Forensic Biology by Richard Li

1. Chapter 4: Serology Concepts Forensic Biologyby Richard Li

2. Serological Reagents • 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)

3. no memory • memory

4. Serological Reagents • Innate Immunity: • Barrier defense: • In mammals, epithelial cells including skin, mucous membranes • Saliva, mucous, and tears • Acidic stomach juices • Acidic sweat

5. Serological Reagents • 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

6. Serological Reagents • Innate Immunity: • Internal defenses: • Inflammatory response

7. Serological Reagents • 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)

8. Serological Reagents • 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

9. Serological Reagents • Acquired Immunity: • Most antigens are multivalent • Have more than one epitope

10. Serological Reagents • 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

12. Serological Reagents • 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

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

14. Serological Reagents • 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

15. Serological Reagents • 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

16. Polyclonal antibodies

17. Serological Reagents • 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

18. Serology Reagents • 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

20. The Strength of Antigen-Antibody Binding • 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

21. The Strength of Antigen-Antibody Binding • 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

22. AFFINITY AVIDITY

23. Antigen-Antibody Binding Reactions • 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

24. affinity constant Primary antigen-antibody reaction

25. Antigen-Antibody Binding Reactions • Secondary Reactions • Precipitation • Agglutination • Complement Fixation Form the basis for many serologic assays in forensic labs Used less frequently in forensic serology

26. Antigen-Antibody Binding Reactions • 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 Li

27. Antigen-Antibody Binding Reactions

28. Antigen-Antibody Binding Reactions • Prozone: • There is far more antibody than antigen • Cross-linking does not occur because there is not enough antigen

29. Antigen-Antibody Binding Reactions • Zone of Equivalence: • Reached as more antigen is added • Cross-linking occurs between neighboring complexes

30. Antigen-Antibody Binding Reactions • Post Zone: • Antigen is in excess • There is not enough antibody for cross-links to form

31. Antigen-Antibody Binding Reactions • 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

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