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].

IMIN 372 Experiment 3

Immunization and the Immune Response as a Function of Time

Dr. James StaffordCW319-A Biological Sciences Building

[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:

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

v

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