Concepts, Considerations and Control Experiments for Immunolabeling Karl Garsha, Imaging Technology Group, Beckman Institute for Advanced Science and Technology, UIUC
[email protected] Introduction The exquisite specificity of antigen-antibody interactions has led to the exploitation of antibodies to detect the presence and location of a wide variety of antigens in prokaryotic cells and eukaryotic cells and tissues. The success of an immunodetection experiment depends on the reactivity and quality of the reagents employed as well as the careful attention of the investigator to the labeling conditions. Protocols for immunolabeling vary widely due to experimental goals as well as the differences between antigens and their recognition by antibody. The methods used to prepare a sample for microscopic visualization may confound immunolabeling: some antigenic epitopes (the small structural element recognized by an antibody) are destroyed by temperature extremes or the organic solvents commonly used to process histological samples. Different fixatives may destroy epitopes or make them less accessible. The choice of blocking agent may impact on the level of non-specific background staining. The pH of various solutions used in the labeling procedure may alte r the charge properties and hence the binding properties of antibodies to antigens. Indeed, volumes have been written on the subjects of immunohistochemistry, immunocytochemistry and immunolabeling; a detailed review of the many applications of antibody technology to microscopy and/or general protocols for immunolabeling is beyond the scope of this article. Rather, it is hoped that the following information will supplement the reader’s existing knowledge of basic immunostaining technique by providing insight into some of the more subtle considerations which may go into a well designed and executed immunolabeling experiment. For success in immunolabeling it is crucial to carefully optimize the labeling conditions; in order to do this it is important to understand some basic concepts regarding antigen-antibody interactions. It is also important for an investigator to be aware of important control experiments which will aid in troubleshooting poor labeling results and increase confidence in the data obtained with the optimized labeling protocol. Polyclonal Antibodies vs. Monoclonal Antibodies Soluble immunoglobulins (antibodies) function as antigen binding proteins which circulate in the blood of most vertebrates; they serve as effectors of the immune system by searching out and neutralizing antigens or marking them for elimination. Most antigens are complex and contain many different antigenic determinants, or epitopes, and so the immune system generally responds by producing antibodies to several of them (Figure 1). This response requires the
recruitment of several antibody producing cells (B-cells); each B-cell (and its progeny) produces antibodies which specifically bind to a single epitope (antibody binding region) on the antigen. Each B-cell produces a monoclonal antibody, the collective output of these monoclonal B-cells is considered to be a polyclonal antibody response to an immunizing antigen. The polyclonal antibody produced in vivo is beneficial to the organism, however polyclonal antisera has numerous disadvantages for in vitro applications such as immunolabeling experiments, which demand precise control of antibody quantity, properties and specificity. Figure 1. Monoclonal antibodies binding to different epitopes.
For most research purposes, monoclonal antibodies, which are derived from a single B-cell clone and thus specific for a single epitope, are preferable. The method for preparing monoclonal antibody was devised by Kohler and Milstein (1975) and their work was later recognized with a Nobel Prize in 1984. The basic approach involves fusing an antibody producing B-cell with a myeloma cell. The resulting hybridoma possesses the immortal growth properties of the myeloma cell and secretes the antibody encoded by the genetic material of the B-cell. The resulting clones of the hybridoma cell secrete large amounts of monoclonal antibody, and may be cultured indefinitely. Once antibody secreting hybridomas are obtained, they are screened for the desired antigen specificity. Monoclonal antibodies are generally well characterized, an important consideration for optimizing immunolabeling studies. Antibody Structure and Classification Antibodies are glycoproteins, and they can function as potent antigens to induce an antibody response. For example, antibodies from a rabbit may be used to immunize a goat to produce anti-rabbit antibodies. These goat anti-rabbit
antibodies may be used as a reagent to detect the binding of mouse antibodies in vitro. Such a detection scheme is known as indirect labeling, and an advantage of this approach is that the signal resulting from the binding of the primary antibody is amplified (Figure 2). A disadvantage of indirect labeling is that nonspecific antibody staining is often more prevalent. Proper choice of antibody reagents is critical to the success of this approach. Secondary labeling reagents may be specific for the species (xenotype) from which the primary antibody is derived, the class (isotype), the subclass (allotype) a nd the light chain of the primary antibody. Highly specific secondary antibody reagents may reduce nonspecific staining significantly (as compared to secondary reagents which are specific for the xenotype and isotype of the primary antibody alone). Thus, it is advantageous to be familiar with the antibody isotype, allotype and light chain type of the primary antibody reagent for purposes of selecting the most specific secondary antibody labeling reagent. Figure 2. Signal amplification through indirect labeling.
