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Immunology Chapter 6, Lecture 2. Richard L. Myers, Ph.D. Department of Biology Southwest Missouri State Temple Hall 227 Telephone: 417-836-5307 Email: rlm967f@mail.smsu.edu. Agglutination reactions. Antibody reacts with a “particulate” antigen similar to a precipitation reaction

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immunology chapter 6 lecture 2
ImmunologyChapter 6, Lecture 2
  • Richard L. Myers, Ph.D.
  • Department of Biology
  • Southwest Missouri State
  • Temple Hall 227
  • Telephone: 417-836-5307
  • Email: rlm967f@mail.smsu.edu
agglutination reactions
Agglutination reactions
  • Antibody reacts with a “particulate” antigen
    • similar to a precipitation reaction
    • must watch antibody excess (prozone effect)
    • incomplete antibodies may block agglutination
  • Hemagglutination
    • agglutination involving RBCs
    • used in blood typing
    • IgM is the most effective agglutinating Ab
agglutination
Agglutination
  • Bacterial agglutination
    • antibodies can be made to agglutinate bacteria
    • can determine the Ab (agglutination) titer
    • this aids in diagnosis of bacterial infection
    • an example is typhoid fever (S. typhi)
  • Passive agglutination
    • simple and sensitive
    • can detect Ab as low as 0.001 mg/ml
agglutination1
Agglutination
  • Agglutination inhibition
    • very sensitive assay for antigen
    • best example of the pregnancy test
      • uses antibody to HCG coated latex particles
    • another example is detection of Ab to viruses that will hemagglutinate RBCs
      • Ab interfere with hemagglutination
      • example is testing for immunity to rubella virus
radioimmunoassay
Radioimmunoassay
  • A very sensitive test (0.001 mg/ml)
  • Used to quantitate hormones, serum proteins, drugs, vitamins and other things
  • Involves competitive binding
    • radiolabeled antigen and unlabeled antigen to a high-affinity Ab
    • Ag usually labeled with 125I
    • Ab cannot distinguish between labeled and unlabeled Ag
elisa or eia
ELISA or EIA
  • ELISA/EIA is similar to RIA but requires an enzyme rather than a radioisotope
    • can be indirect, sandwich or competitive ELISA
  • An enzyme conjugated to an antibody reacts with a colorless substrate to generate a colored product
western blotting
Western blotting
  • Method for identification of specific protein
    • Southern blotting identifies DNA
    • Northern blotting identifies mRNA
  • Protein mixture electrophoresed on PAGE
    • with SDS, a dissociating agent
  • Proteins then transferred to a membrane
  • Proteins of interest are detected by flooding membrane with radiolabeled monoclonals
immunofluorescence
Immunofluorescence
  • Antibody molecules are “tagged” with a fluorescent dye (fluorochrome)
    • fluorescein-labeled antibodies are popular
    • protein A from S. aureus freqeuntly used
    • another is biotin-avidin labeling
  • Immunofluorescence used to identify cells
    • can be direct or indirect
slide16
FACS
  • Fluorescence-activated cell sorter (FACS) used to identify and separate subpopulations of lymphocytes
    • process called flow cytometry
  • It is possible to analyze three fluorochromes on a single stained sample
assignment
Assignment
  • Begin reading Chapter 7, Organization and Expression of Ig Genes
  • Review question 4, (pg 192)