1. Chapter 4: Serology Concepts

2.  Animals are constantly under attack by pathogens (viruses, bacteria, protists, and fungi)  Animals have evolved ways to defend themselves  Innate immunity (all animals)  Acquired immunity (vertebrates only) 2

3. 3 no memory memory

4.  Innate Immunity:  Barrier defense: ▪ In mammals, epithelial cells including skin, mucous membranes ▪ Saliva, mucous, and tears ▪ Acidic stomach juices ▪ Acidic sweat 4

5.  Innate Immunity:  Internal defenses: ▪ Phagocytic white blood cells (leukocytes) ▪ Neutrophils and macrophages ▪ Receptor-mediated recognition of fragments of molecules characteristics of a set of pathogens ▪ E.g. flagellin, lipopolysaccharides, dsRNA ▪ Engulf invaders and degrade in lysosomes ▪ Antimicrobial peptides and proteins ▪ E.g. interferons, complement system 5

6.  Innate Immunity:  Internal defenses: ▪ Inflammatory response 6

7.  Acquired Immunity:  Involves antigen recognition by lymphocytes  Antigens: foreign substances produced by invading organisms, usually proteins or polysaccharides ▪ Immunogen-natural antigen ▪ Usually macromolecules such as glycolipids (ABO system) and glycoproteins (Rh, Lewis systems) ▪ Haptens- small molecules that are not natural antigens ▪ Can conjugate with larger molecules to artificially elicit antibody formation (cocaine, amphetamines) 7

8.  Acquired Immunity:  B and T lymphocytes have specific antigen receptors embedded in plasma membrane ▪ 100,000 per cell ▪ Each cell expresses only one type of receptor  The receptors recognize a small accessible portion of the antigen called an epitope 8

9.  Acquired Immunity:  Most antigens are multivalent ▪ Have more than one epitope 9

10.  Acquired Immunity:  Receptors on B cells are Y-shaped ▪ 2 light chains and 2 heavy chains  Disulfide bridges hold the chains together  Transmembrane region near one end of each heavy chain anchors receptor in cell membrane  A short tail region at the end of the heavy chain extends into the cytoplasm 10

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12.  Acquired Immunity:  Sometimes B cells give rise to plasma cells that secrete a soluble form of the antigen receptor  The secreted receptor is called an antibody or immunoglobulin (Ig)  Found in serum portion of the blood  Five classes: IgG, IgA, IgM, IgD, and IgE 12

13. 13 Fragment antigen binding (Fragment crystallizable) Lower part of heavy chains = constant domains

14.  Antigens and antibodies are important tools in forensic serology  Antibodies to specific proteins can be raised in animals and used in the identification of bodily fluids  Hemoglobin in blood  Prostate specific antigen (PSA) in semen  Amylase in saliva 14

15.  Polyclonal Antibodies:  Generated by introducing an antigen (e.g. human hemoglobin) into a host animal (e.g. rabbit)  The animal’s B cells express receptors that bind various epitopes on the antigen  Some of these B cells become plasma cells and the receptors are released into the blood serum, forming a group of soluble antibodies  Blood is removed from the host animal and allowed to clot; serum contains antibodies 15

16. 16 Polyclonal antibodies

17.  Monoclonal Antibodies  Preparation of antibodies from a single plasma cell; all antibodies are identical and recognize the same epitope ▪ Plasma cells harvested from innoculated host animal’s spleen ▪ Plasma cells are immortalized by fusing them with myeloma cells to form hybridoma cells ▪ Pools of hybridoma cells are diluted into single clones and allowed to proliferate ▪ Clones are screened for antibody of interest 17

18.  Antiglobulins: Immunoglobins that are, themseves, immunogens  Antiglobulins recognize a common epitope on the immunoglobulins produced by a particular animal  E.g. Goat anti-mouse antiglobulins are raised in goat against mouse immunoglobulins ▪ Have important uses in forensic science ▪ Use in immunochromatographic assays we will perform in lab

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20.  Mediated by the interaction between epitope of the antigen and the binding site of the antibody  Strongest binding occurs only if the shape of the epitope fits the binding site  Depends on:  Affinity  Avidity 20

21.  Affinity- the strength of the interaction between a single epitope on the antigen and a single binding site on the corresponding antibody  Depends on specificity of antibody for antigen  Cross-reactions can occur  Avidity- the overall strength of binding between an antibody and an antigen  Overall synergistic strength of all interactions 21

22. 22 AFFINITYAVIDITY

23.  Two types used in forensic serology:  Primary ▪ Initial binding of a single epitope of an antigen and single binding site of an antibody to form an antigen- antibody complex ▪ Most sensitive techniques for detecting amounts of antigen and antibody in a sample  Secondary ▪ Less sensitive but easier to perform ▪ Three types: precipitation, agglutination, and complement fixation 23

24. 24 Primary antigen-antibody reaction affinity constant

25.  Secondary Reactions  Precipitation  Agglutination  Complement Fixation 25 Form the basis for many serologic assays in forensic labs Used less frequently in forensic serology

26.  Precipitation  May occur when a soluble antigen is incubated with its antibody  The antigen-antibody complex cross-link to one another to form an insoluble precipitate  Whether or not a precipitation reaction occurs depends on the relative concentrations of antigen and antibody ▪ Precipitin Curve Forensic Biology by Richard Li26

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28.  Prozone:  There is far more antibody than antigen  Cross-linking does not occur because there is not enough antigen 28

29.  Zone of Equivalence:  Reached as more antigen is added  Cross-linking occurs between neighboring complexes 29

30.  Post Zone:  Antigen is in excess  There is not enough antibody for cross-links to form 30

31.  Agglutination  If antigens are located on the surface of cells or carriers, the interaction of antibodies and antigens will cause the cells or carriers to aggregate and form larger complexes  If the antigen is located on a red blood cell, reaction is called hemagglutination  A visible clump is observed in the test tube if an agglutination reaction has occurred 31

32. 32 Agglutination Red blood cell expressing A antigen Antibody to A antigen in blood serum of person with Type B blood

33. 33 Blood GroupAntigensAntibodiesCan give blood to Can receive blood from ABA and BNoneABAB, A, B, 0 AABA and ABA and 0 BBAB and ABB and 0 0NoneA and BAB, A, B, 0 0