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Immunology Chapter 6, Lecture 2

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

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  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Assignment • Begin reading Chapter 7, Organization and Expression of Ig Genes • Review question 4, (pg 192)

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