IMMUNOLOGICAL METHODS Dr. Gülderen Yanıkkaya Demirel

OUTLINE of THIS LECTURE Definition of Immunology Innate and Adaptive Immunity Antigen/Antibody Immunological Methods

Innate Immunity Ancient system - present in invertebrates naïve, immediate, everyday immunity Molecules recognize common features of pathogens Lectin Phagocytes, large lymphocytes (NK cells) Complement

Adaptive Immunity specialized, late, long term immunity Newer system - present in fish, birds, human specialized, late, long term immunity Molecules recognize specific features of pathogens Antibodies B and T cells - small lymphocytes Immunological memory

Innate and Adaptive Responses Pathogen independent Immediate (hours) Neutrophils Macrophages Mast cells Eosinophils Basophils NK cells “Large Lymphocytes” = NK cells Adaptive Pathogen-dependent Slower (days) Dendritic cells B cells T cells (CD4 or CD8) “Small Lymphocytes” = B & T cells Both systems “talk” to each other to modulate response Both systems use leukocytes = white blood cells

IMMUNOLOGICAL METHODS depend on Ag Ab interactions

WHAT IS AN ANTIGEN ? An antigen is any substance that elicits an immune response and is then capable of binding to the subsequently produced antibodies. Antigens are generally proteins or polysaccharides, but other substances such as nucleic acids can also be antigens.

How, Why and Where are Antibodies Produced How, Why and Where are Antibodies Produced? (Massively condensed version) Produced in vertebrate cells in response to a ‘non-self’ substance termed an antigen. The process begins with antigen presenting cells roaming the bloodstream. They engulf ‘non-self’ molecules and then display portions of the engulfed molecules on their outer plasma membranes. After many recognition steps B-cells are stimulated to produce specific antibodies. The antibodies then go off and bind to the foreign substance thereby marking the substance for destruction.

Recognition modules of Adaptive immunity B-cell receptor (BCR) T cell receptor (TCR) B cells T cells TCR is membrane bound Antibody is a secreted form of BCR

IMMUNOLOGICAL METHODS Agglutination/Hemaglutination Immunoprecipitation Immunodiffusion Immunoelectrophoresis RIA/ELISA Immunoflorescence Immunohistochemistry Flow Cytometry Molecular Methods PCR Sequence Analysis Immunomics Gene transfection

AGGLUTINATION/HEMAGGLUTINATION Definition - tests that have as their endpoint the agglutination of a particulate antigen Agglutinin/hemagglutinin Y +  Qualitative agglutination test Ag or Ab

AGGLUTINATION/HEMAGGLUTINATION Quantitative agglutination test Titer Prozone 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 4 Patient 1 2 3 5 6 7

AGGLUTINATION/HEMAGGLUTINATION Definition Qualitative test Quantitative test 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Applications Blood typing Bacterial infections Fourfold rise in titer Practical considerations Easy Semi-quantitative

Radial Immunodiffusion Method Ab in gel Ag in a well Ag Ab in gel Ag Concentration Diameter2 Interpretation Diameter of ring is proportional to the concentration Quantitative Ig levels

p. 152

Immunoelectrophoresis Method Ags are separated by electrophoresis Ag - + Ag Ab Ag Ab Interpretation Precipitin arc represent individual antigens

IMMUNE ELECTROPHORESIS

ELISA – Enzyme Linked Immuno Sorbent Assay

RIA – Radio Immuno Assay

ELISPOT

Immunofluorescence Direct Y Ab to tissue Ag is labeled with fluorochrome Ag Y Fluorochrome Labeled Ab Tissue Section

Immunofluorescence Indirect Y Qualitative to Semi-Quantitative Ab to tissue Ag is unlabeled Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab Qualitative to Semi-Quantitative

ImmunoHistochemistry

ImmunoHistochemistry Arrows indicate localized CD3+ lymphocytes in tissue Antibodies against surface receptors react with tissue on slide. Secondary antibody with chromgenic probe recognizes first antibody. Addition of substrate allows deposition of material and histological detection/localization of cell populations within tissue.

Mixed population containing CD3+ cells Add FITC-antibody specific for Laser Excitation Light Scatter Detector FITC Fluor CD3+ CD3+ CD3+ CD3+ CD3+ CD3+

p. 163

Immunoelectron microscopy

AUTOANTIBODIES

dsDNA

LYMPHOCYTE PROLIFERATION Reactive Lymphocytes Mitogen (ConA) added Antigen added

MAGNETIC BEAD SORTING p. 160

CELL SORTING

MOLECULAR METHODS

Western blotting

GENE TRANSFECTION

SUGGESTED READING Abbas Immunology 2012 edition Kuby’s Immunology Janeway Immunology Temel İmmunoloji, İmmun sistemin İşlev ve Bozuklukları, 2007.