1. 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 Homepage: http://creative.smsu.edu/biology/ myersr/index.html TopClass: http://creative.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

5. 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  g/ml

6. 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

9. Radioimmunoassay A very sensitive test (0.001  g/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 125 I –Ab cannot distinguish between labeled and unlabeled Ag

10. 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

12. 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

13. 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

16. 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

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