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 492-9258 stafford@ualberta.ca

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 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)

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

BASIC STRUCTURE

Production of Antibodies 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 Y no anti-sheep RBC Abs with anti-sheep RBC Abs

Hemagglutination with anti-SRBC Antibodies 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 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

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 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 http://web.indstate.edu/thcme/ PSP/labtests/complementfix.htm

v 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