MICR 270 Chapter 5: Micr 270 module 5 online notes
1. What is the purpose of indirect ELISA?
● To detect and quantify substances
2. Describe the 7 steps of an indirect ELISA
● Bottom of well is coated with antigen
○ Antigen has to be recognized by antibody you wish to measure
● Well is washed to remove excess antigen
● Sample containing antibody is added to well
○ If antibody is present, they will bind to antigens
● Well is washed to remove excess antibodies
● Enzyme conjugated secondary antibody is added to the well
○ It will bind to the Fc portion of the primary antibody
○ The secondary antibody has to specifically recognize antibodies from a different
animal
● Well is washed to remove excess secondary antibodies
● Substrate of enzyme attached to the secondary antibody is added
○ Reaction of substrate (chromogen) and enzyme will produce a colored product
that can be measured through absorbance
3. What is the purpose of performing Flow Cytometry?
● To detect and quantify different cell types in a mixed cell suspension
4. How does Flow Cytometry work?
● Narrow stream of cells is passed single file through a laser
● Forward and side scattered light is measured to detect size and shape of granules
● Cell suspension is treated with antibody that recognizes the antigen and is coupled to a
fluorescent molecule
● Laser light source is designed to excite the fluorescent molecule
● Specific colored light is emitted only by those cells bound to the antibody
● Used to perform complete blood counts
5. What can monoclonal antibodies be used for in terms of cancer?
● Research
● Diagnosis
● Therapy
6. What 2 things does monoclonal antibodies measure?
● Immunotoxins
○ Long term objectives is to use immunotoxins to target and eliminate tumor cells
to treat cancer
● Radiolabeled antibodies
○ Can be used to diagnose tumors earlier than other methods
7. How are monoclonal antibodies created?
● Created in labs by hybridomas
8. What are 2 characteristics of hybridomas?
● Immortal
● Produce unlimited quantities of an identical antibody
9. What 2 cells are hybridomas a fusion of?
● Plasma cell
○ Produce specific antibodies
● Cancerous cell (Myeloma)
○ Immortal growth
10. What are the 4 types of Vaccines?
● (KLiTS)
● Live attenuated vaccine
● Killer inactivated vaccine
● Toxoid vaccine
● Subunit vaccine
11. Describe live attenuated vaccines, explain the pros and cons and provide examples
● Modified strain of disease causing agent that has lost pathogenic ability
● Can still replicate in host
● Pros
○ Can generate cell-mediated immunity
○ Prolonged exposure to disease causing agent
● Cons
○ Potential to revert back to virulent form
○ Requires cold transport
● Examples
○ Smallpox vaccine
○ Oral polio vaccine
○ Measles vaccine
12. Describe killer-inactivated vaccines, explain the pros and the cons, and provide examples
● Contains a strain of the disease causing agent that has been inactivated by heat,
chemicals or radiation
● Ability to generate immune response but cannot replicate
● Pros
○ Safe, cannot revert back to virulent form
○ Easy to store and transport
● Cons
○ Requires multiple booster shots
○ Must be injected
● Examples
Document Summary
To detect and quantify substances: describe the 7 steps of an indirect elisa. Bottom of well is coated with antigen. Antigen has to be recognized by antibody you wish to measure. Well is washed to remove excess antigen. Sample containing antibody is added to well. If antibody is present, they will bind to antigens. Well is washed to remove excess antibodies. Enzyme conjugated secondary antibody is added to the well. It will bind to the fc portion of the primary antibody. The secondary antibody has to specifically recognize antibodies from a different animal. Well is washed to remove excess secondary antibodies. Substrate of enzyme attached to the secondary antibody is added. Narrow stream of cells is passed single file through a laser. Forward and side scattered light is measured to detect size and shape of granules. Cell suspension is treated with antibody that recognizes the antigen and is coupled to a fluorescent molecule.