Immunology Chapter 6, Lecture 2 Richard L. Myers, Ph.D. Department of Biology Southwest Missouri...
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Transcript of Immunology Chapter 6, Lecture 2 Richard L. Myers, Ph.D. Department of Biology Southwest Missouri...
![Page 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/1.jpg)
ImmunologyChapter 6, Lecture 2
• Richard L. Myers, Ph.D.
• Department of Biology
• Southwest Missouri State
• Temple Hall 227
• Telephone: 417-836-5307
• Email: [email protected]
![Page 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/2.jpg)
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
![Page 3: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/3.jpg)
![Page 4: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/4.jpg)
![Page 5: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/5.jpg)
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
![Page 6: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/6.jpg)
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
![Page 7: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/7.jpg)
![Page 8: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/8.jpg)
![Page 9: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/9.jpg)
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 125I– Ab cannot distinguish between labeled and
unlabeled Ag
![Page 10: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/10.jpg)
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
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![Page 12: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/12.jpg)
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
![Page 13: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/13.jpg)
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
![Page 14: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/14.jpg)
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![Page 16: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/16.jpg)
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
![Page 17: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/17.jpg)
![Page 18: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070400/56649f125503460f94c25a36/html5/thumbnails/18.jpg)
Assignment
• Begin reading Chapter 7, Organization and Expression of Ig Genes
• Review question 4, (pg 192)