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ELISA
(IHC Method)
Enzyme Linked Immunosorbant Assay
ELISA
Enzyme-Linked • Mammalian immune system
Immunosorbant
Assay • Antibody specificity
• Biology’s “magic bullet”
• Evolved over millions of years
• Harness nature’s tool kit
• Imagine the applications!
ELISA
Immuno
Explorer Kit • Lab completed in a 45 min period
Features • Supplies for 48 students (12 workstations)
• Comprehensive and flexible curriculum
• Compelling real-world links
• Cost effective
• Classroom Safe
• Striking results
What Can
You Teach
With The • Hands-on Immunology
ELISA Kit? • One lesson integrates multiple standards
– Health sciences
– Immunology
– Immune response – antibody/antigen interactions
– Disease – infection, detection, transmission
– Biowarfare – defense against acts of aggression
• Links to research, medicine, and consumer
products
• Modern way to teach classic and contemporary
science
• Teach more with less!
Immune
Response C. Macrophage
A. Pathogen
D. Macrophage
B. B cells
F. T cell E. Macrophage
G. B cell
J. Antibodies
attach to pathogen
H. Memory B cells
I. Plasma cells
ELISA
Antibody
Structure
Heavy
chain
Disulfide Light
bonds chain
ELISA ANIMATION
ELISA
Enzyme-Linked
Immunosorbant
Assay
ELISA Kit
Workstation
Inventory 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
ELISA Kit
Results
What Are
The
Reagents? PBS: Phosphate buffered saline – provides stable
And buffered environment to maintain antibody
What structure
Function Do
They Tween 20: Nonionic detergent – removes non-
Perform?
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? Antigen: Chicken gamma globulin
And
What Primary antibody: Polyclonal anti-chicken
Function Do antibody made by rabbits
They
Perform? 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 Controls:
Issues •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 (2 out of 3 ain’t bad)
Washing: eliminates non-specific binding and
reduces background
Ways The ELISA Kit Can Be Used
Protocol Type of ELISA Real-World Application
Tracking outbreaks of
I disease
HIV, SARS, smallpox & anthrax
Pregnancy, drugs, GMO, BSE
II Detecting antigens
(and all the above)
Detecting antibodies in HIV, Lyme disease, smallpox
III serum and West Nile virus
Protocol I: Tracking Disease Outbreaks
Real-World Application - HIV
Step 1 - Share Transmission & Detection
‘body fluid’
samples to Tube Actual Tube Simulated Tube
simulate disease Description Contents Contents
transmission Student Antigen or PBS Sample derived from
samples patient’s blood
Step 2 - ELISA to Primary Primary antibody Antibody against HIV
detect infection antibody from mice
Secondary Secondary HRP-linked antibody
antibody antibody against mouse
antibodies
Results – Positive control Antigen Heat-inactivated HIV
Infection of about virus
50% of students Negative PBS HIV negative human
control serum
Protocol II: Antigen Detection ELISA
Real-World Application –
Protocol - ELISA Pregnancy Test
on simulated urine
samples Tube Actual Tube Simulated Tube
Description Contents Contents
Student Antigen or PBS Patient’s urine sample
samples
Primary Primary antibody Antibody against hCG
antibody hormone from mice
Secondary Secondary HRP-linked antibody
antibody antibody against mouse
antibodies
Positive control Antigen Purified hCG hormone
Negative PBS Urine from woman
control that is not pregnant
Protocol III: ELISA Antibody Test
Real-World Application – Lyme
Protocol - ELISA Disease
to detect exposure
to a disease Tube Actual Tube Simulated Tube
Description Contents Contents
Do you have Purified antigen Antigen B. Bergoferi purified
antibodies to the proteins
disease? Student Primary antibody Patient’s serum
samples or wash buffer samples
Secondary Secondary HRP-linked antibody
antibody antibody against B. bergoferi
Positive control Primary antibody Serum from patients
with Lyme disease
Negative Wash buffer Serum negative for
control Lyme disease
Real-world Uses
– Disease diagnosis
Applications
– Dipstick tests/ELISA
Applications – Basic Research – Immunostaining
of Antibodies – Immunotherapy – Western blotting
Bio-Rad’s HIV-2
ELISA Kit
Bio-Rad’s HIV
Western Blot Kit
Example:
Pregnancy
Test