Antibodies are composed of four polypeptide chains, two light chains and two heavy chains (Figure 3). Each light chain is composed of two domains, one variable domain (V L) and one constant domain (C L). There are two types of light chains, lamda (λ) and kappa (κ). In humans, 60% of the light chains are κ, and 40% are λ, whereas in mice, 95% of the light chains are κ and only 5% are λ. A single antibody molecule contains either κ light chains or λ light chains, but neve r both.
The heavy chain serves to determine the functional activity of the molecule in vivo. There are five different heavy chain isotypes present in mammals such as humans and mice: IgG, IgA, IgM, IgE and IgD. IgG isotypes can be further subdivided, for instance, in mice IgG isotypes are divided into IgG1 , IgG2a, IgG2b and IgG3 subclasses, or allotypes. Figure 3. Basic antibody monomer structure.
For a well characterized monoclonal antibody, the species, isotype, allotype and light chain component will be known. For instance, a mouse derived (murine) monoclonal antibody might be designated as IgG2b κ, reflecting the antibody isotype, allotype and light chain type. A well matched secondary probe would be specific not just for murine IgG antibodies, but for murine IgG2bκ antibodies in particular. Strength of Antigen-Antibody Interactions The antigen-antibody interaction is a bimolecular association that does not lead to an irreversible chemical alteration in either the antibody or the antigen. This interaction is therefore reversible. Interactions that contribute to antibody binding include hydrogen bonds, ionic bonds, hydrophobic interactions and van der Waals forces. The strength of each of these interactions is weak compared to a covalent bond, and consequently, a large number of non-covalent interactions are required to stabilize antibody binding to an antigen. Each of these noncovalent interactions operates over a very small distance (1 angstrom or less) and so a strong Ag-Ab interaction depends on a very close fit between the antigen and antibody. This fit requires a high degree of complementarity between antigen and antibody, and this requirement is the basis of the specificity that characterizes antigen-antibody interactions. The strength of the sum of the noncovalent interactions between a single antigen binding site (F v ) 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 disassociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer. Antibody affinity for a particular antigen may be measured quantitatively using methods such as equilibrium dialysis and Scatchard analysis, but the important concept to note is that not all antibodies
have the same affinity for their respective ligands. Different monoclonal antibodies to the same antigen may bind with vastly different affinities. When possible, it is important to take the affinity of the antibody for its respective ligand into consideration when designing immunolabeling experiments. The affinity at one binding site does not always reflect the true strength of the antibody-antigen interaction. When complex antigens containing multiple, repeating epitopes are mixed with antibodies containing multiple binding sites, the interaction of an antibody 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. High avidity can compensate for low affinity. For example, secreted pentameric IgM often has lower affinity than IgG, but the high avidity of IgM, resulting from the pentameric structure, enables it to bind antigen effectively. Cross-Reactivity Although Ag-Ab reactions are highly specific, in some cases antibody elicited by one antigen can cross-react with an unrelated antigen. Such cross-reactivity occurs if two different antigens share an identical epitope or if antibodies specific for one epitope also bind an unrelated epitope possessing similar chemical and structural properties. In the latter case, the antibody’s affinity for the crossreacting epitope is usually less than that for the original epitope. When using a monoclonal antibody developed towards a protein of interest from one species to probe for a similar protein in a related species it is important to realize that the affinity of the antibody binding phenomenon will be affected by the degree of structural conservation between the antigenic epitopes from the two species. Interestingly, cross-reactivity is the basis for