1 / 33

Forensic Biology by Richard Li

Chapter 4: Serology Concepts. Forensic Biology by Richard Li. 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).

hestia
Download Presentation

Forensic Biology by Richard Li

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

  11. 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

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

  13. 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

  14. 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

  15. Polyclonal antibodies

  16. 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

  17. 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

  18. 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

  19. 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

  20. AFFINITY AVIDITY

  21. 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

  22. affinity constant Primary antigen-antibody reaction

  23. 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

  24. 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

  25. Antigen-Antibody Binding Reactions

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

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

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

  29. 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

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

More Related