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3.5: Extension - Antibodies in Quantitation and In Vivo Detection

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    64709
  • Enzyme-Linked Immunosorbent Assay (ELISA) 

    (work derived from the Human Atlas Project)

    Since the very first use of antibodies for the detection of antigens, many different technologies have been developed that make use of the antibodies' capability to bind to other molecules. During the 1950s, the scientists Yalow and Berson developed a method where radioactivity is used to determine the amount of an analyte in a solution. This so called 'radioimmunoassay' (RIA), for which Yarlow received the Nobel prize in 1977, was a very sensitive method for the detection of hormones but using radioactivity for antigen detection is not safe and suitable for a general use. Hence, an alternative procedure was developed by linking enzymes to antibodies instead of a radioactive molecule, and by adhering molecules to surfaces. In one of the nowadays most common applications today are measuring the quantity of a biomolecule in a sample by "enzyme-linked immunosorbent assay" (ELISA). This term originally refers to the use of an enzyme to report an interaction between an antibody and its binding partner (Gan & Patel, 2013). The foundation for Perlmann and Engvall in Sweden (Engvall & Perlmann, 1971) as well as Schuurs and van Weemen from the Netherlands (Van Weemen & Schuurs, 1971), who built assays with immobilized and enzyme-modified reagents in the early 1970s. Today, scientists also use colored molecules (so called fluorophores) that re-emit light upon excitation to visualize antibody-antigen interactions. Many variants of experimental procedures have been developed, and it is common to build assays using more than one antibody to detect a target of interest (see Figure 3.xx C-D). To further enhance the possibilities offered by the immunoassay format, applications based on microarrays have been developed and which allow to measure more than one molecule in a single reaction chamber (see below).

    ELISA Assay Design

    The use of antibodies allows designing experiments in many different ways for the intended analysis. To achieve the best possible results from the experiment, different reagents, additives, and solutions have to be tested for their optimal combination and concentration, incubation times and the number of wash cycles need to be evaluated and adjusted. This is to avoid unwanted interactions, which disturb the analysis from detecting the target of interest. Moreover, the mode of how a target is identified and detection can be performed in a number of ways, as described in Figure \(\PageIndex{1}\) below.

    Figure \(\PageIndex{1}\): Different setups for ELISA and Other Immunossays. (CC BY-SA 3.0; The Human Atlas Project)

    In ELISA assays, the antibodies may (A) detect an immobilized antigen, (B) capture a labeled antigen, (C) capture an unlabeled antigen and use a second, labeled antibody to detect the captured antigen, or (D) use a third antibody for detection, or even use two antibodies for detection (E). Direct labeling of the antibody or antigen as in (A), (B), and (C) is the simplest and fastest method for detection. Using a secondary antibody as detection method, as shown in (D) and (E), will further increase the sensitivity and selectivity of the analysis. The method used in (D) also allows greater flexibility, whereas method (E) further increases the specificity, as three antibodies must bind the antigen in order to produce a reporter molecule. Out of the presented assays, the most commonly used concepts are shown in (C) and (D).

     

    Microarrays

    Multiplexing

    A new era in immunoassays started with the development of a technology called microarrays. The term microarray most commonly describes the ordered organization of small volume droplets that have dried on a small surface area. The reaction dimensions are miniaturized so that many assays can be performed in multiple samples in parallel, several thousands of different features may be presented to the surrounding solution. This means that scientists can measure a large number of molecules with one single experiment. There is the possibility to use microscope glass slides and specialized robotics that deposit very small drops of liquid (1 nl = 0.000000001 liter) on the glass surface in an ordered fashion. This leaves behind spots of less than one millimeter in diameter (0.15 mm). Another common technique for multiplexing is to use even smaller and color-coded particles (diameter of 0.005 mm). These particles can be coated with antibodies to fish out the analyte from the solution.

    Sensitivity

    In many applications it is important to measure very small amounts (sometimes only traces) of a molecule in a given sample. In order to achieve the required sensitivity, the conditions of the experiment need to be adjusted to suit the antibodies, the detection system, and the type of samples. In addition, there is progress being made on using better colors,signal amplification specialized lasers and filters, as well as miniaturization (Ekins & Edwards, 1997).

    Specific examples

    There are many examples of how ELISA assays may be used in basic research and in clinical diagnostics. One specific example is the sensitive sandwich-type enzyme-linked immunoassay used to determine the amount of the protein prostate-specific antigen (PSA), which is a biomarker used to detect prostate cancer (Kuriyama et al., 1980).

    Microarray assays on the other hand, have previously received a lot of attention for their use in parallel analysis of DNA and RNA molecules. To translate their advantages to assays for the analysis of proteins with antibodies, new protocols and routines had to be developed and established. Nowadays, there are multiplexed techniques for measuring the amount of proteins in different sample types (e.g. cells, blood serum, urine), to determine how proteins are modified in biological processes (e.g. phosphorylation), or to describe specific protein-protein interactions. Another example is the analysis of antibodies circulating in blood from patients. Microarray-based applications have also been built for purified antibodies and to study the antibody binding characteristics – an important aspect when using binding reagents as research reagents. Such protein microarrays can either consist of proteins, protein fragments, or small peptides to test the specificity of the binding reagent. Protein microarrays can reveal the interactions to entire proteins or larger protein fragments, while peptide microarrays show to which particular parts (epitopes) of the proteins the antibodies bind. A typical epitope mapping result is shown in Figure \(\PageIndex{2}\) below (Edfors et al., 2014). 

    Figure \(\PageIndex{2}\): Epitope Mapping of Polyclonal Antibodies. Polyclonal antibodies binding to a peptide array where the result displays four distinct linear epitopes and the consecutive overlapping peptides which are bound. X-axis: peptides, Y-axis: mean fluorescence intensity (MFI). (Edfors et al., 2014)  Image from The Human Atlas Project

    Synthesizing millions of overlapping peptides with only one amino acid residue shift on such arrays enables the mapping of antibody binding regions at high resolution. This gives very detailed information of the linear (continuous) epitopes recognized by an antibody. Just like with proteins, protein fragments or other antigens, the assembly of peptides on arrays may also be used for studies of antibody reactivity in plasma samples from patients with infectious and autoimmune diseases.

    Immunohistochemistry - Detecting Proteins in Vivo

    (work derived from the Human Atlas Project)

    Immunohistochemistry (IHC) is a powerful microscopy-based technique for visualizing cellular components, for instance proteins or other macromolecules in tissue samples. The strength of IHC is the intuitive visual output that reveals the existence and localization of the target-protein in the context of different cell types, biological states, and/or subcellular localization within complex tissues.

    The IHC technique was invented during the 1940s (Coons, Creech, & Jones, 1941) and is routinely used as an important tool in health care and pathology for e.g. diagnostic purposes or to stratify patients for optimized treatment regimes. IHC is also widely used in research where molecules of interest are analyzed to study their roles in both healthy and diseased cells and tissues on the molecular, cellular or tissue level. There are many different ways to perform visualization of targets in tissues using IHC or IHC-based methods, and numerous protocols exist for different applications and assays. Even though IHC is generally a robust and established method, new assays often need careful optimization depending on the tissue or on the properties of the target protein, binder-molecule and/or reporter system. Many years of technical development and the hugely increased availability for specific binding-molecules have greatly improved the usefulness and areas of applications for IHC. The progress in the field of IHC-based techniques and reagents has enabled scientists and health care providers with more precise tools, assays and biomarkers. In addition, technical advances have enabled e.g. highly sensitive simultaneous detection of multiple proteins in the same sample, and the detection of protein-protein interactions.

    The classical IHC assay is illustrated in Figure \(\PageIndex{3}\) below and involves detection of epitopes expressed by a single protein-target within a tissue sample using a "primary antibody" capable of binding those epitopes with high specificity. After the epitope-antibody binding event, a "secondary antibody" capable of binding the primary antibody with high specificity is added. The secondary antibody is coupled to a reporter molecule and after the antibody-antibody binding event, a chemical substrate is added which reacts with the reporter molecule to produce a colored precipitate at the site of the whole epitope-antibody complex.

