MICB 212 Chapter 7 Notes

Antibodies as Tools in Research and Medicine

-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

Diagnostic Uses of Antibodies

-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)

Polyclonal Antisera

-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

Monoclonal Antibodies

-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

ELISA

-enzyme-linked immunosorbent assay

-sensitive method for detecting presence of antibodies or antigens in fluids and determining their concentrations

Testing for Presence of Antibody

-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

Testing for Presence of Antigen

-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

Immunofluorescence

-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

Summary

-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