ELISA AND RELATED TECHNOLOGIES CORE MODULE 1 : Laboratory Methods and Instrumentation Dr Brian...
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![Page 1: ELISA AND RELATED TECHNOLOGIES CORE MODULE 1 : Laboratory Methods and Instrumentation Dr Brian Jones, Clinical Immunology Division bmjones@ha.org.hk.](https://reader030.fdocuments.us/reader030/viewer/2022032521/56649d585503460f94a374bd/html5/thumbnails/1.jpg)
ELISA AND RELATED TECHNOLOGIES
CORE MODULE 1 : Laboratory Methods and Instrumentation
Dr Brian Jones, Clinical Immunology [email protected]
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ENZYME-LINKED IMMUNOSORBENT ASSAY
• valuable tools for use in clinical labs• can measure antibodies or antigens• inexpensive, rapid, quantitative, specific• sensitive (pg/ml)• expensive equipment not required (but helps!)• can be automated
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BASIC FORMAT
Solid phase = 96 / 384-well microplate
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Analyte = antibody Analyte = antigen
Incubate, wash
1. Coat solid phase withantigen when analysing antibody
antibody when analysing antigen
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2. Block free binding sites. Incubate. Wash.
Analyte = antibody Analyte = antigen
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3. Add sample. Incubate. Wash
Analyte = antibody Analyte = antigen
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4. Add conjugate. Incubate. Wash.
Analyte = antibody Analyte = antigen
E E E E
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5. Add substrate
6. Incubate, stop, measure colour change
Colourless
ENZYME
OD
CONCENTRATION
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COATING THE PLATE
• protein-binding 96 (384)-well polystyrene plate eg Immulon-2 (Dynatech)
• buffer = 0.1M Na2CO3/NaHCO3 pH 9.6 0.1M tris-HCl pH 7.6 0.01M PBS pH 7.3 etc.
• antigen or antibody at 0.5 - 20 g/ml
• 100 l/well, 4oC overnight
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WASHING THE PLATE
• buffer + 0.05% Tween 20
• 200 l/well
• 3 - 6 washes with 1 minute soak
• automated washer or• “flood and flick” (biohazard) or• multichannel pipette for dispensing, manifold connected to vacuum pump (for safe disposal of wash fluid)
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BLOCKING THE PLATE
• 0.25% - 2% bovine serum albumin 2% non-fat dried milk 5 - 10% foetal calf serum in buffer + 0.05% Tween 20
• 100 l/well, 37oC, > 60 min
• Wash x3 with buffer-Tween 20
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SAMPLE• Dilute in buffer-Tween 20
• include known positive and negative samples
• standards……. recombinant protein international standard antibody double-dilute from 10 pg/ml - 10 ng/ml
• 100 l/well, duplicates
• 2 - 4 hours 20/37oC or overnight 4oC
• 3 - 6 washes with buffer-Tween 20
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CONJUGATE• For assays of (human) antibodies use : anti-(human) Ig-enzyme IgG / A / M / E / subclass-specific • For assays of antigens use enzyme-conjugated antibody:
against a different epitope to the one recognized by capture antibody
often monoclonal capture antibody polyclonal detection antibody
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AMPLIFICATION
E
Directly conjugated developingantibody may give weak signal
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amplify with
unlabelled (rabbit) anti-(human) Ig followed by
anti-(rabbit) Ig-enzyme
EE
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or
Biotin-labelled anti-Ig followed by
streptavidin-enzyme
E-S B S-E
ES
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SUBSTRATESSee Sigma catalogue for list of conjugates and substrates
Orthophenylene diamine Tetramethyl hydrochloride (OPD) benzidine (TMP)
Horse radish peroxidase (HRP) Orange, 490 nm Yellow, 450 nm Spectrophotometer
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Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate
Alkaline phosphatase
Yellow, 405 nm Methyl umbelliferone Spectrophotometer
365 nm 445 nm Fluorimeter
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INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
• screening hybridoma supernatants• detecting clinically important antibodies - autoantibodies - anti-pathogens - anti-allergens
1. Antigen
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INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
2. Sample (human) antibody
1. Antigen
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INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
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INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E4. Substrate
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
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ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
Useful when pure antigen not available or antigen coats poorly
1. Specific antibody
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ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
2. Impure antigen eg tissue homogenate
1. Specific antibody
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ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
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ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
4. Sample (human antibody)
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
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ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
E E5. Anti-human Ig-enzyme
4. Sample (human antibody)
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
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ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
6. Substrate
5. Anti-human Ig-enzyme
4. Sample (human antibody)
3. Wash pure antigen
2. Impure antigen
1. Specific antibody
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ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
eg. hormones drugs tumour antigens cytokines
1. Anti-analyte
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ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
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ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
3. Anti-analyte-enzyme
E E
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ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
3. Or: anti-analyte-biotin followed by streptavidin-enzyme
E-S B S-E
ES
E S E-S B S-E
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ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
4. Substrate
3. Or: anti-analyte-biotin followed by streptavidin-enzyme
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COMPETITION ELISA TO DETECT ANTIGENS
1. Analyte
(antigen-coated plate)
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COMPETITION ELISA TO DETECT ANTIGENS
2. Anti-analyte-E + sample
1. Analyte
Low [analyte] High [analyte]
E E E
E EE
EE
E
E
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COMPETITION ELISA TO DETECT ANTIGENS
3. Wash
2. Anti-analyte-E + sample
1. Analyte
Low [analyte] High [analyte]
E E E
E
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COMPETITION ELISA TO DETECT ANTIGENS
3. Wash
2. Anti-analyte-E + sample
1. Analyte
Low [analyte] High [analyte]
4. Substrate
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COMPETITION ELISA TO DETECT ANTIGENS
1. Anti-analyte
(antibody-coated plate)
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COMPETITION ELISA TO DETECT ANTIGENS
2. Analyte-E + sample
1. Anti-analyte
Low [analyte] High [analyte]
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COMPETITION ELISA TO DETECT ANTIGENS
3. Wash
2. Analyte-E + sample
1. Anti-analyte
Low [analyte] High [analyte]
E E E E E E E
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COMPETITION ELISA TO DETECT ANTIGENS
3. Wash
2. Analyte-E + sample
1. Analyte
Low [analyte] High [analyte]
4. Substrate
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MICROPARTICLE ENZYME IMMUNOASSAY (ABBOTT AxSYM)
Automated measurement down to 1 ng/mlEg. tumour markers (AFP, CEA, PSA, CA15.3) immune activation marker 2M IgE
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AP
Microparticlecoated withanti-analyte
Sample/Control/Standard
Anti-analyte- alkaline- phosphatase
Automated mixing fluorimetryincubation reference curve washing calculation
Methyl umbelliferol phosphate
Methyl umbelliferone
365 nmmercuryarc lamp
445 nmfluorimeter
AP
Glass fibre matrix
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ALLERGEN-SPECIFIC IgE: Pharmacia UNICAP system
Anti-IgE--galactosidase
Sample
“CAP” = allergen-coated cellulose disc
E E E
