Transcript A “Y”

IMIN 372 Experiment 3
Immunization and the Immune
Response as a Function of Time
Dr. James Stafford
CW319-A Biological Sciences Building
492-9258
[email protected]
HEMAGGLUTINATION & HEMOLYSIS ASSAYS
MONITORING THE PRIMARY HUMORAL IMMUNE
RESPONSE
1. Overview of antigens and antibodies.
2. Humoral immune response kinetics.
3. Application of agglutination assays.
4. Applications of hemolysis, including Complement
assays.
ANTIGENS / ADJUVANTS
DEFINITIONS
-Antigens are molecules that elicit an immune response in the body.
-Antigens can be:
Proteins
Polysaccharides - sugars such as mannose.
Lipoproteins - conjugates of lipids (fats) with proteins.
-Adjuvants are agents that may stimulate the immune system and
increase the response to a vaccine, without having any specific
antigenic effect on its own.
-Adjuvants can be:
Oils
Aluminum salts
Virosomes
TYPES OF ANTIGENS
EXOGNEOUS ANTIGENS
Antigens that enter the body from the environment
Inhaled Antigens
-Proteins on cat hairs
-Dust
-Pollen
ASTHMA ATTACK
Ingested Antigens
-Shellfish proteins
-Peanuts
ALLERGIC RESPONSES
Antigens Introduced Beneath Skin
-Splinter
-Injected vaccine
IMMUNIZATION
TYPES OF ANTIGENS
ENDOGENOUS ANTIGENS
Antigens that are generated within cells of the body
Proteins encoded by the genes of viruses (foreign)
Abnormal or altered proteins
-Encoded by mutant genes
(e.g. mutated proteins produced by cancer cells)
ANTIBODIES
Also referred to as Immunoglobulins (or Ig)
-Proteins produced by plasma cells (activated B-cells).
-One of the major proteins found in the serum.
-Antibodies are used by the immune system to identify and
neutralize foreign objects like bacteria and viruses.
-Five different classes of antibodies in mammals;
-IgA, IgD, IgE, IgG and IgM
-also subclasses of antibodies; IgG
IgG1, IgG2, IgG3, IgG4
-Antibodies can be found at mucosal sites and in milk.
-Secretory antibodies (IgA and IgM)
ANTIBODY STRUCTURE
Basic Structure - A “Y” Shape
2 Light
2 Heavy
IDENTICAL
-S-S-
IDENTICAL
Covalently held
together by
interchain
disulfide bonds
BASIC STRUCTURE
HUMORAL RESPONSE
Production of Antibodies
The Antibody Responses (Kinetics)
IgG produced
IgM
produced
The Primary Antibody Response
The Secondary Antibody Response
The Antibody Response
Measuring The Antibody Response
Hemagglutingation & Hemolysis
-First observed in the 17th century -early attempts to perform blood transfusions-
Hemagglutination
Cross-linking of RBC by antibodies
Hemolysis
Lysis of RBC’s by antibodies and complement
Discovery of ABO blood Types (Karl Landsteiner)
3 types of sugars found
on RBC's....
Similar to bacterial
Antigens (i.e. LPS)
Kuby 16-13
Human RBC's before and after adding incompatible serum
agglutinated cells
as clumps settle out of solution
Hemagglutination
in the absence of cross-linking Abs the SRBCs will eventually
settle together into a 'button' in U-shaped wells
x-linking or agglutination spreads out the RBCs
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no anti-sheep RBC Abs
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with anti-sheep RBC Abs
Hemagglutination with anti-SRBC Antibodies
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scored in 96 well plate
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wells must have a U-shaped bottom to see this effect
Hemagglutination - Modifications
Can couple (or attach) selected antigens to SRBC surface
-useful for antibody screening experiments.
Replace SRBC’s with bacteria
Replace SRBC’s with latex beads covalently bound by antigen.
Coat particles with antibody instead of antigen
(Reverse Agglutination)
Reverse Agglutination of latex beads with covalently bound
antibody specific for Streptococcus group A Antigen
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no bacteria
with Streptococcus added
UC Irvine, Medical school
Complement Fixation and Hemolysis
Complement Fixation and Hemolysis
Hemolysis in
96-well plates
Indiana state Univ, School of Medicine
The Complement Fixation
Assay Indicator System
If patient has been exposed
to pathogen - then all C'
is fixed in presence of Ag...
no C' available to lyse RBC
http://web.indstate.edu/thcme/
PSP/labtests/complementfix.htm
If patient has not been exposed
to pathogen - then C'
is available to lyse RBC
Ag specific
v
http://web.indstate.edu/thcme/PSP
/labtests/complementfix.htm
DILUTION SERIES
Serial dilutions - each successive dilution is derived from previous
e.g. a 2-fold dilution series
1 volume buffer is placed in each well, then an equal volume of
solution to be diluted is passed in succession down the wells
original
solution
1/2
1/4
1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 1/2048
The TITRE corresponds to the most dilute concentration at which desired
effect is still seen.
Usually the dilution factor is stated - e.g. a titre of 512 ≈ 1/512 dilution