the presence of antibodies to the ABO blood group antigens. The antibodies to blood group antigens are induced in an individual not by exposure to red blood cell antigens but by exposure to cross-reacting microbial antigens present on common intestinal bacteria. These cross-reacting microbial antigens induce the formation of antibodies in individuals lacking similar antigens on their red blood cells. The blood group antibodies, although elicited by microbial antigens, will cross-react with similar oligosaccharides on red blood cells. This example illustrates the importance of confirming that positive reactivity in an immunolabeling experiment reflects the presence of the antigen of interest. Positive Controls for Immunolabeling Positive controls are valuable tools to confirm the reactivity of the antibody to antigen under the labeling conditions specific to a particular protocol. If the antigen of interest is available in purified form, it can be adsorbed onto a nitrocellulose membrane. This membrane may then be blocked using a blocking reagent. Ideally the blocking reagent of choice will have similar charge properties to the primary antibody. This “dot-blot” may then be probed for the presence of
the antigen with the primary antibody under conditions which approximate the intended experimental conditions (Brada and Roth, 1984). Published reports of immunofluorescent staining can provide useful information regarding different experimental protocols for generating cells or tissues for use as positive staining controls. In systems where the presence of the antigen of interest is being tested, cells or tissues that are well documented to possess the antigen should be used as positive controls under identical labeling conditions to the experimental system. In order to explore the possibility that a monoclonal antibody is crossreacting with an irrelevant antigen in the experimental system, a monoclonal antibody to an alternate epitope on the antigen of interest may be used. Using monoclonal antibodies to different epitopes on the same antigen will strengthen the evidence that the antigen being detected is, in fact, the antigen of interest. Negative Controls for Immunolabeling Once a positive signal is obtained, proving that signal truly reflects the distribution of the molecule of interest is still a matter of some difficulty. One or more of the following controls can be used to discriminate specific from nonspecific staining. In order to assess the degree to which non-specific binding of the labeling antibody plays in the generation of a signal, an isotype control may be used. In the most basic form of this test, the negative control specimen is exposed to an antibody of the same isotype as the experimental antibody probe, but the control antibody has an irrelevant specificity. Oftentimes isotype control antibodies specific for plant antigens are used in animal systems and vice versa. Suppliers of monoclonal antibodies oftentimes offer well characterized isotype control antibodies. Ideally, the isotype control antibody will be of the same species, and have the same heavy chain and light chain antigens as the probe antibody. For instance, a murine IgG2b κ monoclonal antibody probe should have a monoclonal murine IgG2b κ isotype control. Using a poly-clonal antibody (ie., affinity-purified murine IgG taken from pooled serum) for an isotype control in an experimental system which uses a monoclonal antibody may give misleading results. This is because there is an increased chance for cross-reactivity present when using polyclonal isotype control. In a ligand-blocking control, the antibody probe is allowed to react with an excess of purified antigen. This pre-blocked antibody is then incubated with the intended target. Theoretically, antibody binding to the target should be attenuated. In practice, however, the degree to which this is true is related to the affinity and avidity of the antibody for its ligand. Recall that antibody binding is reversible. If a monoclonal antibody has a low affinity for the ligand, an equilibrium between the amount of antibody bound to antigen from the preblocking step and antibody bound to antigen localized in the cell or tissue being tested may be established. This phenomenon can make interpretation of the results of a pre-blocking control less than straightforward in some cases.
