Lecture 5: Antigen-Immunoglobulin interactions, Experimental Systems, Monoclonal Antibodies.
Chapter 5, Benjamini et al.
5.1 Antibody-Antigen Affinity:
Antibodies form specific complexes with their antigen via non-covalent interactions. Nevertheless, the large number of van der Waals interactions, hydrogen bonds, electrostatic interactions and the hydrophobic effect that are possible within the epitope generally lead to very high affinities for protein-antibody interactions. Note that even with the high specificity of Antibody-antigen interactions it is possible to have cross-reactivity. That is more than one antigen can bind to a given antibody.
The only way to measure the affinity is to experimentally determine a binding curve. This involves measuring the amount of antibody-antigen complex as a function of the concentration of the free antigen. There are two common methods of performing this measurement:
Analysis of Binding Data (See Biochemistry Lecture notes for Review):
The affinity, or association, constant for binding is: KA=k1/k-1; where k1 is the forward rate constant while k-1 is the reverse rate constant, the rate at which the antibody-antigen complex dissociates. Differences in binding affinities are usually due to differences in k-1.
The fractional saturation, Y, is defined as:
Where [AL] is the antibody (A) ñ antigen (L) complex, [A] is the free antibody. Using the definition of the dissociation constant, KA=[AL]/[A][L] to express [AL] in terms of [A], gives:
The fractional saturation ranges from 0 ([L]=0) to 1 ([L] >>1/KA). The KA is 1/[L] that gives Y=0.5.
The dissociation constant is also used: KD=1/KA. The KD is the ligand concentration that gives ‡ fractional saturation. You should be able to determine KD from a binding curve.
The KA can be obtained directly from a binding curve. However, it is possible to linearize the above equation to give a linear equation:
Therefore a plot of Y/[L] versus Y should be straight line with a slope of ñ1/KD.
The avidity constant of an antibody refers to the effect of multiple binding sites (e.g. in pentameric IgM). It is equal to the product of the individual binding constants (or equivalently, sum of binding free energies). Thus pentimeric antibodies have high avidity, even if they have low affinity.
5.2 Detection of Antibody-Antigen Complexes.
5.2.1 Precipitation Reactions: These methods rely on the ability of the antibody to form large crosslinked antibody-antigen complexes that precipitate out of solution. These methods are becoming less frequently used as solid-phase assays become more prevalent. A fixed amount of antigen is mixed with a set of serial dilutions of serum.
The dilution of serum that gives the largest amount of precipitation is referred to as the titer of the serum.
These reactions can also occur in gels. Antigen and antibody are added to wells in an agar gel. As the two molecules diffuse towards each other they will sample different concentrations. A some location in the gel the concentration of both are just right for aggregation, leading to a precipitate in the gel. This method does not require any knowledge about the level of antibody in the serum.
5.2.2 Agglutination Reactions: These are similar to precipitation reactions, except that they involve the precipitation of cells. Usually, indirect crosslinking is used to form the aggregation. A secondary antibody is added that binds to the primary antibodies that have bound to their epitope on the surface of the cell.
This type of test is referred to as the indirect Coombs test. For example, the presence of anti-Rh antibodies in an Rh- mother can be detected by incubating Rh+ red blood cells with the serum form the mother, followed by the addition of antibodies (e.g. from a rabbit) that will bind to the human IgG and lead to precipitation.
5.2.3 Direct Binding Assays: In assays of these types, the antibody, or antigen are immobilized on a solid surface and the amount of bound antigen, or antibody, respectively is detected by radioactivity or by detection of color changes in the case of enzyme-linked detection schemes.
Western Blot: This is one of least quantitative measure, but is useful for detecting antigen in the presence of a complex mixture of proteins, or if the antibody available is polyclonal. A standard SDS gel is run to separate proteins according to size. These proteins are transferred to a membrane of nitrocellulose, forming an image of the gel. The gel is then washed with the primary antigens that will recognize the desired antigens. These antibodies binding to the immobilized and denatured protein. The membrane is treated with a secondary antibody that recognizes the constant region of the primary antibody. The secondary antibody is linked to alkaline phosphatase, horseradish peroxidase, or p-nitrophenyl phosphatase, all of which give colored products.
Radioimmunoassay: This is usually used to detect antigen. Antigen specific antibodies are immobilized on plastic or on beads and allowed to bind a known amount of radioactive antigen. The bound radioactivity is then measured. Serum (or other fluid), containing an unknown amount of the antigen (unlabeled of course) is added to the antibody and radioactive antigen. The amount of radioactivity bound to the antibody will decrease because the unlabeled material can compete for binding. The fractional saturation of antibody with radioactive antigen(L*) in the presence of unlabeled antigen (L) is given by the following:
ELISA (Enzyme-Linked Immunosorbent Assay):
Indirect ELISA (Detection of Antibody)
Sandwich ELISA (Detection of Antigen)
Immunofluorescence: Fluorescence is used to detect the presence of the complex.
Fluorescence-Activated Cell Sorter
Magnetic Cell Sorter
Similar to fluorescent cell sorter, except small magnetic beads are bound to the surface of the cell and these cells are separated from others using a magnet.
5.3 Generation of Monoclonal Antibodies
Animals (e.g. rabbits) are not the best source of antibodies for several reasons, here are some of the scientific ones:
Consequently, considerable effort was placed into developing cultured lines of B-cells that would secrete high levels of soluble immunoglobulins. Unfortunately, B-cells do not do well in cell culture and die with a few days. This problem was solved by fusing the B-cells with an immortal cell line (myeloma cells) to produce an immortal antibody producing cell line. The basic steps in this procedure are:
Inoculate a mouse with the antigen
The key tricks in this procedure were to obtain myeloma cells that did not secrete their own antibodies and to devise a selection scheme for hybridomas that selected against the myeloma cells. It is not necessary to select against the plasma cells from the spleen since they die anyhow.
Selection for hybirdomas:
Therefore, growth of the cells on HAT media (hypoxanthine, aminopterin, thymidine) will select for immortal cells that have acquired the HGPRT gene via fusion with a plasma cell.
Uses of Monoclonal Antibodies:
Humanized Monoclonal Antibodies:
When patients are treated with mouse monoclonal antibodies they rapidly develop antibodies against the constant region of the mouse (murine) antibody. There are at least three solutions to this problem: