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
Source: Biorad
ELISA technique
Is a biochemical technique used mainly in immunology to detect the
presence of an antibody or an antigen in a sample.
 Requirements for ELISA test

Source: Invitrogen and Biotek

Stages in ELISA
1.The adsorption of either antigen or antibody to the micro-titer plate.
2.The addition of the test sample and subsequent reagents.
3.The incubation of reactants (formation of antigen-antibody complex).
4.The separation of bound and free reactants by washing.
5.The binding of enzyme to the target antigen or antibody.
6.The addition of substrate (production of a visible signal).
7.The visual or spectrophotometric reading of the assay.
ELISA Enzyme-
Linked
Immunosorbant
Assay
 Reagents:
Yellow tubes Test samples 2
Violet tube (+) Positive control 1
Blue tube (-) Negative control 1
ELISA Kit Workstation Green tube (PA) Primary antibody 1
Inventory Orange tube (SA) Secondary antibody1

Lab Equipment and Supplies:

Microplate strips, pipettor, pipette tips,
transfer pipette, wash buffer, paper towels,
marking pen
ELISA Kit Quick Guide
ELISA Kit
Results
 PBS: Phosphate buffered saline –
provides stable buffered environment
What Are The to maintain antibody structure
Reagents? Tween 20: Nonionic detergent –
And removes non-specifically bound
What Function Do proteins to reduce background and
blocks protein binding sites on the
They Perform? polystyrene
Microplates: Polystyrene – proteins
absorb (bind) by hydrophobic bonds
to the polystyrene
 Antigen: Chicken gamma globulin

What Are The Primary antibody: Polyclonal anti-chicken

antibody made by rabbits
Reagents?
Secondary antibody: Polyclonal anti-rabbit
And antibody made by goats linked
What Function Do (conjugated) to horseradish peroxidase
(HRP)
They Perform?
Enzyme substrate: 3,3’,5,5’ –
tetramethylbenzidine (TMB) – a colorless
solution that when oxidized by HRP turns
blue
 Controls:
•Protocols I and II
Positive control = antigen
Negative control = PBS

•Protocol III
ELISA Kit Protocol Positive control = primary antibody
Negative control = wash buffer
Issues
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 Transmission &
Step 1 - Share 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 – Pregnancy Test
Protocol - ELISA
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 Disease
Protocol - ELISA
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
 Uses Applications
– Disease diagnosis – Dipstick tests/ELISA
– Basic Research – Immunostaining
– Immunotherapy – Western blotting

Real-world
Applications of
Antibodies

Bio-Rad’s HIV-2
ELISA Kit

Bio-Rad’s HIV
Western Blot Kit
Example:
Pregnancy Test