    Figure \(\PageIndex{3}\): The Basic Principle of Immunohistochemistry.  mage from The Human Atlas Project

    In the schematic illustration, a formalin-fixed paraffin embedded tissue section is stained using a primary antibody directed towards a specific protein target. A solution containing the primary antibody is added to the tissue section and the antibodies are allowed some time to find and bind to their target. After this step, unbound and surplus antibodies are washed away and the secondary antibody is added. The secondary antibody, which carries a linker molecule with horseradish peroxidase (HRP) enzymes, is also allowed some time to bind to the primary antibody, followed by another washing step. After this, 3,3' Diaminobenzidine (DAB) is added. The HRP enzyme transforms the DAB substrate into a brownish precipitate that is deposited in the tissue at the site of the reaction, thus producing a visual representation of where the primary antibody first bound its target.

     

    Tissue preparation

    The tissue plays a central role in the experiment and it is important that it is processed so that epitopes and proper morphology is preserved. The most common processing for IHC is to prepare formalin-fixed paraffin-embedded (FFPE) tissue blocks. The purpose of formalin fixation is to produce chemical cross-linking of proteins within the tissue. This terminates all cellular processes and freezes the cellular components at the place and in the conformation they were in at the time of fixation and also prevent degradation. After adequate fixation, the tissue is further processed and ultimately embedded in paraffin blocks, which are then sectioned into thin slices (usually 4-10µm) using a microtome. The sections are transferred to glass slides and allowed to adhere prior to further processing.

    Other methods for fixation besides formalin are sometimes used. These include other types of aldehydes or using different alcohol solutions. The best choice of fixative is very much dependent on the assay. A common alternative to FFPE is to prepare frozen tissue samples. In this case, the tissue is embedded in a cryoprotective medium and frozen, and fixation is performed post-sectioning. Frozen tissues are sectioned in cryostats and have the advantage of short processing times and of better preservation of sensitive epitopes, but can often be inferior to FFPE tissues in terms of preserving histological morphology.

    Antigen (epitope) retrieval

    A concern associated with cross-linking fixatives like formalin, or too long time spent in fixative medium is the masking of epitopes, which can obstruct the primary antibody from binding to its target. Especially with FFPE samples, there is often a need to revert some of the chemical crosslinking and "retrieve" the epitopes before proceeding to the actual IHC. There are several antigen retrieval protocols available and the main strategies include treating the tissue slide with heat, digestive enzymes, detergents, or combinations thereof. The most common method for antigen retrieval in FFPE samples is to pressure-boil the tissue slides in an acidic citrate buffer for around 15-20 minutes.

    Antibody binding

    The quality and specificity of the binding molecule is crucial for any IHC based technique, and the choice of binder can directly affect the outcome, reliability, and possibly also the interpretation of the assay. Antibodies are by far the most common type of binding-molecule used for IHC, and although most antibodies are able to adequately detect the correct molecule of interest, they may also vary greatly in their specificity for their intended target. Antibodies with high specificity are therefore more reliable when interpreting "on-target" binding, since they produce little or no "off-target" binding or "background". Antibodies that are less specific can produce more off-target binding, and the resulting background will possibly interfere with the correct interpretation of the true on-target signals. There are two main types of antibodies; polyclonal antibodies which is a heterogeneous mix of antibodies that bind different epitopes on the target and monoclonal antibodies that all bind the same epitope. Polyclonal antibodies are often very potent due to their ability to detect and bind multiple epitopes on the same target. However, the epitopes they bind are often poorly defined and with multiple and varying epitope-specificities comes the increased likelihood of off-target binding events and background noise. However, the potency of polyclonal antibodies can be advantageous since the concentration of binding events around the on-target molecule usually outweighs potential background noise. A drawback is that polyclonal antibodies are usually limited resources since they are derived from animal sera. Monoclonal antibodies, by contrast, have more continuity since they can be produced in hybridoma cell lines. Monoclonal antibodies are also often well defined in terms of epitope binding, but can still generate results that are hard to interpret if the specificity is low or if the target epitope is present in low abundance.

