Antibodies
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Name the classes of antibodies (immunoglobulins)
• produced by B-cells (lymphocytes that mature in the bone marrow) • differ by structure and physiological function -IgG is the most abundant
what does IgG consist of
4 subunits (polypeptide chains) -> 2 heavy, 2 light 12 immunoglobulin folds (antiparallel beta sheets) carbohydrate moeity
what is the immunoglobulin fold?
N terminal domain of human immunoglobulin - the polypeptide chain is folded into a sandwhich of 3 and 4 stranded antiparallel beta sheets
what is an epitope?
specific part of an antigen recognised by an antibody
where is the antigen binding site located?
at the tip of Fab fragment in a crevice between VL and VH domains
what is divalent bonding?
IgG can bind two identical antigens simultaneously
why do antibodies cross link antigens to form a lattice?
speeds up removal of antigen and triggers B-cell proliferation
why can’t normal lymphocytes be cultured indefinitely?
because lymphocytes will not grow continuously unless they are immortalised
who developed the method for generating monoclonal antibodies?
Milstein and Kohler
how are lymphocytes immortalised to produce monoclonal antibodies?
by fusing them with myeloma (cancer) cells to form hybridome cells
what are hybridoma cells?
fused cells combining the antibody-producing ability of lymphocytes with the immortality of myeloma cells
why are only hybridoma cells able to grow in selective medium?
the selective medium allow survival only of fused (hybrid) cells, not unfused lymphocytes or myeloma cells
how are monoclonal antibodies obtained from hybridoma cells?
single hybridoma cells are isolated, grown into clones, tested for antibody production, and positive clones are cultured to produce large quantities of the desired antibody
what are three major laboratory or medical uses of monoclonal antibodies?
• protein purification and identification in the lab • disease diagnosis • therapeutic treatment of diseases
why are antibodies labelled or tagged in laboratory experiments?
to identify, quantify or isolate specific proteins of interest
give three main applications of antibody labelling
• determining whether a specific protein is present in a mixture • measuring how much of the protein is present • separating or purifying a specific protein
what is ELISA?
Enzyme-Linked Immunosorbent Assay
what is the purpose of ELISA?
to detect and quantify specific proteins or antibodies in a sample
what are the main steps of an ELISA?
• immobilise the first antibody on a solid support • incubate with a sample containing the target protein • add a second antibody linked to an enzyme • wash and measure enzyme activity - the amount of product formed reflects the protein concentration
what type of monoclonal antibody is used to treat rheumatoid arthritis and Crohn’s disease?
a fully humanised IgG monoclonal antibody