IMIN 372 Experiment 3
Immunization and the Immune Response as a Function of Time
Dr. James StaffordCW319-A Biological Sciences Building
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:
ProteinsPolysaccharides - 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:
OilsAluminum saltsVirosomes
TYPES OF ANTIGENS
EXOGNEOUS ANTIGENS
Antigens that enter the body from the environment
Inhaled Antigens-Proteins on cat hairs-Dust ASTHMA ATTACK-Pollen
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
IDENTICAL
2 Heavy
IDENTICAL
-S-S
-
-S-S-
-S-S-
Covalently held together by interchain
disulfide bonds
BASIC STRUCTURE
HUMORAL RESPONSEProduction of Antibodies
The Antibody Responses (Kinetics)
IgM produced
IgG 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-
HemagglutinationCross-linking of RBC by antibodies
HemolysisLysis of RBC’s by antibodies and complement
3 types of sugars foundon RBC's....
Similar to bacterial Antigens (i.e. LPS)
Kuby 16-13
Discovery of ABO blood Types (Karl Landsteiner)
Human RBC's before and after adding incompatible serum
agglutinated cellsas clumps settle out of solution
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no anti-sheep RBC Abs with anti-sheep RBC Abs
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
Hemagglutination with anti-SRBC Antibodies
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scored in 96 well plate
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
UC Irvine, Medical school
no bacteria with Streptococcus added
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Complement Fixation and Hemolysis
Complement Fixation and Hemolysis
Hemolysis in 96-well plates
Indiana state Univ, School of Medicine
http://web.indstate.edu/thcme/PSP/labtests/complementfix.htm
If patient has been exposed
to pathogen - then all C'
is fixed in presence of Ag...
no C' available to lyse RBC
The Complement FixationAssay Indicator System
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
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DILUTION SERIES
Serial dilutions - each successive dilution is derived from previous
e.g. a 2-fold dilution series1 volume buffer is placed in each well, then an equal volume of solution to be diluted is passed in succession down the wells
1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 1/2048
originalsolution
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
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