . Pipette calibration . Replicate mixing . Capture antibody mixing well . volume . Troubleshooting Case 6. Standard , sample .. Sample type . Validation sample type spike&recovery test (validation test) . protocol . Sample target . reference . Out of range .. diluent . Insert sample dilution . Sample matrix effects dilution . Troubleshooting Case 7. Standard .. (Standard curve flat non-linear). Plate reader setting.