1 / 18

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

oliver
Télécharger la présentation

Immunology Chapter 6, Lecture 2

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. 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)

More Related