The AntigenThe basic principle of any immunochemical technique is that a specific antibody will combine with its specific antigen to give an exclusive antibody-antigen complex.
An ANTIGEN is defined as “any foreign substance that elicits an immune response (e.g., the production of specific antibody molecules) when introduced into the tissues of a susceptible animal and is capable of combining with the specific antibodies formed”. Antigens are generally of high molecular weight and commonly are proteins or polysaccharides. Polypeptides, lipids, nucleic acids and many other materials can also function as antigens. Immune responses may also be generated against smaller substances, called haptens, if these are chemically coupled to a larger carrier protein, such as bovine serum albumin, keyhole limpet hemocyanin (KLH) or other synthetic matrices. A variety of molecules such as drugs, simple sugars, amino acids, small peptides, phospholipids, or triglycerides may function as haptens. Thus, given enough time, just about any foreign substance will be identified by the immune system and evoke specific antibody production. However, this specific immune response is highly variable and depends much in part on the size, structure and composition of antigens. Antigens that elicit strong immune responses are said to be strongly immunogenic.
The small site on an antigen to which a complementary antibody may specifically bind is called an epitope. This is usually one to six monosaccharides or 5–8 amino acid residues on the surface of the antigen. Because antigen molecules exist in space, the epitope recognized by an antibody may be dependent upon the presence of a specific three-dimensional antigenic conformation (e.g., a unique site formed by the interaction of two native protein loops or subunits), or the epitope may correspond to a simple primary sequence region. Such epitopes are described as conformational and linear, respectively. The range of possible binding sites is enormous, with each potential binding site having its own structural properties derived from covalent bonds, ionic bonds and hydrophilic and hydrophobic interactions.
For efficient interaction to occur between the antigen and the antibody, the epitope must be readily available for binding. If the target molecule is denatured, e.g., through fixation, reduction, pH changes or during preparation for gel electrophoresis, the epitope may be altered and this may affect its ability to interact with an antibody. For example, some antibodies are ineffective in Western blot but very good in immunohistochemistry because in the latter procedure, a complex antigenic site might be maintained in the tissue, whereas in the former procedure the process of sample preparation alters the protein conformation sufficiently to destroy the antigenic site and hence eliminate antibody binding. Thus, the epitope may be present in the antigen’s native, cellular environment, or only exposed when denatured. In their natural form they may be cytoplasmic (soluble), membrane associated, or secreted. The number, location and size of the epitopes depends on how much of the antigen is presented during the antibody making process.
If a gene product of interest is present in extremely low concentrations, one may choose to use known nucleotide sequence information to derive a corresponding peptide for generating sequence-specific antibodies. In some instances, peptide antigens have advantages over whole protein antigens in that the antibodies generated may be targeted to unique sequence regions. This is especially useful when investigating proteins that belong to families of high sequence homology.
Characteristics of a Good Antigen Include:
- Areas of structural stability and chemical complexity within the molecule.
- Significant stretches lacking extensive repeating units.
- A minimal molecular weight of 8,000–10,000 Daltons, although haptens with molecular weights as low as 200 Da have been used in the presence of a carrier protein
- The ability to be processed by the immune system.
- Immunogenic regions which are accessible to the antibody-forming mechanism.
- Structural elements that are sufficiently different from the host.
- For peptide antigens, regions containing at least 30% of immunogenic amino acids: K, R, E, D, Q, N.
- For peptide antigens, significant hydrophilic or charged residues.
|An ANTIBODY is defined as “an immunoglobulin capable of specific combination with the antigen that caused its production in a susceptible animal.” They are produced in response to the invasion of foreign molecules in the body. Most antibodies exist as one or more copies of a Y-shaped unit, composed of four polypeptide chains. Each Y contains two identical copies of a heavy chain, and two identical copies of a light chain, named as such by their relative molecular weights. Nonmammalian vertebrate antibodies are similar in structure to mammalian IgG and carry the designation IgY (for yolk-derived). Mammalian antibodies can be divided into five classes: IgG, IgM, IgA, IgD and IgE, based on the number of Y units and the type of heavy chain. Heavy chains of IgG, IgM, IgA, IgD, and IgE, are known as g, µ, a, d, and e, respectively. The light chains of any antibody can be classified as either a kappa (.) or Antibody Structure lambda (.) type (based on small polypeptide structural differences); however, the heavy chain determines the subclass of each antibody. |
The subclasses of antibodies differ in the number of disulfide bonds and the length of the hinge region. The most commonly used antibody in immunochemical procedures is of the IgG class because they are the major immunoglobulin (Ig) released in serum.
