Medical Sciences 3900F/G Lecture Notes - Lecture 5: B-Cell Receptor, Plate Reader, Immunoglobulin Heavy Chain
3 May 2018
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ELISA ONLINE MODULE
Scenario
- You are working as a physician in a chronic would-healing clinic and you are interested to se if a new
drug that you have prescribed to helping resolve wounds in your patients. To evaluate whether the
drug is working, you want to measure the levels of the cytokine VEGF a peptide in your patient’s
blood. How would you proceed?
- How could you measure the levels of the cytokine peptide?
What is an ELISA?
- Enzyme linked immunosorbent assay
o Need enzyme for the detection to occur
o Immunosorbent – looking at components of the immune system
▪ Antibodies – key role in ELISA
- Biochemical technique that can be very sensitive
o Allows for the detection of antibody or antigen in very small amounts in a sample
o A lot of amplification of occurs
o Allows for detection of something in minute sizes, which would other wise not be able to be
detected (ADVANTAGE)
- Antigen-antibody binding is linked to an enzyme-mediated colour change that can be detected
o What the ELISA detects
o Without enzyme, can not detect any of our sample
- Creating colour change on plate that is going to be read on the plate reader
- What is a sample?
o Blood sample from a patient, detecting AIDS/HIV, malaria, pregnancy
o Cell culture media
▪ NOT detecting the cells, but something the cell might secrete in the media
▪ What we are doing in this lab
o Antigen or antibody from animal model blood
What can an ELISA measure?
- Antigen: proteins, peptides, hormones
o E.g. thyroxin (protein type of hormone)
- Antibody
o Measuring whether or not an immune response to particular antigen or infection has
occurred
Antigens/Antibodies
- Antibody made by B cells
o Secreted form of the B cell receptor
- C terminus of the antibody:
o IF going to be secreted, it is hydrophilic
o When it is the B cell receptor form of antibody (inserted into the plasma membrane), it is
hydrophobic
find more resources at oneclass.com
find more resources at oneclass.com
- Heavy and light chain peptides
o Have to do with the size
▪ Light chain = smaller
▪ Heavy chain = smaller
- Each part (heavy & light) have variable region and constant region
o Variable region: bind antigen
o Constant region: same between isotypes in same species
Antibodies have 2 main tasks:
- 1. Binding to a number of antigens via variable region of antibody
o any given antibody binds to one specific epitome of one specific antigen
o ONE ANTIGEN PER ANTIBODY
o Important for the specificity of the antibodies, to bind specific antigens
- 2. Constant region interacts with effectors and cells, and therefore performs the other main
functions of antibodies
o Recognized by different cells and effectors in the body
o IF it is variable and not constant, it would not be recognized or be able to carry out its
specific function (ex. kill a specific cell)
- Antibodies help mediate the immune response
o Done through the constant regions
Antibodies/Antigens
- Antigen: a molecule that can bind specifically to an antibody
o Can get antibodies generated for all types of antigens
- Epitope/antigenic determinant: the part of an antigen that actually binds to the antibody
o For a given antigen, there are a number of different epitopes or points on antigen that could
bind to antibody
o Ex. could bind middle, left right – different antibody for each region
- Antigen could have a number of different antibodies that are targeting it, recognizing different
epitopes of that particular antigen
The big idea – sandwich ELISA
- Add something in ELISA, incubate it, wash it off (REEPEAT)
- Ex. Want to detect the amount of thyroxine in patient’s blood
- In a 96-well plate, you have your capture antibody coded onto the wells (each well)
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
You are working as a physician in a chronic would-healing clinic and you are interested to se if a new drug that you have prescribed to helping resolve wounds in your patients. To evaluate whether the drug is working, you want to measure the levels of the cytokine vegf (cid:523)a peptide(cid:524) in your patient"s blood. Enzyme linked immunosorbent assay: need enzyme for the detection to occur. Immunosorbent looking at components of the immune system: antibodies key role in elisa. Antigen-antibody binding is linked to an enzyme-mediated colour change that can be detected: what the elisa detects, without enzyme, can not detect any of our sample. Creating colour change on plate that is going to be read on the plate reader. Antigen: proteins, peptides, hormones: e. g. thyroxin (protein type of hormone) Antibody: measuring whether or not an immune response to particular antigen or infection has occurred. Antibody made by b cells: secreted form of the b cell receptor.
