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In Search of the Body’s Antibodies:
Investigate Antibodies Using Enzyme
Linked Immunosorbent Assay (ELISA)
Module developed at Boston
University School of Medicine
Presented by Dr. Dan Murray
The human body has developed
intricate means of defense against
infections, tissue damage, and
abnormal body cells.
Outline
•Non-specific immunity
•Specific immunity
•Immunoglobulin Structure and Function
•Enzyme Linked Immunosorbent Assay
Non-specific Immunity
Two Lines of Defense
•
Non-specific (innate) Immunity
–
–
•
Body’s response is effective against a
variety of “attackers”
Involves antimicrobial cells and proteins
Specific (acquired) Immunity
–
–
Body’s response is tailored for a
specific “attacker”
Involves antibodies
Non-specific (innate) Defenses
•
Mediated by host cells
–
–
•
Phagocytosis (by phagocytes)
Non-phagocytic cells
Mediated by host proteins
–
–
Complement system
Interferons
Each of these play a role following a
microbial infection and/or a wound to tissue.
Phagocytosis
Ingestion of infecting microbes by phagocytic
white blood cells (i.e., leukocytes)
•
•
Neutrophils – short-lived; 60-70%
of leukocytes
Macrophages – long-lived; develop
from monocytes
Non-phagocytic Cells
Killing is by means other than
phagocytosis
•
•
Eosinophils – effective against
larger parasites; attach to
parasite and discharge
destructive enzymes
http://www.som.tulane.edu/classware
/pathology/Krause/Blood/BL11a.html
Natural Killer Cells – destroy
infected cells or precancerous
cells by destroying the cell
membrane
http://www.cat.cc.md.us/courses/
bio141/lecguide/unit3/nknomhc.html
Complement System
•
•
•
Made up of about 20 serum
proteins
Form pores in microbial cells that
cause them to lyse
Also functions in Specific Immunity
Interferons
•
•
Proteins secreted by virus-infected
cells
Inhibit virus reproduction in
neighboring cells
Specific Immunity
Antibody-Antigen Interaction
•
Antigen - any agent capable of eliciting an
immune response
–
–
•
Isolated molecules
Molecules on surface of cell or virus
A specific antibody molecule will be able to
recognize a specific epitope of an antigen
–
Antibody binds to antigen
Clonal Selection
•
•
•
•
•
The proliferation of lymphocyte cells due to
activation by an antigen
Useful in primary (first exposure to antigen)
and secondary (subsequent exposure to
same antigen) immune responses
Results in production of many antibodies
against the antigen
Primary immune response – 10-17 days
before maximum response is mounted
Secondary immune response – 2-4 days for
maximum response
Clonal Selection
• B-lymphocyte
binds antigen
• Stimulates
reproduction of Bcells
• B-cell differentiates
into memory cells
and plasma cells
–Plasma cells
produce soluble
antibody
–Memory cells
display antibody on
surface
Immunoglobulins:
Structure and Function
Immunoglobulin Structure
• Heavy & Light
Chains
• Disulfide bonds
– Inter-chain
– Intra-chain
Disulfide bond
Carbohydrate
CL
VL
CH2
CH1
VH
Hinge Region
CH3
Immunoglobulin Structure
Disulfide bond
• Variable &
Constant Regions
– VL & CL
– VH & CH
Carbohydrate
CL
VL
• Hinge Region
CH2
CH1
VH
Hinge Region
CH3
Immunoglobulin Fragments:
Structure/Function Relationships
Ag Binding
Complement Binding Site
Binding to Fc
Receptors
Placental Transfer
Enzyme-Linked Immunosorbent
Assays (ELISA)
ELISA
Used for Ab detection
• Immobilize Ag
• Incubate with patient sample
• Add antibody-enzyme
conjugate
• Amount of antibody-enzyme
conjugate bound is proportional
to amount of Ab in the sample
• Add substrate of enzyme
• Amount of color is
proportional to amount of Ab
in patient’s sample
Ab-enzyme
conjugate
X
Ab in
patient’s
sample
Immobilized
Y
Ag
ELISA
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Patient #
1
2
+ Control
 Control
1/2
Dilutions of patient sample are placed in
adjacent wells of microtiter plate
More intense color = more Ab present
Antibody-Antigen Interaction
Nature of Ag/Ab Reactions
• Lock and Key Concept
http://www.med.sc.edu:85/chime2/lyso-abfr.htm
• Non-covalent Bonds
– Hydrogen bonds
– Electrostatic bonds
– Van der Waal forces
– Hydrophobic bonds
• Multiple Bonds
• Reversible
Source: Li, Y., Li, H., Smith-Gill, S. J.,
Mariuzza, R. A., Biochemistry 39, 6296, 2000