Enzyme linked immunosorbent

assay (ELISA)

Assoc. Prof. Dr Noraziah Mohamad Zin

Block C, Fifth floor
noraziah.zin@ukm.edu.my
Introduction
• Precipitation & agglutination
• A/g & A/b complexes is the endpoint
• Qualitative/semi qualitative

• Indicator labeled
• A/b or A/g labeled with molecule
• Quantitative
• Radioactive isotope, fluorochromes, enzymes
Enzyme-linked approach
• Catalyst for chemical reactions
• It is not be altered
• Interaction A/g-A/b not affected
• Alkaline phosphatase, horseradish peroxidase (HRPand p-
nitrophenyl phosphatase
• Substrate dependent: colored product or chemiluminescence
Enzyme-linked
immunosorbent assay (ELISA)
Standard curve in
imunoassay
• Clinical test: The conc of A/g or A/b in biological
sample need to be known, by standard curve
• Can be used to read the conc of unknown
• Y-axis: readout (A655);X-axis: known conc of A/b
or A/g
• To ensure the values obtained fall within the
range, the signal arising from wide range of A/g
or A/b conc is used
Sandwich and competitive ELISA
Principles of immunoassay
• 2 types
• Competitive:labelled & non labelled A/g compete the same site
on A/b
• Non competitive:A/g & A/b are allowed to interact without
deliberate introduction of competing molecules
• Either way, the indicator label will be attached to A/g or A/b
Principles of immunoassay-
cont..
• Non competitive
• Indirect ELISA: to detect A/b
• A/g is affixed to solid phase
• Incubation with nonsps protein (BSA, casein milk,gelatin) or
detergent (Tween), to block nonsps binding site;
• Addition of A/b specific for A/g
• Sandwich ELISA: to detect A/g
• A/b is affixed to solid phase
• A/g is sandwiched in between the 2 A/b
• As above
Experimental design
• Antibody capture ELISA
• Screening antiserum for specific A/b
• Steps involved
1. A/g coating
2. Addition of primary (1st) A/b and washing
3. Addition of 2nd A/b and washing
4. Addition of substrate & stop solution
5. Read at A655
Enzyme-Linked Immunosorbant
Assay
Reagents:
Yellow tubes Test samples 2
Violet tube (+) Positive control 1
Blue tube (-) Negative control 1
Green tube (PA)Primary antibody 1
Orange tube (SA) Secondary antibody 1

Lab Equipment and Supplies:

Microplate strips, pipettor, pipette tips,
transfer pipette, wash buffer, paper towels,
marking pen
Experimental design-cont..
• A/g coating (sample or control).5 min, RT
• Stock: 1 ug/ml (1000 ng/ml), make it 2 fold dilution
• Addition of primary (1st) A/b and washing
• Addition of secondary (2nd) A/b and washing
ELISA Kit Results
 What Are The Reagents?
PBS: Phosphate buffered saline – provides stable
buffered environment to maintain antibody
structure

Tween 20: Nonionic detergent – removes non-

specifically bound proteins to reduce background
and blocks protein binding sites on the polystyrene

Microplates: Polystyrene – proteins absorb (bind)

by hydrophobic bonds to the polystyrene
What Are The Reagents?What
Function Do They Perform?
Antigen: Chicken gamma globulin

Primary antibody: Polyclonal anti-chicken antibody

made by rabbits

Secondary antibody: Polyclonal anti-rabbit antibody

made by goats linked (conjugated) to horseradish
peroxidase (HRP)

Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine

(TMB) – a colorless solution that when oxidized by HRP
turns blue
ELISA Kit Protocol Issues
Controls:
•Protocols I and II
Positive control = antigen
Negative control = PBS

•Protocol III
Positive control = primary antibody
Negative control = wash buffer

Triplicate samples: controls against cross contamination

Washing: eliminates non-specific binding and reduces background

 Ways The ELISA Kit Can Be Used
Real-World
Protocol Type of ELISA
Application
Tracking
HIV, SARS, smallpox &
I outbreaks of
anthrax
disease

Pregnancy, drugs, GMO,

II Detecting antigens
BSE (and all the above)

Detecting
antibodies in
III HIV, Lyme disease,
serum
smallpox and West Nile
IV virus
Deternination of
A/g concentration
 Protocol I: Tracking Disease Outbreaks

Real-World Application - HIV

Step 1 - Share Transmission & Detection
‘body fluid’
samples to Tube Actual Tube Simulated Tube
simulate Description Contents Contents
disease Student Antigen or Sample derived
transmission samples PBS from patient’s
blood
Step 2 - ELISA Primary Primary Antibody against
to detect antibody antibody HIV from mice
infection Secondary Secondary HRP-linked
antibody antibody antibody against
Results – mouse antibodies
Infection of about Positive Antigen Heat-inactivated
50% of students control HIV virus
Negative PBS HIV negative
control human serum
Protocol II: Antigen Detection
ELISA

Real-World Application – Pregnancy

Test
Tube Actual Tube Simulated Tube
Description Contents Contents
Protocol – Student Antigen or Patient’s urine
ELISA on samples PBS sample
simulated Primary Primary Antibody against
urine antibody antibody hCG hormone
samples from mice
Secondary Secondary HRP-linked
antibody antibody antibody against
mouse
antibodies
Positive Antigen Purified hCG
control hormone
Negative PBS Urine from
control woman that is
not pregnant
 Protocol III: ELISA Antibody Test
Real-World Application – Lyme Disease

Protocol - ELISA Tube Actual Tube Simulated Tube

Description Contents Contents
to detect exposure
to a disease Purified Antigen B. Bergoferi
antigen purified proteins
Do you have Student Primary Patient’s serum
antibodies to the samples antibody or samples
disease? wash buffer
Secondary Secondary HRP-linked
antibody antibody antibody against
B. bergoferi
Positive Primary patients Serum
control antibody Lyme disease
Negative Wash buffer Serum negative
control for Lyme
disease
Real-world Applications of Antibodies

Uses Applications
– Disease diagnosis – Dipstick tests/ELISA
– Basic Research – Immunostaining
– Immunotherapy – Western blotting

Bio-Rad’s HIV
Western Blot Kit
Example: Pregnancy Test
• How to read semi-quantitatively and quantitatively results?
• Semi-quantitative : perform serial dilution and read the titre.
• Quantitative : read the result using an ELISA reader.