Read this article to learn about the types, production, labelling, affinity and avidity of antibodies.
Antibody Production:
(a) Polyclonal Antibodies:
It is possible to produce antibodies which bind to proteins, peptides, carbohydrate and nucleic acids. In general, most immunochemical methods are devised for use with antibodies that recognize proteins and peptides.
Choice of species for antibody production depends upon the amount of antigen available, amount of antiserum required and quality of antiserum desired. In some case use of closely related species or different strains within single species for derivation of antigen and production of antibody can provide antibodies with specific properties.
The most important consideration for immunogenicity is differences in amino acid sequences. Immunogenicity tends to increase with size. Peptides with molecular weight less than 2000 are poor antigens while with more than 10000 are good antigens as long as they are recognized as foreign in responding animal.
Antibodies that bind to smaller peptides can be produced by linking (conjugating) these substances to larger proteins called as carrier proteins. Thus the Antisera produce will recognize carrier protein too along with peptide. Substances that are not immunogenic alone, but are when conjugated are known as heptane’s. Such substances potentiate the immune response by forming a slow-release depot of antigen, by stimulating T cell help or by aiding antigen presentation.
Single immunization results in production of antisera but it is usually suboptimal, containing antibodies of low avidity and a high proportion of IgM, therefore several “booster” immunizations with the antigen is done so as to boost the antibody production by making the animal hyper-immune. Such “hyper-immune sera” are usually the polyclonal reagent of choice for immunochemical techniques.
(b) Monoclonal Antibodies:
Monoclonal antibodies are secreted by cloned, i.e., monoclonal cells. Mature, antibody secreted lymphocytes from immunized animals can be cloned, but these survive only for very few period in culture, and therefore, do not provide useful amount of antibody.
However, procedures have been developed to separate antibodies from continuously growing (immortal) cells. These involve generation of hybrid cells, transformation of lymphocytes with virus or recombinant DNA.
Antibody Labelling:
Like the fluorochrome as described in above section, antibodies can also be tagged with enzymes, or carbohydrate or biotin for different purposes. Labelling antibodies with enzymes, fluorochromes, or biotin provides a signal for visualization or quantitation of the target molecule. Antibody bound to agarose is useful for separating a target antigen from a complex mixture.
To avoid excessive background staining and to improve sensitivity, only purified antibodies should be used for staining. At the minimum, the IgG fraction, which contains naturally occurring immunoglobulin as well as specific antibody, should be isolated from the antiserum.
This can be done by various methods, usually involving a combination of fractionation and chromatography. Preferably, the antibody should be affinity isolated on a column containing the antigen bound to a solid support. This will eliminate all serum proteins, including immunoglobulin’s that do not specifically bind to the antigen.
Labelling with Enzymes:
Antibodies may be labelled with various enzymes to provide highly specific probes that both visualize the target and amplify the signal by acting on a substrate to produce a coloured or chemiluminescent product. Horseradish peroxidase and alkaline phosphatase are the most commonly used enzymes for this purpose. Antibody-enzyme conjugates may be used for detecting proteins in immuno-histology and immuno-cytology, immuno-blotting, and ELISA.
Labelling with Fluorochromes:
Fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and R-phycoerythrin (PE), represent the fluorescent probes most commonly cited. Labelling proteins with fluorochromes provides a coloured reagent that can be observed directly. The intensity and narrow wavelength of the fluorochromes make them useful in immunohistochemistry and immunocytochemistry, using both visible and fluorescence microscopy, and in flow cytometry.
Labelling with Biotin:
Biotinylated antibodies are widely used in systems where signal amplification is desired. Biotin binds avidin with a high degree of affinity and specificity. Avidin, ExtrAvidin®, or streptavidin labelled with enzymes or fluorochromes can bind biotinylated antibodies, amplifying the signal and allowing detection of antigens present in small amounts. This system may be used in immunohistochemistry and immunocytochemistry, immuno-blotting, ELISA, and flow cytometry.
Labelling with Gold:
Antibodies conjugated to colloidal gold are used primarily in electron microscopy (EM) because of the electron-dense nature of the gold particles. Gold-antibody conjugates may also be used for immunohistochemistry and immunocytochemistry, and immuno-blotting. In some cases silver enhancement may be used to amplify the signal.
In general, 5 nm particles are recommended for intracellular staining because they are able to penetrate the cell membranes more easily, and for high resolution EM because the small size allows more exact localization of antigen. 10 nm particles are recommended for cell surface staining and for light microscopy because the larger size makes the stain more visible. 20 nm particles are recommended for blotting and for some histochemical applications. These are suggestions only — the end user will have to determine the best particle size for each application.
Attaching Antibodies to Agarose:
Antibodies attached to agarose have a variety of applications. They can be used to isolate proteins and other compounds from sera or from cell and tissue homogenates by affinity chromatography for quantitation or further analysis. They have also been used for immuno-precipitation of proteins from cell lysates, and for reduction of serum immunoglobulin in autoimmune diseases and transplantation experiments.
Antibody Fragmentation:
In some assays it is preferable to use only the antigen binding (Fab) portion of the antibody. For these applications, antibodies may be enzymatically digested to produce either an Fab or an F (ab’)2 fragment of the antibody. To produce an F (ab’)2 fragment, IgG is digested with pepsin, which cleaves the heavy chains near the hinge region.
