Immunological Techniques and Body Fluid Analysis

New Immunological Techniques

  • DNA or RNA based Techniques:
    • PCR amplification tests
    • mRNA profiling
    • 12s ribosomal RNA
    • Mitochondrial DNA sequence analysis

Key Definitions

  • Antigen (Ag):
    • Foreign substance that triggers antibody production; causes an immune response in vitro.
  • Antibody (Ab):
    • Substance-specific marker produced by the host's immune system to neutralize antigens.

Types of Antigens

  • Autologous Antigens:
    • Antigens from the same organism that induces immune response; not foreign to the host.
  • Homologous Antigens:
    • Antigens used to prepare antiserum; effective in immunization.
  • Heterologous Antigens:
    • Antigens different from the one used to create antiserum or vaccine.

Immune Responses

  • Primary Response:
    • First response of the immune system to a new antigen.
  • Secondary Booster Memory Response:
    • Faster response due to existing antibodies upon a second exposure to the antigen.

Antisera and Antibodies

  • Antiserum:
    • Purified blood serum containing a spectrum of antibodies.
  • Monoclonal Antibodies (mAb):
    • Specific antibodies produced by a cell line; bind to specific areas of the antigen.
  • Polyclonal Antibodies:
    • Antiserum containing antibodies reacting with multiple proteins at various sites.

Cross-Reactivity

  • Cross-Reactivity:
    • Ability of an antibody to react with closely related species or proteins.
  • Choosing Antigens:
    • Select antigens foreign to the test animal's immune cells, accessible to the immune system, and with appropriate size/complexity.

Testing Methods

  • Ouchterlony Double Diffusion Method:
    • Antigen and antibodies diffuse towards the zone of equivalence, where precipitin lines form.
  • Crossover Electrophoresis:
    • Utilizes an electric field to drive antigen-antibody interactions.
  • Chromatography Method:
    • Uses dye-tagged mobile antibodies to reveal antigen-antibody interactions.
  • Precipitation Reaction:
    • Forms visible complexes through extensive cross-linking of antigens and antibodies.

Ouchterlony Double Diffusion Test Results

  • Positive Result:
    • White lines indicating antigen recognition by antiserum.
  • Negative Result:
    • Absence of lines indicates no recognition.

Advantages and Disadvantages of Detection Methods

  • Ouchterlony Double Diffusion
    • Advantages: Allows multiple samples, less reagents, sensitive.
    • Disadvantages: Time-consuming, can lose sample concentration.
  • Tube Precipitin Test
    • Advantages: Quick and reliable.
    • Disadvantages: Prone to contamination; requires more antiserum.
  • Crossover Electrophoresis
    • Advantages: Sensitive, fast, inexpensive.
    • Disadvantages: Complex apparatus required.
  • Immunochromatographic Assays
    • Advantages: Simple, inexpensive, sensitive.
    • Disadvantages: False positives/negatives are possible, high-dose hook effect.

PCR Based Methods

  • Common Techniques:
    • Meow Plex, Genotypes Panel 11, Powerplex DNA Amplification Kits, Quantifier Human Quantification Kits, AMPFLSTR PCR Amplification Kits.
  • Advantages: Sensitive, no need for serum validation.
  • Disadvantages: Expensive, complicated, time-intensive.

Salivary Tests

  • Components of Saliva:
    • 97-99% water, mucin, histidine-rich proteins, salts, enzymes, antimicrobial functions.
  • Production: Parotid, submandibular, and sublingual glands.
  • Key Enzyme: Amylase, used in forensic tests (e.g., ALS).
  • Presumptive Tests for Saliva:
    • Phadebas, starch-iodine, PRA paper, RSID saliva test, SALIgAE.
  • Phadebas Test: Cross-reacts with feces, breast milk; positive = blue color.
  • Starch-Iodine Test: Positive = color change from blue to orange/yellow when amylase is present.

Urine Tests

  • Components of Urine:
    • Water, urea, inorganic ions, uric acid, creatinine, and more.
  • Presumptive Tests for Urine:
    • Urease, creatinine, sulfate tests, and RSID urine test.
  • Urease Test: Ammonia production indicates positive result through color change.

Fecal Analysis

  • Composition:
    • Approximately 75% water; solids include bacteria, fiber, fats, and proteins.
  • Presumptive Tests for Feces: Sight/smell, microscopic examination, chemical tests (e.g., Edelman's test).
  • Edelman’s Test: Extracts urobolin; positive results yield color changes under UV and white light.

Vaginal Fluid Tests

  • Key Compounds:
    • Glycogenated epithelial cells, peptides, lactic acid, immunoglobulins.
  • Presumptive Tests: Microscopic examination, immunohistochemical techniques.

Sweat Composition and Testing

  • Components of Sweat:
    • Organic compounds, salts, and immunoglobulins.
  • Presumptive Tests: SEM with EDX, immunoassays.

New Technologies for Body Fluid Differentiation

  • Central Dogma:
    • DNA transcription to mRNA, mRNA translation to protein; indicates specific proteins in body fluids.
  • Differentiating Epithelial Cells:
    • Imaging flow cytometry used to distinguish cell types based on morphology and autofluorescence properties.

Summary of Key Tests

  1. Phadebas Test: Detects amylase and identifies saliva.
  2. Urease Test: Indicates urine presence through pH change.
  3. Edelman's Test: Presence of fecal matter through color changes.