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Antibodies as Laboratory Tools

Antibodies as Laboratory Tools. Antibodies Bind Specifically to Antigens. Antigenic Determinants. Antibodies recognize and react with antigenic determinants or epitopes. Figure 17.3. Antibodies in the Body. Amino Acid Side Groups Interact. B. Native Protein. A. Antibody. Antibody.

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Antibodies as Laboratory Tools

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  1. Antibodies as Laboratory Tools

  2. Antibodies Bind Specifically to Antigens

  3. Antigenic Determinants • Antibodies recognize and react with antigenic determinants or epitopes. Figure 17.3

  4. Antibodies in the Body

  5. Amino Acid Side Groups Interact B Native Protein A Antibody Antibody Multiple Antibodies can bind a single protein/Antigen

  6. Clonal Selection Figure 17.8

  7. Polyclonal Antibody Production

  8. Monoclonal Antibody Production Figure 17.11

  9. Antibody Tests Applications Figure 18.13

  10. Fluorescent Antibody Techniques (Direct) Figure 18.10a

  11. Antibody titer: • Is the amount of Ab in serum Figure 17.10

  12. Antibody Titer • Is the concentration of antibodies against a particular antigen Figure 18.5

  13. Enzyme-Linked Immunosorbent Assay (ELISA)

  14. Enzyme-Linked Immunosorbent Assay(Direct ELISA) Figure 18.12a

  15. Enzyme-Linked Immunosorbent Assay (Indirect ELISA) Figure 18.12b

  16. Lab Skill • Columbia Scientists used Bioinformatics to compare the viral sequences of H1N1 Viruses • A gene was found that coded for a protein called, PB1-F2, which correlates with severity of the illness • Your job is to use ELISA to investigate the presence of the H1N1 viral protein associated with high mortality rate and inform the attending physicians

  17. Serum from Patients added to wells that already have the PB1-F2 Antigen bound (indirect ELISA) 1º Ab _ +

  18. Wells are Washed with PBS Buffer Only Antibodies that are able to bind the Antigen will remain _ +

  19. Addition of secondary antibody (2º Ab), which is specific for the 1º Ab species and has been conjugated with an enzyme,Horseradish Peroxidase (HRP) or Alkaline Phosphotase (AP). 2º Ab Enzyme _ +

  20. Only Wells that had Primary Antibody bound will get the labeled Secondary Antibody _ +

  21. Chemical Substrate is added, such that wells containing the Enzyme-linked Antibodies will convert the substrate and a color change occurs Color Change= Antibodies to Antigen are Present _ +

  22. SDS PAGE Separates Proteins by Size - +

  23. B Native Protein A Heat (95C), disulfide reducing agent (-mercaptoethanol), and detergent Sodium DodecylSulfate (SDS) Denatured Protein A A A B Discontinuous Epitope Epitope

  24. Western Blotting Transfer to Membrane Filter Paper Nitrocellulose SDS PAGE Filter Paper + _

  25. Proteins move from Gel to Membrane at Same Location Filter Paper SDS PAGE Filter Paper Nitrocellulose + _

  26. Detection of Specific Protein With Labeled Antibody Nitrocellulose Antibody conjugated with an enzyme such as Horseradish Peroxidase (HRP) or Alkaline Phosphates (AP).

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