The antibody-blocking control is the inverse of the ligand-blocking control. In the antibody-blocking control, the cells or tissue of interest is pre-incubated with probe antibody which has not been conjugated to a signaling molecule. The blocked cell or tissue is then probed with the conjugated antibody probe. In theory, the bound antibody will prevent specific binding of the same antibody which has been conjugated to a signaling molecule, and any signal present after this incubation should be due to non-specific binding. In reality, the same complications as in the ligand-blocking control may arise if the probe antibody does not have high affinity for its respective antigen, or if fixation protocols have negatively influenced the affinity of the antibody for its ligand. Up-scaling these control strategies from direct labeling studies to indirect labeling studies involves exploring the nonspecific binding of the secondary antibody as well as the primary antibody. Because the primary antibodies used in indirect labeling are not usually conjugated directly to a signaling molecule, it is important to assess and minimize the nonspecific binding of the secondary reagent before exploring the nonspecific binding of the primary antibody reagent. In many cases, simply ommitting the primary antibody from the labeling protocol will suffice to give a n indication of non-specific binding of the secondary reagent. This can be taken one step further by omitting the primary antibody probe, and using an isotype control of irrelevant specificity in place of the secondary antibody. In so doing, one might gain a better perspective on the possibility that the secondary antibody is cross-reacting with immunoglobulins which may be present constituitively in the tissue under investigation, or with contaminating immunoglobulins which may be present in a serum derived blocking reagent. Controls for Autofluorescence When using antibodies conjugated to fluorescent molecules for immunolabeling it is important to control the degree to which autofluoresence of the cells or tissue under study contributes to the overall signal obtained using fluorescence microscopy. Many cellular metabolites and structural components exhibit autofluorescence, particularly in plant tissues. Cell culture media can also be intensely autofluorescent, and fixation with glutaraldehyde can induce autofluorescence in a sample as well. Mounting and embedding media used for sectioning work can be another source of autofluorescence, Canada balsam and glycerin-albumen are amongst the worst culprits. The simplest approach to control for the contribution of autofluorescence is to observe an unlabeled specimen prepared under the same conditions as the experimental specimen. This may be a useful way to troubleshoot autofluorescence problems without using up valuable labeling reagents. More sophisticated methods of controlling for autofluorescence have been recently outlined in an excellent article by Knight and Billinton (2001). These methods include the use of optical filters, dual-wavelength correction, computational image correction, fluorescence lifetime imaging microscopy (FLIM), quenching using histological stains, and fluorescence energy resonance transfer (FRET).
Conclusion It is rare that any two experiments employing immunolabeling are completely alike. There are many factors that govern the specific details of an antibody labeling experiment. Much to the chagrin of many investigators, trial and error play a large role in the eventual outcome of an experiment, although this is not generally reflected in the methodology section of published papers. With this in mind, some of the factors that may impact the specificity of an immunolocalization experiment, and aid in the optimization/troubleshooting process have been reviewed. There is a tendency for some investigators to believe (or at least want to believe) that any aggregation of label represents a specific localization, however, controls will often reveal considerable localization artifact due to non-specific binding. The data from immunolabeling experiments is not compelling without sets of positive and negative controls. These control experiments should be conducted simultaneously with the localization attempt. References Arevalo, JH, MJ Taussig, and IA Wilson. 1993. Molecular basis of crossreactivity and the limits of antibody-antigen complementarity. Nature 365:859. Berzofsky, JA, IJ Berkower and SL Epstein. 1991. Antigen-antibody interactions and monoclonal antibodies. In: Fundamental Immunology, 3 rd ed., WE Paul, ed. Raven Press, New York. Brada, D. and J Roth. 1984. “Golden Blot.” Detection of polyclonal and monoclonal antibodies bound to antigens on nitro-cellulose by protein-A gold complexes. Anal. Biochem. 142:79. Goldsby, R, TJ Kindt and BA Osborne. 2000. Immunoglobulins: Structure and Function. In: Kuby Immunology, 4 th ed., W.H. Freeman and Company, New York. Knight, AW and N Billinton. 2001. Distinguishing GFP from Cellular Autofluorescence. Biophotonics International 8,7:42. Kohler, G. and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256:495. Polak, JM and S Van Noorden. 1997. Immunohistochemistry 2 nd ed. BIOS Scientific Publishers, Oxford.
