ELISA experment by 2b3U0m2d

VIEWS: 24 PAGES: 19

									         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

								
To top