One or more of the disulphide bonds that join the heavy chains in the hinge region are preserved, so the two Fab regions of the antibody remain joined together, yielding a divalent molecule (containing two antibody binding sites), hence the designation F (ab’)2. The light chains remain intact and attached to the heavy chain. The Fc fragment is digested into small peptides. Fab fragments are generated by cleavage of IgG with papain instead of pepsin.
Papain cleaves IgG above the hinge region containing the disulphide bonds that join the heavy chains, but below the site of the disulphide bond between the light chain and heavy chain. This generates two separate monovalent (containing a single antibody binding site) Fab fragments and an intact Fc fragment. The fragments can be purified by gel filtration, ion exchange, or affinity chromatography.
Affinity and Avidity:
Antibody Affinity is a Quantitative Measure of Binding Strength:
The combined strength of the non-covalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope. Low-affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer.
The association between a binding site on an antibody (Ab) with a monovalent antigen (Ag) can be described by the equation:
where k1 is the forward (association) rate constant and k-1 is the reverse (dissociation) rate constant. The ratio k1/k-1 is the association constant Ka (i.e., k1/k-1 = Ka), a measure of affinity. Because Ka is the equilibrium constant for the above reaction, it can be calculated from the ratio of the molar concentration of bound Ag-Ab complex to the molar concentration of unbound antigen and antibody at equilibrium as follows:
Ka = [Ag-Ab] / [Ab][Ag]
The value of Ka varies for different Ag-Ab complexes and depends upon both k1, which is expressed in units for litres/mole/second (1/mol/s), and k-1; which is expressed in units of 1/second (inverse second). For small haptens, the forward rate constant can be extremely high; in some cases, k1 can be as high as 4×108 1/mol/s, approaching the theoretical upper limit of diffusion-limited reactions (109 1/mol/s). For larger protein antigens, however, k1 is smaller, with values in the range of 105 1/mol/s.
The rate at which bound antigen leaves as antibody’s binding site (i.e., the dissociation rate constant, k-1) plays a major role in determining the antibody’s affinity for an antigen.
For some purposes, the dissociation of the antigen-antibody complex is of interest:
Ag-Ab ↔ Ab + Ag
The equilibrium constant for that reactions is Kd, the reciprocal of Ka
Kd = [Ab] [Ag]/[Ab-Ag] = 1/Ka
and is a quantitative indicator of the stability of an Ag-Ab complex; very stable complexes have very low values of Kd, and less stable ones have higher values. The affinity constant, Ka, can be determined by equilibrium dialysis or by various newer methods. Because equilibrium dialysis remains for many the standard against which other methods are evaluated, it is described here. This procedure uses a dialysis chamber containing two equal compartments separated by a semipermeable membrane.
Antibody is placed in one compartment, and a radioactively labelled ligand that is small enough to pass through the semipermeable membrane is placed in the other compartment (Fig. 6.17). Suitable ligands include haptens, oligosaccharides, and oligopeptides.
In the absence of antibody, ligand added to compartment B will equilibrate on both sides of the membrane (Fig. 6.17a). In the presence of antibody, however, part of the labelled ligand will be bound to the antibody at equilibrium, trapping the ligand on the antibody side of the vessel, whereas unbound ligand will be equally distributed in both compartments.
Thus the total concentration of ligand will be greater in the compartment containing antibody (Fig. 6.17b). The difference in the ligand concentration in the two compartments represents the concentration of ligand bound to the antibody (i.e., the concentration of Ag-Ab complex). The higher the affinity of the antibody, the more ligand is bound.
Since the total concentration of antibody in the equilibrium dialysis chamber is known, the equilibrium equation can be rewritten as:
Ka = [Ab-Ag] / [Ab] [Ag] = r/c(n – r),
where r equals the ratio of the concentration of bound ligand to total antibody concentration, c is the concentration of free ligand, and n is the number of binding sites per antibody molecule. This expression can be rearranged to give the Scatchard equation:
r/c = Kan – Kar.
Values for r and c can be obtained by repeating the equilibrium dialysis with the same concentration of antibody but with different concentrations of ligand. If Ka is a constant, that is, if all the antibodies within the dialysis chamber have the same affinity for the ligand, then a Scatchard plot of r/c versus r will yield a straight line with a slope of —Ka (Fig. 6.18a). As the concentration of unbound ligand c increases, r/c approaches 0, and r approaches n, the valency, equal to the number of binding sites per antibody molecule.
Most antibody preparations are polyclonal, and Ka is, therefore, not a constant because a heterogeneous mixture of antibodies with a range of affinities is present. A Scatchard plot of heterogeneous antibody yields a curved line whose slope is constantly changing, reflecting this antibody heterogeneity (Fig. 6.19b).
With this type of Scatchard plot, it is possible to determine the average affinity constant, K0, by determining the value of Ka, when half of the antigen-binding sites are filled. This is conveniently done by determining the slope of the curve at the point where half of the antigen binding sites are filled.
Antibody Avidity Incorporates Affinity of Multiple Binding Sites:
The affinity at one binding site does not always reflect the true strength of the antibody-antigen interaction. When complex antigens containing multiple, repeating antigenic determinants are mixed with antibodies containing multiple binding sites, the interaction of an antibody molecule with an antigen molecule at one site will increase the probability of reaction between those two molecules at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity.
The avidity of an antibody is a better measure of its binding capacity within biological systems (e.g., the reaction of an antibody with antigenic determinants on a virus or bacterial cell) than the affinity of its individual binding sites. High avidity can compensate for low affinity. For example, secreted pentameric IgM often has a lower affinity than IgG, but the high avidity of IgM, resulting from its higher valence, enables it to bind antigen effectively.