ELISA AND RELATED TECHNOLOGIES CORE MODULE 1 : Laboratory Methods and Instrumentation Dr Brian Jones, Clinical Immunology Division [email protected]

ELISA AND RELATED TECHNOLOGIES

CORE MODULE 1 : Laboratory Methods and Instrumentation

Dr Brian Jones, Clinical Immunology [email protected]

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

BASIC FORMAT

Solid phase = 96 / 384-well microplate

Analyte = antibody Analyte = antigen

Incubate, wash

1. Coat solid phase withantigen when analysing antibody

antibody when analysing antigen

2. Block free binding sites. Incubate. Wash.


3. Add sample. Incubate. Wash


4. Add conjugate. Incubate. Wash.


E E E E

5. Add substrate

6. Incubate, stop, measure colour change

Colourless

ENZYME

OD

CONCENTRATION

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

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)

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

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

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

AMPLIFICATION

E

Directly conjugated developingantibody may give weak signal

amplify with

unlabelled (rabbit) anti-(human) Ig followed by

anti-(rabbit) Ig-enzyme

EE

or

Biotin-labelled anti-Ig followed by

streptavidin-enzyme

E-S B S-E

ES

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

Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate

Alkaline phosphatase

Yellow, 405 nm Methyl umbelliferone Spectrophotometer

365 nm 445 nm Fluorimeter

INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES

• screening hybridoma supernatants• detecting clinically important antibodies - autoantibodies - anti-pathogens - anti-allergens

1. Antigen


2. Sample (human) antibody

1. Antigen


E E E

3. Anti-(human) Ig-enzyme


1. Antigen


E E E4. Substrate

3. Anti-(human) Ig-enzyme


1. Antigen

ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

Useful when pure antigen not available or antigen coats poorly

1. Specific antibody


2. Impure antigen eg tissue homogenate



3. Wash pure antigen

2. Impure antigen



4. Sample (human antibody)


2. Impure antigen



E E5. Anti-human Ig-enzyme



2. Impure antigen



6. Substrate

5. Anti-human Ig-enzyme



2. Impure antigen


ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

eg. hormones drugs tumour antigens cytokines

1. Anti-analyte


1. Anti-analyte

2. Sample


1. Anti-analyte

2. Sample

3. Anti-analyte-enzyme

E E


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


1. Anti-analyte

2. Sample

4. Substrate

3. Or: anti-analyte-biotin followed by streptavidin-enzyme

COMPETITION ELISA TO DETECT ANTIGENS

1. Analyte

(antigen-coated plate)


2. Anti-analyte-E + sample

1. Analyte

Low [analyte] High [analyte]

E E E

E EE

EE

E

E


3. Wash


1. Analyte


E E E

E


3. Wash


1. Analyte


4. Substrate


1. Anti-analyte

(antibody-coated plate)


2. Analyte-E + sample

1. Anti-analyte



3. Wash


1. Anti-analyte


E E E E E E E


3. Wash


1. Analyte


4. Substrate

MICROPARTICLE ENZYME IMMUNOASSAY (ABBOTT AxSYM)

Automated measurement down to 1 ng/mlEg. tumour markers (AFP, CEA, PSA, CA15.3) immune activation marker 2M IgE

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

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

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

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

• Add standards (recombinant CK 10 pg-10 ng /ml), controls and samples • 4o overnight


• Add biotinylated anti-CK (Pharmingen) 0.5 g/ml

• RT 60 minutes


• Add streptavidin-peroxidase

• RT 30 min


• Add OPD substrate

• 15 min RT, dark

• Stop with N H2SO4

• Read A490

2

1

0

[rCK]

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

S

I

G

N

A

L

[CK]

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

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

Add sample

IL1

IL2

IL5

IL7

IL10

IL16

gmCSF

TNF

IL1

IL4

IL6

IL8

IL12

IL18

IFN

TGF

Add fluorochrome-labelled detection antibody

Flow cytometry

IL1

IL4

IL6

IL8

IL12

IL18

IFN

TGF

DETECTION OF CYTOKINE-SECRETING CELLS BY

ELISPOT ASSAY

Czerkinsky et al, 1983; Sedgwick & Holt, 1983J. Immunol. Methods

Capture anti-CK antibody

96-well culture plate with nitrocellulose bottom

PBM + stimulus, 22-24 hr

WASH

Add biotin-labelled detection anti-CK. 90 min. Wash

B B B

Add streptavidin-alkaline phosphatase. 60 min. Wash

S-AP S-AP S-APB B B

Colourless, soluble BCIP-NBT

insoluble blue product

S-AP S-AP S-APB B B

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

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

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

DEMONSTRATION

Room 508/511, Clinical Pathology Building

ELISA washer, readerUNICAP systemAxSYMELISPOTS

Documents

ELISA AND RELATED TECHNOLOGIES CORE MODULE 1 : Laboratory Methods and Instrumentation Dr Brian Jones, Clinical Immunology Division [email protected]