Substrate = methyl umbelliferyl- -galactoside
Methyl umbelliferone
365nm 445 nm
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12 1
2
HEALTH
CANCER VIRUSES
MYCOBACTERIAHELMINTHS
ASTHMA, ALLERGYLUPUS
RHEUMATOID ARTHRITISMULTIPLE SCLEROSIS UVEITISDIABETES
IL2IL12IFNTNF
IL4 IL5 IL6 IL10
CMI(AB)
Type 1 Type 2
AB(CMI)
CYTOKINES
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Detection of cytokines by ELISA
• Plasma or supernatant of cultured mononuclear cells + activator
• Coat plate with anti-CK (Pharmingen) 0.5 g/ml in bicarbonate buffer, 4o overnight
• Wash x 2 with PBS-T
• Block with PBS + 10% FCS, 2 hours RT
• Wash x2 with PBS-T
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• Add standards (recombinant CK 10 pg-10 ng /ml), controls and samples • 4o overnight
• Wash x3 with PBS-T
• Add biotinylated anti-CK (Pharmingen) 0.5 g/ml
• RT 60 minutes
• Wash x6 with PBS-T
• Add streptavidin-peroxidase
• RT 30 min
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• Wash x8 with PBS-T
• Add OPD substrate
• 15 min RT, dark
• Stop with N H2SO4
• Read A490
2
1
0
[rCK]
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MICROARRAYEg. Novagen ProteoPlex
Std 1
Std 3 Std 4
Std 2
Std 6
#2
#4
#6
#8
#10
Std 5
#1
#3
#5
#7
#9
IL1
IL2
IL5
IL7
IL10
IL16
gmCSF
TNF
IL1
IL4
IL6
IL8
IL12
IL18
IFN
TGF
Quadruplicate capture anti-cytokine antibody spotsOverlay with standards, samples. Incubate, washAdd fluorophore-detection antibody. Incubate, washScan
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S
I
G
N
A
L
[CK]
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Std 1
Std 3 Std 4
Std 2
Std 6
#2
#4
#6
#8
#10
Std 5
#1
#3
#5
#7
#9
IL1
IL2
IL5
IL7
IL10
IL16
gmCSF
TNF
IL1
IL4
IL6
IL8
IL12
IL18
IFN
TGF
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CYTOMETRIC BEAD ASSAYS
Mixture of beads with(a) Anti-cytokine capture antibody (b) Individual fluorescence properties
IL1
IL2
IL5
IL7
IL10
IL16
gmCSF
TNF
IL1
IL4
IL6
IL8
IL12
IL18
IFN
TGF
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Add sample
IL1
IL2
IL5
IL7
IL10
IL16
gmCSF
TNF
IL1
IL4
IL6
IL8
IL12
IL18
IFN
TGF
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Add fluorochrome-labelled detection antibody
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Flow cytometry
IL1
IL4
IL6
IL8
IL12
IL18
IFN
TGF
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DETECTION OF CYTOKINE-SECRETING CELLS BY
ELISPOT ASSAY
Czerkinsky et al, 1983; Sedgwick & Holt, 1983J. Immunol. Methods
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Capture anti-CK antibody
96-well culture plate with nitrocellulose bottom
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PBM + stimulus, 22-24 hr
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WASH
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Add biotin-labelled detection anti-CK. 90 min. Wash
B B B
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Add streptavidin-alkaline phosphatase. 60 min. Wash
S-AP S-AP S-APB B B
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Colourless, soluble BCIP-NBT
insoluble blue product
S-AP S-AP S-APB B B
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Unstimulated PBM
PHA, Con A, anti-CD3, antigen, alloantigen (T-cells)
LPS, SAC (monocytes)
Type 1 cytokines (CMI, proinflammatory) - IFN, IL2, IL12, TNF
Type 2 cytokines (antibody, anti-inflammatory) - IL4, IL6, IL10
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CK ELISPOTS - NORMAL RANGES (n = 60)(CK-secreting cells/106 PBM)
IFN IL2 IL4 IL10 IL12 IL6 TNF
unstim 0 0 0 0-6300
0-42 0-70000
0-88000
PHA 450-3500
3000-6900
850-5000
2100-11600
144-1068
19000-105000
27000-108000
Con A 2000-10900
1300-6500
340-4300
SAC 2600-9700
79- 558
19000-92000
19000-97000
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CASE STUDY: POTENTIAL OF CYTOKINE PROFILING IN CLINICAL PRACTICE
• 48 year old woman with frequent life-long respiratory and intestinal infections
• Cytokine profile : IL6, TNF, IL10, IFN, IL12, normal IL4
• rIL12 in vitro normalized IFN
• rIFN in vitro normalized IL12
• Anti-IL10 in vitro normalized IFN and IL12
• Treatment with rIFN, rIL12, anti-IL10 not possible
• Thymosin-1 normal IL6, IFN, IL4; near-normal IL12, TNF; IL10
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DEMONSTRATION
Room 508/511, Clinical Pathology Building
ELISA washer, readerUNICAP systemAxSYMELISPOTS