1. Immunization and the Immune Response as a Function of Time
IMIN 372 Experiment 3
Immunization and the Immune Response as a Function of Time
Dr. James Stafford
CW319-A Biological Sciences Building

2. 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.

3. 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

4. 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

5. 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)

6. 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)

7. Basic Structure - A “Y” Shape
ANTIBODY STRUCTURE
Basic Structure - A “Y” Shape
2 Light
IDENTICAL
2 Heavy
IDENTICAL
-S-S-
Covalently held together by interchain disulfide bonds

8. BASIC STRUCTURE

9. Production of Antibodies
HUMORAL RESPONSE
Production of Antibodies

10. The Antibody Responses (Kinetics)
IgG produced
IgM
produced

11. The Primary Antibody Response

12. The Secondary Antibody Response

13. The Antibody Response

14. 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

15. 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

16. Human RBC's before and after adding incompatible serum
agglutinated cells
as clumps settle out of solution

17. 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 Y
no anti-sheep RBC Abs
with anti-sheep RBC Abs

18. Hemagglutination with anti-SRBC Antibodies
scored in 96 well plate
+ + +
+ + + - - - - -
wells must have a U-shaped bottom to see this effect

20. 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)

21. Reverse Agglutination of latex beads with covalently bound
antibody specific for Streptococcus group A Antigen Y Y
Red blood cells are not the only particulate antigens which can be used for agglutination assays.  In this example, microscopic latex particles are coated with an antibody specific for a bacterial (streptococcal Group A) antigen.  In the absence of antigen, the antibody-coated particles exhibit a smooth milky distribution (left).  In the presence of the antigen (a small amount of cultured bacteria) the particles clump into the pattern seen at the right.  This "reverse agglutination" (so named because it is the antibody, not the antigen, which is immobilized on the particles) is one of many kinds of tests used to identify different bacteria.  Y Y Y Y
no bacteria with Streptococcus added
UC Irvine, Medical school

22. Complement Fixation and Hemolysis

23. Complement Fixation and Hemolysis

24. Hemolysis in
96-well plates
Indiana state Univ, School of Medicine

25. 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
In it's simplest form the test is used to detect a patient serum antibody, so an ANTIGEN that is recognized by that antibody is the first reagent shown. If the antibody is present in the patient's serum it binds to the antigen, and the complement reagent is completely consumed in the reaction. (The test can also be used to look for antigen in the serum by modifying the reagents used).
The complement fixation assay indicator system uses sheep red blood cells (SRBC) and anti-SRBC antibody.
If the antibody specific for the antigen in the assay is present in the patient's serum, then complement is completely consumed in the reaction and there is none left to bind to the SRBC/anti-SRBC complexes.
A Test Positive For Ab =
NO HEMOLYSIS
PSP/labtests/complementfix.htm

26. v If patient has not been exposed to pathogen - then C'
is available to lyse RBC
Ag specific v
/labtests/complementfix.htm

27. 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/ /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