ELISA AND RELATED

TECHNOLOGIES

CORE MODULE 1 :
Laboratory Methods and Instrumentation

Dr Brian Jones, Clinical Immunology Division

[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

1. Coat solid phase with
antigen when analysing antibody
antibody when analysing antigen

Analyte = antibody Analyte = antigen

Incubate, wash
2. Block free binding sites. Incubate. Wash.

Analyte = antibody Analyte = antigen

3. Add sample. Incubate. Wash

Analyte = antibody Analyte = antigen

4. Add conjugate. Incubate. Wash.

E E E E

Analyte = antibody Analyte = antigen

5. Add substrate
6. Incubate, stop, measure colour change

ENZYME

Colourless

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

Directly conjugated developing

antibody may give weak signal
 amplify with

E
E

unlabelled (rabbit) anti-(human) Ig

followed by
anti-(rabbit) Ig-enzyme
 or
E
S
E-S B S-E

Biotin-labelled anti-Ig
followed by
streptavidin-enzyme
 SUBSTRATES
See 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
 INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES

2. Sample (human) antibody

1. Antigen
 INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES

E E E
3. Anti-(human) Ig-enzyme

2. Sample (human) antibody

1. Antigen
 INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES

4. Substrate
E E E
3. Anti-(human) Ig-enzyme

2. Sample (human) antibody

1. Antigen
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

Useful when pure antigen not available

or antigen coats poorly

1. Specific antibody
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

2. Impure antigen
eg tissue homogenate

1. Specific antibody
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

3. Wash  pure antigen

2. Impure antigen

1. Specific antibody
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

4. Sample (human
antibody)

3. Wash  pure antigen

2. Impure antigen

1. Specific antibody
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

5. Anti-human Ig-enzyme
E E
4. Sample (human antibody)

3. Wash  pure antigen

2. Impure antigen

1. Specific antibody
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
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

eg. hormones
drugs
tumour antigens
cytokines

1. Anti-analyte
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

2. Sample

1. Anti-analyte
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

E E
3. Anti-analyte-enzyme
2. Sample

1. Anti-analyte
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

3. Or: E E
anti-analyte-biotin S S
followed by E-S B S-E E-S B S-E
streptavidin-enzyme
2. Sample

1. Anti-analyte
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

4. Substrate

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

1. Anti-analyte
COMPETITION ELISA TO DETECT ANTIGENS
(antigen-coated plate)

1. Analyte
COMPETITION ELISA TO DETECT ANTIGENS

Low [analyte] High [analyte]

E E E
E

E
E
E E E 2. Anti-analyte-E
E
+ sample

1. Analyte
COMPETITION ELISA TO DETECT ANTIGENS

Low [analyte] High [analyte]

3. Wash

E E E 2. Anti-analyte-E E
+ sample

1. Analyte
COMPETITION ELISA TO DETECT ANTIGENS

Low [analyte] High [analyte]

4. Substrate

3. Wash

2. Anti-analyte-E
+ sample

1. Analyte
COMPETITION ELISA TO DETECT ANTIGENS
(antibody-coated plate)

1. Anti-analyte
COMPETITION ELISA TO DETECT ANTIGENS

Low [analyte] High [analyte]

2. Analyte-E
+ sample

1. Anti-analyte
 COMPETITION ELISA TO DETECT ANTIGENS

Low [analyte] High [analyte]

3. Wash

2. Analyte-E
E E E E E + sample E E

1. Anti-analyte
COMPETITION ELISA TO DETECT ANTIGENS

Low [analyte] High [analyte]

4. Substrate

3. Wash

2. Analyte-E
+ sample

1. Analyte
MICROPARTICLE ENZYME IMMUNOASSAY
(ABBOTT AxSYM)

Automated measurement down to 1 ng/ml

Eg. tumour markers (AFP, CEA, PSA, CA15.3)
immune activation marker 2M
IgE
 Sample/ Anti-analyte-
Control/ alkaline- Methyl umbelliferol phosphate
Standard phosphatase
AP

Methyl umbelliferone
AP

Microparticle Glass fibre matrix 365 nm 445 nm

coated with mercury fluorimeter
anti-analyte arc lamp
Automated

mixing fluorimetry
incubation reference curve
washing calculation
 ALLERGEN-SPECIFIC IgE:
Pharmacia UNICAP system
365nm 445 nm

Substrate = methyl umbelliferyl- -galactoside

Methyl umbelliferone

E E E
Anti-IgE--galactosidase

Sample

“CAP” = allergen-coated
cellulose disc
 CYTOKINES

Type 1 Type 2
IL2 HEALTH IL4
CMI IL12 IL5 AB
IFN IL6
(AB) TNF IL10
(CMI)

RHEUMATOID ARTHRITIS CANCER

MULTIPLE SCLEROSIS VIRUSES
UVEITIS MYCOBACTERIA
DIABETES 2 1
HELMINTHS
ASTHMA, ALLERGY
LUPUS
1 2
 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

• 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
• Wash x8 with PBS-T 2

• Add OPD substrate

• 15 min RT, dark 1

• Stop with N H2SO4

• Read A490 0
[rCK]
MICROARRAY Quadruplicate capture anti-cytokine antibody spots
Eg. Novagen ProteoPlex Overlay with standards, samples. Incubate, wash
Add fluorophore-detection antibody. Incubate, wash
Scan
Std 1 Std 2

Std 3 Std 4 IL1 IL1

IL2 IL4
Std 5 Std 6
IL5 IL6
#1 #2
IL7 IL8
#3 #4
IL10 IL12
#5 #6 IL16 IL18

#7 #8 gmCSF IFN

#9 #10 TNF TGF

S
I
G
N
A
L

[CK]
Std 1 Std 2

IL1 IL1
Std 3 Std 4
IL2 IL4
Std 5 Std 6
IL5 IL6
#1 #2
IL7 IL8
#3 #4 IL10 IL12

#5 #6 IL16 IL18

#7 #8 gmCSF IFN

TNF
#9 #10 TGF
CYTOMETRIC BEAD ASSAYS

IL1 IL1

IL2 IL4

IL5 IL6
Mixture of beads with
IL7 IL8 (a) Anti-cytokine capture antibody
IL10 IL12 (b) Individual fluorescence properties
IL16 IL18

gmCSF IFN

TNF TGF
IL1 IL1

IL2 IL4

IL5 IL6

IL7 IL8
Add sample
IL10 IL12

IL16 IL18

gmCSF IFN

TNF TGF
Add fluorochrome-labelled
detection antibody
 IL1

IL4
Flow cytometry
IL6

IL8

IL12

IL18

IFN

TGF
DETECTION OF CYTOKINE-
SECRETING CELLS BY
ELISPOT ASSAY

Czerkinsky et al, 1983; Sedgwick & Holt, 1983

J. Immunol. Methods
96-well culture plate with nitrocellulose bottom

Capture anti-CK antibody

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-AP

B B B
 Colourless, soluble BCIP-NBT

insoluble blue product

S-AP S-AP S-AP

B 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- 3000- 850- 2100- 144- 19000- 27000-

3500 6900 5000 11600 1068 105000 108000
Con A 2000- 1300- 340-
10900 6500 4300
SAC 2600- 79- 19000- 19000-
9700 558 92000 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, reader

UNICAP system
AxSYM
ELISPOTS