The classical Y shape of IgG is composed of the two variable, antigen specific F(ab) arms, which are critical for actual antigen binding, and the constant Fc “tail” that binds immune cell Fc receptors and also serves as a useful “handle” for manipulating the antibody during most immunochemical procedures. The number of F(ab) regions on the antibody, corresponds with its subclass, and determines the valency of the antibody (loosely stated, the number of “arms” with which the antibody may bind its antigen). Direct-conjugated antibodies are labeled with an enzyme or fluorophore in the Fc region. The Fc region also anchors the antibody to the plate in ELISA procedures and is also seen by secondary antibodies in immunoprecipitation, immunoblots and immunohistochemistry. These three regions can be cleaved into two F(ab) and one Fc fragments by the proteolytic enzyme papain, or into just two parts: one F(ab’)2 and one Fc at the hinge region by the proteolytic enzyme pepsin. Fragmenting IgG antibodies is sometimes useful because F(ab) fragments (1) will not precipitate the antigen; and (2) will not be bound by immune cells in live studies because of the lack of an Fc region. Often, because of their smaller size and lack of crosslinking (due to loss of the Fc region), Fab fragments are radiolabelled for use in functional studies. Interestingly, the Fc fragments are often used as blocking agents in histochemical staining.
Properties of Immunoglobulins
|Class/ Subclass||Heavy Chain||Light Chain||Molecular Weight (kDa)||Structure||Function|
|l or κ||150 to 600||Monomer to tetramer||Most produced lg; protects mucosal surfaces; resistant to digestion; secreted in milk.|
|lgD||d||l or κ||150||Monomer||Function unclear; Works with lgM in B-cell development. mostly B cell bound|
|lgE||e||l or κ||190||Monomer||Defends against parasites; causes allergic reactions|
|l or κ||150||Monomer||Major lg in serum; good opsonizer; moderate complement fixer (lgG3); can cross placenta|
|lgM||µ||l or κ||900||Pentamer||First response antibody; Strong complement fixer; Good opsonizer|
Antigen-Antibody InteractionThe specific association of antigens and antibodies is dependent on hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Waals forces. These are all bonds of a weak, non-covalent nature, yet some of the associations between antigen and antibody can be quite strong. Like antibodies, antigens can be multivalent, either through multiple copies of the same epitope, or through the presence of multiple epitopes that are recognized by multiple antibodies. Interactions involving multivalency can produce more stabilized complexes, however multivalency can also result in steric difficulties, thus reducing the possibility for binding. All antigen-antibody binding is reversible, however, and follows the basic thermodynamic principles of any reversible bimolecular interaction:
where KA is the affinity constant, Ab and Ag are the molar concentrations of unoccupied binding sites on the antibody or antigen respectively, and Ab–Ag is the molar concentration of the antibody-antigen complex.
The time taken to reach equilibrium is dependent on the rate of diffusion and the affinity of the antibody for the antigen, and can vary widely. The affinity constant for antibody-antigen binding can span a wide range, extending from below 105 mol–1 to above 1012 mol–1. Affinity constants can be affected by temperature, pH and solvent. Affinity constants can be determined for monoclonal antibodies, but not for polyclonal antibodies, as multiple bondings take place between polyclonal antibodies and their antigens. Quantitative measurements of antibody affinity for antigen can be made by equilibrium dialysis. Repeated equilibrium dialyses with a constant antibody concentration but varying ligand concentration are used to generate Scatchard plots, which give information about affinity valence and possible cross-reactivity.
Affinity describes the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity. Avidity is perhaps a more informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: antibody-epitope affinity; the valence of both the antigen and antibody; and the structural arrangement of the interacting parts.
Ultimately these factors define the specificity of the antibody, that is, the likelihood that the particular antibody is binding to a precise antigen epitope. Cross-reactivity refers to an antibody or population of antibodies binding to epitopes on other antigens. This can be caused either by low avidity or specificity of the antibody or by multiple distinct antigens having identical or very similar epitopes. Cross-reactivity is sometimes desirable when one wants general binding to a related group of antigens or when attempting cross-species labeling when the antigen epitope sequence is not highly conserved in evolution.
Immunochemical techniques capitalize upon the extreme specificity, at the molecular level, of each immunoglobulin for its antigen, even in the presence of high levels of contaminating molecules. The multivalancy of most antigens and antibodies enables them to interact to form a precipitate. Examples of experimental applications that use antibodies are Western blot, Immunohistochemistry and Immunocytochemistry, Enzyme-Linked Immunosorbent Assay (ELISA), Immunoprecipitation, and Flow Cytometry. Each discussed in more detail in later sections of this publication.
Monoclonal & Polyclonal AntibodiesWhen designing experimental procedures, it is important to differentiate between monoclonal and polyclonal antibodies, as these differences are the foundation of both advantages and limitations of their use.
