-antibodies have many uses in clinic, research laboratories and diagnostic services
-antibodies can be produced by
immunizing animals (rabbits, mice, goats)
cells (hybridomas) that grow in tissue culture
-antibodies can be purified and used for detecting and quantifying the antigens that they recognize
-antibodies have many uses in medicine
-ex:
detection of pathogens (bacteria, viruses, toxins) in patient samples
detection of antibodies in blood that indicate exposure to an antigen (covid test)
detection and measurement of hormone levels (thyroid hormones, pregnancy tests)
analysis of blood cells and immune cells (blood and tissue typing, enumeration of cell types
therapeutic medication for medical conditions (certain cancers, psoriasis, Crohn’s disease)
-animal (rabbit, goat, etc) can be immunized with an antigen (virus, snake venom) and antibodies can be isolated from animal’s serum
serum — liquid portion of blood that remains after blood has been allowed to clot
contains a mixture of antibodies that recognize different epitopes on the antigen that was injected
-most antigens have more than one epitope and will activate many B cells
each B cell recognizes a different epitope
-each activated B cell proliferates to give rise a clone of cells
some clones will differentiate to become plasma cells that secrete antibodies that recognize the specific epitope
-since there are many different types of plasma cell clones secreting antibodies into the serum → mixture of antibodies purified from serum is polyclonal antiserum
not a standardized reagent → limited supply of polyclonal antibody
-sometimes necessary to detect only one epitope on a pathogen
ex: if you want to detect the presence of a mutant virus strain that differs from wild type strain in only one epitope → polyclonal antiserum is not helpful
polyclonal antiserum that recognized all the epitopes on the virus won’t be able to distinguish virus mutant from wild type virus
-process of producing a monoclonal antibody starts by repeatedly immunizing a mouse with an antigen to get a good secondary immune response
-B cells specific for different epitopes on the antigen will become activated, proliferate and differentiate into clones of antibody secreting plasma cells
-remove mouse’s spleen → antibody secreting plasma cells are immortalized by fusing them with myeloma cell that grows in culture
myeloma — plasma cell tumour that is unable to make its own antibodies
-B cells fuse to myeloma cells using polyethylene glycol to create a hybridoma
cell that has characteristics of B cell (ability to make antibody and grow in presence of certain drugs) & myeloma (immortality)
-plasma B cels only live in culture for a few days before they die
hybridomas can grow and divide indefinitely in tissue culture dishes
-since plasma cells that were fused to myeloma cells were a mixture of various B cell clones → mixture of immortal hybridomas making antibodies against different epitopes
-to obtain clones of hybridoma that is making antibody to desired epitope → single cell placed in each well of a 96 well tissue culture plate
cells in each well divide and form clones of identical cells
-hybridoma clones making antibodies against desired epirope are then selected
taking a small amount of culture medium from each well and using ELISA assay to see if antibodies secreted into medium by hybridoma cells bind to epitope of interest
-selected hybridoma clones can grow indefinitely in culture and used to make large amounts of monoclonal antibody
antibody secreted into culture medium can be collected and purified and hybridoma cells can be frozen for long term storage
-enzyme-linked immunosorbent assay
-sensitive method for detecting presence of antibodies or antigens in fluids and determining their concentrations
-ELISA screens population of hybridomas to see if any of them make an antibody to antigen of interest
-coat ELISA plate with antigen
plastic of ELISA plate is treated so proteins can bind to it
-small volume of culture medium from hybridoma is added to wells of ELISA plate
-after incubation, ELISA plate is washed
if antibodies that bound to epitope on antigen were present → they remain bound to well
antibodies against other antigens will be washed away so no antibody will be bound in those wells
-a secondary antibody is used to determine which wells have antibodies bound to antigen
an antibody that recognizes the heavy chain of another antibody
-antibodies produced by hybridomas are mouse IgG antibodies
since hybridomas are derived from mouse B cells
-to detect mouse IgG bound to wells
