ELISA experment_2

8/3/2019 ELISA experment_2

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ELISA

(IHC Method)

Enzyme Linked Immunosorbant Assay


2/19

ELISA

Enzyme-LinkedImmunosorbantAssay

Mammalian immune system

Antibodyspecificity

Biologys magic bullet

Evolved over millions ofyears

Harness naturestool kit

Imagine the applications!


3/19

ELISAImmunoExplorer KitFeatures

Lab completed in a 45 min period

Supplies for 48 students(12 workstations)

Comprehensive and flexible curriculum

Compelling real-world links

Cost effective

Classroom Safe

Striking results


4/19

WhatCanYou TeachWith TheELISA Kit?

Hands-on Immunology

One lesson integrates multiple standards

Healthsciences

Immunology

Immune response antibody/antigen interactions

Disease infection, detection, transmission

Biowarfare defense against acts of aggression

Linksto research, medicine, and consumerproducts

Modern wayto teach classic and contemporaryscience

Teach more with less!


5/19

ImmuneResponse

A.Pathogen

C.Macrophage

D.Macrophage

E.MacrophageF.T cell

B.B cells

G.B cell

H.Memory B cells

I.Plasma cells

J.Antibodies

attach to pathogen


6/19

ELISAAntibodyStructure

Lightchain

Heavy

chain

Disulfidebonds


7/19

ELISAANIMATION


8/19

ELISAEnzyme-LinkedImmunosorbantAssay


9/19

ELISA KitWorkstationInventory Reagents:

Yellow tubes Test samples 2Violet tube (+) Positive control 1

Blue tube (-) Negative control 1Green tube (PA) Primary antibody 1Orange tube (SA) Secondary antibody 1

Lab Equipment and Supplies:

Microplate strips, pipettor, pipette tips,

transfer pipette, wash buffer, paper towels,

marking pen


10/19

ELISA KitResults


11/19

WhatAreTheReagents?AndWhatFunction DoTheyPerform?

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


12/19

WhatAreTheReagents?AndWhatFunction DoTheyPerform?

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


13/19

ELISA KitProtocolIssues

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 (2 out of 3 aint bad)

Washing:eliminates non-specific binding andreduces background


14/19

Ways The ELISA KitCan Be Used

Protocol Type of ELISA Real-World Application

ITracking outbreaks of

diseaseHIV, SARS, smallpox & anthrax

II Detecting antigensPregnancy, drugs, GMO, BSE

(and all the above)

IIIDetecting antibodies in

serum

HIV, Lyme disease, smallpox

and West Nile virus


15/19

Protocol I: Tracking Disease Outbreaks

Step 1 - Sharebody fluidsamples to

simulate diseasetransmission

Step 2 - ELISA todetect infection

Results

Infection of about

50% of students

Tube

Description

Actual Tube

Contents

Simulated Tube

Contents

Student

samples

Antigen orPBS Sample derived from

patients blood

Primary

antibody

Primary antibody Antibody against HIV

from mice

Secondary

antibody

Secondary

antibody

HRP-linked antibody

against mouseantibodies

Positive control Antigen Heat-inactivated HIV

virus

Negative

control

PBS HIV negative human

serum

Real-World Application - HIV

Transmission & Detection


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Protocol II:Antigen Detection ELISA

Protocol - ELISAon simulated urinesamples Tube

Description

Actual Tube

Contents

Simulated Tube

Contents

Student

samples

Antigen orPBS Patients urine sample

Primary

antibody

Primary antibody Antibody against hCG

hormone from mice

Secondary

antibody

Secondary

antibody

HRP-linked antibody

against mouseantibodies

Positive control Antigen Purified hCG hormone

Negative

control

PBS Urine from woman

that is not pregnant

Real-World Application

Pregnancy Test


17/19

Protocol III:ELISAAntibody Test

Protocol - ELISAto detect exposureto a disease

Do you haveantibodies to thedisease?

Tube

Description

Actual Tube

Contents

Simulated Tube

Contents

Purified antigen Antigen B. Bergoferipurified

proteins

Student

samples

Primary antibody

or wash buffer

Patients serum

samples

Secondary

antibody

Secondary

antibody

HRP-linked antibody

against B. bergoferi

Positive control Primary antibody Serum from patients

with Lyme disease

Negative

control

Wash buffer Serum negative for

Lyme disease

Real-World Application Lyme

Disease


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Real-worldApplicationsofAntibodies

Uses Disease diagnosis

Basic Research

Immunotherapy

Applications Dipstick tests/ELISA

Immunostaining

Western blotting

Bio-Rads HIV-2

ELISA Kit

Bio-Rads HIV

Western Blot Kit


19/19

Example:PregnancyTest

ELISA experment_2

Documents

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