Many of the antibodies used in immunochemical techniques are raised by repeated immunization of a suitable animal, e.g., rabbit, goat, donkey, or sheep, with a suspension of the appropriate antigen. Serum is harvested at the peak of antibody production. Specific IgG concentrations of approximately 1 to 10 mg/mL serum can be obtained by this method. Weakly antigenic molecules may require the addition of an adjuvant, which allows for the slow release of the antigen making it more readily trapped by macrophages. Smaller molecules such as drugs must be coupled to more antigenic structures (carrier proteins) to stimulate an immune response.
One characteristic of large antigen molecules is that they induce the activation of many antibody-producing B cell clones in the immunized animal. This polyclonal mixture of resulting antibodies may then recognize a variety of epitopes on the antigen, which can be an especially useful feature in some experimental procedures. Because these polyclonal mixtures of antibodies react with multiple epitopes on the surface of the antigen, they will be more tolerant of minor changes in the antigen, e.g., polymorphism, heterogeneity of glycosylation, or slight denaturation, than will monoclonal (homogenous) antibodies.
Depending upon the antigen that is used to create the antibody, one may use polyclonal antibodies to identify proteins of high homology to the immunogen protein or to screen for the target protein in tissue samples from species other than that of the immunogen. Along the same lines, it is especially important when working with polyclonal antibodies to educate one’s self about the immunogen that has been used for production of the polyclonal antibody and the potential for undesired cross-reactivity within one’s sample. Peptide immunogens are often used to generate polyclonal antibodies that target unique epitopes, especially for protein families of high homology.
A homogeneous population of antibodies (i.e. monoclonal antibodies) can be raised by fusion of B lymphocytes with immortal cell cultures to produce hybridomas. Hybridomas will produce many copies of the exact same antibody. This impressive phenomenon has been instrumental in the development of antibodies for diagnostic applications. Because monoclonal antibodies react with one epitope on the antigen, however, they are more vulnerable to the loss of epitope through chemical treatment of the antigen than are polyclonal antibodies. This can be offset by pooling two or more monoclonal antibodies to the same antigen.
Some Useful Properties of Polyclonal Antibodies
- Polyclonal antibodies often recognize multiple epitopes, making them more tolerant of small changes in the nature of the antigen.
- Polyclonal antibodies are often the preferred choice for detection of denatured proteins.
- Polyclonal antibodies may be generated in a variety of species, including rabbit, goat, sheep, donkey, chicken and others, giving the users many options in experimental design.
- Polyclonal antibodies are sometimes used when the nature of the antigen in an untested species is not known.
- Polyclonal antibodies target multiple epitopes and so they generally provide more robust detection.
- Because of their specificity, monoclonal antibodies are excellent as the primary antibody in an assay, or for detecting antigens in tissue,
- and will often give significantly less background staining than polyclonal antibodies.
- When compared to that of polyclonal antibodies, homogeneity of monoclonal antibodies is very high. If experimental conditions are kept constant,
- results from monoclonal antibodies will be highly reproducible, between experiments.
- Specificity of monoclonal antibodies makes them extremely efficient for binding of antigen within a mixture of related molecules, such as in the case of affinity purification.
Antibody FormatsPolyclonal antibodies are often available in relatively unpurified formats, described as “serum” or “antiserum”. Antiserum refers to the blood from an immunized host from which the clotting proteins and RBCs have been removed. The antiserum, as its name suggests, still possesses antibodies/immunoglobulins of all classes as well as other serum proteins. In addition to antibodies that recognize the target antigen, the antiserum also contains antibodies to various non-target antigens that can sometimes react non-specifically in immunological assays. For this reason, raw antiserum is often purified, to eliminate serum proteins and to enrich the fraction of immunoglobulin that specifically reacts with the target antigen.
Antiserum is commonly purified by one of two methods: Protein A/G purification or antigen affinity chromatography. Protein A/G purification takes advantage of the high affinity of Staphylococcus aureus Protein A or Streptococcus Protein G for the immunoglobulin Fc domain. While Protein A/G purification eliminates the bulk of the serum proteins from the raw antiserum, it does not eliminate the non-specific immunoglobulin fraction. As a result the Protein A/G purified antiserum may still possess undesirable cross reactivity. See the Appendix for Protein A/G binding affinities.
Antigen affinity purification takes advantage of the affinity of the specific immunoglobulin fraction for the immunizing antigen against which it was generated. Unlike Protein A/G purification, antigen affinity purification results in the elimination of the bulk of the non-specific immunoglobulin fraction, while enriching the fraction of immunoglobulin that specifically reacts with the target antigen. The resulting affinity purified immunoglobulin will contain primarily the immunoglobulin of desired specificity.
Monoclonal antibodies may be grown in cell cultures and collected as hybridoma supernatants, or grown in mice or rats and collected as relatively unpurified ascites fluid. These can be purified through the use of Protein A/G or specific antigen affinity chromatography as with polyclonal antibodies.