use secondary antibody that recognizes the constant region of antibody you want to detect
becomes covalently linked to an enzyme → allows detection of antibodies bound to antigen in well
enzyme that is linked to secondary antibodies turns from colourless substrate into a coloured product
-secondary antibodies are added to walls and poured out then washed
unbound material washed away
secondary antibody only binds to wells where mouse IgG from hybridoma medium had bound to antigen
some primary antibodies bound to antigen will have some secondary antibody bound
-colourless substrate is added and secondary antibodies are present
enzyme converts substrate into a coloured product
only wells that had antibodies bound to antigen will show colour
-ELISA can be used to screen presence of soluble proteins (cytokines or hormones)
-use unlabelled primary antibody to coat wells of 96 well ELISA plate
-add known amounts of sample (IL-4) to some wells and diluted patient serum to other wells
during incubation — IL-4 binds to antibody coated on wells
dump out fluid and wash wells after 1 hour
-enzyme-linked antibody (secondary antibody) added that recognizes a different epitope on the IL-4 than the antibody bound to well (primary antibody)
each antibody recognizing a different epitope ensure that 2 antibodies aren’t competing for same spot on antigen and have ample room to bind
dump out fluid and wash wells after 1 hour
-if no antigens present in sample → secondary antibodies would have nothing to bind to and gets washed away
-add substrate for enzyme → amount of coloured product produced is proportional to how much IL-4 was bound
more IL-4 bound to well → more enzyme linked antibody that binds → more substrate that turns into coloured product
-antibodies that are covalently linked to small fluorescent molecules are used to detect proteins on cells
-allow fluorescent antibodies to bind to cells
antibodies will only bind if cells bear antigen (epitope) that antibody recognizes
-sample is rinsed to wash away unbound antibodies after adding antigen-specific antibodies
shine light on sample → correct wavelength of light will cause fluorescent molecules to give off light of different wavelength
-light emitted by fluorescent molecule can be detected with microscope (fluorescence microscopy) or fluorescence-activated cell sorter (FACS)
-cells with fluorescent antibodies bound to them can be seen with fluorescence microscope
-FACS can count number of cells with fluorescent antibodies bound to them
-medical uses for antibodies
detection of pathogens
detection of antibodies in blood
detection and measurement of hormone levels
-polyclonal antisera have antibodies that recognize epitopes on antigen
unuseful for detecting only one epitope
-monoclonal antibodies derived from one clone B cell used to recognize a specific epitope
useful in ELISAs and FACS analysis
-to make monoclonal antibodies
immunize mouse repeatedly with antigen strong enough that a immune response is provoked
spleen of mouse is removed to recover plasma cells and gets crossed with immortal myeloma cells to form hybridomas
hybridomas — immortality of myeloma cell & antibody secreting power of plasma cell
hybridomas are isolated and tested against desired epitope using ELISA assay
selected hybridoma kept in culture and used to make lots of monoclonal antibodies
-ELISA tests can be used to detect presence of antibodies or antigens and to quantify amounts
can detect presence of antibody that recognizes a certain antigen
antigen attached to well and chosen because its bound to primary antibody
if primary antibody is present and bound to antigen → secondary antibody with enzyme will attach
secondary antibody is conjugated with enzyme and will cause an added substrate to change colour → indicates presence of antibodies
can detect presence of certain antigen
wells are coated with primary antibody that recognizes antigen of choice
if antigen is present → attach to primary antibody
secondary antibody will recognize and bind to antigen
secondary antibody is compounded with enzyme that causes colour change to indicate antigen presence
-fluorescence microscopy can detect antibodies covalently linked to fluorescent molecules
possible to target only certain molecules on a sample → viewed under fluorescent microscope
-FACS is a machine that counts and sorts cells
antibodies with different fluorescent molecules attached or cells without any fluorescence are sorted and category counted
efficient and useful in sorting cells in populations
ex. counting T cells in a blood sample