Immunological Applications. Chapter 8: Background

Transcription

1 Immunological Applications Chapter 8: Background

2 The Immune System Types of Immunity Innate The natural immunity present at birth Acquired A specific response to foreign substances. Some cells remember invaders and can respond quickly to future infections Immunizations trigger this response so that recipients are prepared for future infections 2

3 The Immune System Antigens Something that elicits an immune response Antibodies Can be viruses, bacteria, toxins, drugs, DNA, RNA, and other chemicals Proteins produced by white blood cells that bind to antigens Epitope A single shape on the antigen that an antibody will bind to 3

4 The Immune Response Pathogen has antigen on surface, is contacted by macrophage and engulfed through phagocytosis The macrophage displays antigen on surface with major histocompatibility complex (MHC) T cells relay the message to B cells B cells start producing antibodies and become plasma cells Some become memory B cells 4

5 The Immune System 5

6 Antibodies Antibodies are also called immunoglobulins (Ig) Types IgA: first line of defense with invaders found in tears, saliva, milk IgD: initiates B cells and activates other immune cells IgE: allergic reactions IgG: most abundant, produced when infected IgM: found in serum, responsible for primary immune response 6

7 Immune Response Primary response Initial production of antibodies when infected Response is slow Secondary response Response after an initial exposure Much more rapid and robust 7

8 Antibodies as Tools Two main types of antibodies produced to be used as tools in biotech Polyclonal antibodies are produced by injecting an animal with an antigen and harvesting the serum that contains the antibodies 8

9 Antibodies as Tools Monoclonal antibody production Extract B cells from an inoculated animal Fuse with a tumor cell to make immortal Clone the immortal cells and collect antibodies 9

10 Antibodies as Tools Antibodies to deliver treatment to specific cells Humanized recombinant antibodies combine part of a human and part of a mouse antibody sequence 10

11 Immunoassays Tests that use antibodies as a component Most common is pregnancy test Urine containing hcg is added to strip Mobile colored antibodies bind and move They bind to a stationary set against hcg Or continue to bind to a control set 11

12 Other Types of Assays Enzyme-linked immunosorbant assays (ELISA) Ouchterlony or double diffusion assays Western blots Immunohistochemical and immunocytological assays performed on slides or in tissues for microscopic analysis Multiplex assays that use beads and fluorescent sorting also involve antibodies 12

13 ELISA Uses antibodies to detect an antigen or antibody of interest by exploiting the tendency of proteins to adsorb (stick) to a plastic microplate Can detect either antigens or antibodies Can be either direct (using only a primary antibody) or indirect (using a secondary antibody to detect the first antibody) 13

14 ELISA Types Direct ELISA Antigens from sample coat the well Enzyme-linked antibodies bind to the antigen of interest if present Colorimetric substrate is added Enzyme linked to the antibody cleaves the substrate, turning the solution blue, which indicates a positive test 14

15 ELISA Types Indirect ELISA Antigens from sample coat the well Primary antibodies bind to the antigen of interest if present Enzyme-linked secondary antibody is added which binds to the primary antibody Substrate color change indicates a positive test 15

16 ELISA Types Direct sandwich ELISA Primary antibodies are bound to wells Sample is added that may contain the antigen Enzyme-linked secondary antibody is added which binds to the antigen if present Substrate color change indicates a positive test 16

17 ELISA Types Capture serum antibody Purified antigens are bound to wells Serum sample is added that may contain antibodies to the purified antigen Enzyme-linked secondary antibody is added which binds to serum antibodies if present Substrate color change indicates a positive test 17

18 Instruments for ELISA Microplate readers Use spectrophotometric methods to measure the amount of absorbance by the developed substrate 18

19 Ouchterlony or Double Diffusion Assay Antibodies are placed into the center well and diffuse outward Antigens placed in the surrounding wells diffuse inwards Interactions form a precipitation event 19

20 Western Blotting SDS-PAGE results can be used to estimate the mass of proteins, but cannot identify them directly Antibodies can be used in the same way as an ELISA test when the proteins from an SDS- PAGE experiment are transferred to a membrane Electroblotting is the general method used 20

21 Western Blotting Once proteins have been transferred to the membrane, the technique is similar to the indirect ELISA 21

22 Western Blot Detection Colorimetric Visible color left after substrate interaction Chemiluminescent Substrate can emit light when enzyme reacts Sometimes different molecules linked to the secondary antibody emit different colors. This allows for multiple detections 22

23 Other Applications for Western Blots Viral protein detection Diagnosis of HIV infection 23

24 Other Applications for Antibodies Immunohistochemistry and immunocytochemistry When antibodies are used to study tissues and cells Immunofluorescence is when an antibody is labeled with a fluorescent molecule and will emit a different wavelength of light than the antibody being stimulated 24

25 Other Applications for Antibodies Multiplex beads Beads that have a color themselves can be used If different antibodies are linked to various colors then as they move past a detector, the color of the bead showing an interaction can be used to determine what was bound 25

26 Other Applications for Antibodies Fluorescent-activated cell sorting (FACS) Different antibodies are labeled with different fluorescent molecules enabling sorting due to a charge that is applied to the cell 26

27 Chapter 8 Summary Background The Immune System Types of Immunity Immune Response Antibodies Structure Types Responses Antibodies as Tools Treatment Immunoassays Other Applications that Utilize Antibodies 27