antibody based techniques Flashcards
lecture 12
What are antibodies and their characteristics?
Secreted forms of B cell receptors (BCR)
Bind native antigens (Ag), which can be virtually anything
Secreted in high amounts from differentiated B cells/plasma cells (easy to purify)
Can bind almost all biological materials (proteins, CHO, nucleic acids)
Their binding can be well characterized (as they’re monoclonal) and is often of very high affinity (SHM)
What are early approaches to using antibodies?
Following immunization with a pathogen or purified Ag, antibodies are found in the fluid phase of blood (plasma)
Serum is plasma once the blood clot is removed; serum from an immunized person/animal is known as antiserum
Antiserum contains antibodies that bind the Ag, along with other soluble blood components but does not contain cells or clotting proteins
What does antiserum contain?
Many different antibodies secreted by different B cells (clones)
Antibodies that bind the same Ag, even if the Ag was a single purified protein
Different antibodies may bind different regions (epitopes) of the same Ag
How can antibodies be purified from antiserum?
Gel filtration chromatography (separates based on molecular weight)
Affinity chromatography
What are the limitations of using antisera?
Purified antiserum (polyclonal antiserum) is useful, but separating individual antibodies is better
Reproducibility issues: once the antiserum is used, another individual needs immunizing, and antibodies generated will never be exactly the same
What are monoclonal antibodies and how are they generated?
Fusion of mouse myeloma cells (that no longer made antibodies) with mouse plasma cells making antibodies of known specificity to generate hybridomas
Hybridomas can continuously produce a single antibody, are immortal, and grow in culture forever
How are antibodies used in research?
Purifying, isolating, and identifying biological molecules of interest using affinity chromatography
Identifying the location of a protein within a cell using immunofluorescence microscopy
How are antibodies used in diagnostics?
Purifying, isolating, and identifying biological molecules of interest based on specific antibody/antigen interactions
What is the process of Western blotting?
HIV: Dissociate in SDS, lyse the virus
SDS-PAGE: Separate viral proteins based on their molecular weight
Transfer proteins to a membrane and incubate with a monoclonal antibody
Detect bound antibody with enzyme-linked anti-IgG
What are the types of ELISA and their characteristics?
Direct ELISA: Detects non-linear and linear epitopes; simple and less sensitive
Indirect ELISA: More sensitive; uses a secondary antibody
Sandwich ELISA: Most sensitive; uses a capture antibody and a different labeled secondary antibody
What is flow cytometry and how is it used?
Technique for characterizing cells based on light scattering properties
Measures light scatter from individual cells in a mixed population rapidly
Can add antibodies labeled with fluorescent dyes to cells for identification
Describe the process of generating monoclonal antibodies
Immunize with Ag A, harvest spleen
Mix spleen cells with myeloma cells
Fuse cells with PEG, transfer to HAT medium
Select and clone hybridoma that makes antibody specific for Ag A
How are secondary antibodies produced?
Collect mouse sera containing many different mouse antibodies
Purify mouse antibodies
Immunize rat, collect rat anti-mouse antiserum
Purify rat anti-mouse polyclonal antibodies
What are the techniques used for purifying antibodies from serum?
Gel filtration chromatography (separates based on molecular weight)
Affinity chromatography (better method)
What is the importance of myeloma cells in monoclonal antibody production?
Myeloma cells are immortal and can be fused with plasma cells to create hybridomas
Hybridomas continuously produce a single antibody and grow in culture indefinitely
