ELISA AND RELATED

TECHNOLOGIES

CORE MODULE 1 :
Laboratory Methods and Instrumentation

Dr Brian Jones, Clinical Immunology Division

bmjones@ha.org.hk
 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 (TMB)

Horse radish peroxidase (HRP)

Orange, 490 nm Yellow, 450 nm

Spectrophotometer
 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