Unpurified antibody preparations vary significantly in specific antibody concentration. If the specific antibody concentration of a given unpurified antibody preparation is unknown, one may refer to the following “typical ranges” as a guideline for estimation:
- Polyclonal Antiserum: Specific antibody concentrations will typically range from 1–3 mg/mL.
- Hybridoma Supernatant: Specific antibody concentrations will typically range from 0.1–10.0 mg/mL.
- Ascites Fluid (unpurified): Specific antibody concentrations will typically range from 2–10 mg/mL.
- Antibody concentrations of purified preparations should be determined through standard protein assays prior to the addition of stabilizing proteins such as BSA.
Often for signal amplification and detection purposes, purified antibodies are conjugated to enzymes, fluorophores, or haptens such as Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP), Rhodamine, FITC, or biotin. The various antibody conjugates have differential stabilities and require different buffers and storage conditions to retain their maximal activity over time. The following table lists the standard antibody buffers and storage conditions for purified Millipore antibodies and antibody conjugates. Note that these are general guidelines and that one should always consult the datasheet accompanying the antibody for specific storage conditions for that antibody.
General Technical Guidelines
The use of proper controls will help eliminate false positive and false negative results and aid in the interpretation of experimental data. They will also be invaluable in troubleshooting throughout the experimental design process. Whenever possible, both negative and positive control samples should be included in an assay. A positive control sample may be any tissue, cell line, purified protein, etc. that is known to contain the antigen of interest and to have previously been determined to be positive by a reliable method. A negative control sample is one that is known to be devoid of the antigen of interest.
In addition to sample controls, one should also use reagent controls. Remembering to change only one experimental variable at a time, one should run separate controls for primary and secondary antibodies. Because antibodies from different animal bleeds or purification batches may have significantly different titers, each new batch of antibody must be standardized before use in an existing assay.
It should be noted that an integral part of good laboratory practice is to keep complete documentation of all dilutions, diluents, incubation times, lot numbers, preparation dates of all reagents and procedural steps. This kind of information is invaluable in efficient assay development.
Handling of Reagents
In order to preserve maximum reactivity, reagents should be stored according to manufacturer instructions whenever possible (e.g., held minimally at room temperature when storage at 2–8°C is indicated). It is a good rule of thumb to store antibodies in tightly sealed containers in a non-frost-free refrigerator/freezer away from tissue fixatives and cross-linking reagents. Undiluted antibodies should always be aliquoted prior to storage at –20°C to minimize repeated freeze/thaw cycles, which can cause antibody denaturation. Storing an antibody in concentrated form will prevent or minimize degradation. Unless a stabilizing protein such as BSA (1% w/v) has been added, antibodies should not be stored for extended periods at their working dilutions. If antibodies will be stored at 2–8°C for more than two to three days, it is advisable to add a bacteriostat, such as 0.05% sodium azide or 0.1% thimerosal.
Note: Sodium azide will inhibit the activity of horseradish peroxidase
As with all laboratory reagents, consult a Material Safety Data Sheet (MSDS) for additional handling precautions.
Antibody and Titer
As mentioned above, the rate of binding between antibody and antigen is dependent on the affinity constant, which in turn can be affected by temperature, pH, and solvent composition. Varying the relative concentrations of antibody and antigen in solution can also control the extent of antibody-antigen complex formation. In most cases, however, the concentration of antigen in a sample cannot be adjusted. Typically, therefore, the optimal working concentration (dilution) of the antibody must be determined empirically for a given set of experimental conditions. For any assay, the optimum titer is that concentration (dilution) which gives the strongest reaction for positive tests with minimum background reaction (e.g., for negative controls). The optimal antibody concentration must be determined experimentally for each assay and is typically determined by using a dilution series.
The optimal antibody concentration is best determined by first selecting a fixed incubation time, and preparing a series of experimental dilutions to test. Dilutions are usually expressed as the ratio of the more concentrated stock solution to the total volume of the desired solution. For example a 1:10 dilution of antibody is created by mixing one part of antibody stock solution with nine parts of diluent, giving a total of ten parts. For further dilutions, see the Appendix.
Datasheet protocols may suggest approximate dilutions for antibody use. When using an antibody for the first time, or when working with a new batch of antibody, it is advisable to try a dilution series to determine the optimal antibody dilution to use. For example, if a product data sheet suggests using a 1:500 dilution, one may wish to make dilutions of 1:50, 1:100, 1:500, 1:1,000 and 1:10,000, to determine the optimal dilution for one’s unique assay conditions. Especially in the case of polyclonal antisera, antibody concentrations may be significantly different from animal to animal, or from one serum bleed to the next, and this kind of initial titration is essential in reducing interassay variation.