    Careful optimization and titration of antibody concentration for each assay is needed, since the result is dependent not only on the antibody's specificity and affinity for the target, but also on the concentration and availability of on-target and potential off-target epitopes present in the sample. Adding too much antibodies to the sample will increase the number of possible low-affinity off-target binding events once the on-target epitope(s) are saturated with binders. By lowering the antibody concentration, off-target binding events become rarer as they usually have lower affinity than on-target binding events. The risk when attempting to reduce background while using a low-affinity antibody is that the on-target signals are concomitantly weakened to the point of providing a false negative result.

    Other types of binder molecules sometimes used in IHC-based techniques include affibodies, peptides, antibody fragments or other small molecules.

    Detection systems

    The whole purpose of performing IHC is to obtain a visual representation of where the target can be found within the experimental tissue, and preferably also gain information about the target's expression pattern among heterogeneous cell populations and/or subcellular localizations. This is exemplified in Figure \(\PageIndex{4}\) below, which illustrates how different antibodies are used to visualize different cellular or tissue compartments within a complex tissue. To visualize the target-antibody interaction, some kind of detection system that produces an observable stain or signal is needed. The most common method for introducing a detection system to the experiment is to use a secondary antibody that carries a pre-bound reporter molecule, i.e. enzyme or fluorophore. Secondary antibodies are usually targeted specifically towards antibody molecules from a different animal species. For example, if the primary antibody is raised in a rabbit, then the secondary antibody must be raised in another animal and targeted specifically towards rabbit antibodies.

    Figure \(\PageIndex{4}\): Visualizing different protein targets in complex tissues.   Image from The Human Atlas Project

    The right column shows a magnification of the corresponding images in the left column. In the IHC image, consecutive sections of human esophagus stained using four different antibodies allows for direct comparison of different protein expression patterns within the tissue and within subcellular compartments. The top images are only counterstained for hematoxylin for comparison. The p63 antibody stains cell nuclei in a population of cells that reside in the basal part of the esophageal epithelium. The EGFR (Epidermal growth factor receptor) antibody appears to stain the same cell population as p63, but stains cellular membranes instead of nuclei. The G6PD (Glucose-6-phosphate dehydrogenase) antibody stains the cytoplasm of a wider repertoire of esophageal epithelial cells and also cells residing in the connective tissue. The Laminin (LAMB2) antibody stains only cells and structures in the connective tissue underlying the esophagus.

    Image from The Human Atlas Project

    For FFPE tissue samples the most common detection method is to use enzymatic reactions to generate a colored precipitate at the site of antibody binding. The secondary antibodies then carry an enzyme, e.g. horseradish peroxidase (HRP) or alkaline phosphatase (AP), that are capable of converting chromogens like 3,3' Diaminobenzidine (DAB) or 5-bromo-4-chloro-3-indolyl phosphate/ p-nitroblue tetrazolium chloride (BCIP/NBT) into brown or bluish precipitates that are deposited in the tissue at the site of the reaction. Chromogenic stains are observable in light-microscopy and are usually very stable over long periods of time, which is beneficial if the experiment needs to be archived or reviewed at a later time point.

    For frozen tissue sections it is more common to use fluorophore-linked secondary antibodies that emit a specific color (usually green, red, or blue) when excited by the correct wavelengths of light. Moreover, fluorophores are usually not stable for long periods of time. However, the benefit of using fluorophores is that they provide an easy method for performing double-labeling experiments where several antibodies towards multiple targets are assayed in the same sample. The secondary antibodies need to be targeted towards different primary antibodies and also to be coupled to different fluorophores. The different secondary antibodies are then observed separately by exciting them sequentially with different wavelengths of light. These different excitation results are saved as separate images (or color channels) and may later be overlaid to infer protein co-localizations etc.

    Using reporter-carrying secondary antibodies for detection is in itself an amplification step since several secondary antibodies are able to bind a single primary antibody, but sometimes further amplification steps are desired to increase the signal and sensitivity of the experiment. In such cases, the secondary antibody may instead carry "linker molecules", for instance biotin polymers, which are able to recruit a larger number of reporter molecules in subsequent steps. This strategy for amplifying signals is useful for both enzymatic and fluorescent detection methods.

    Counterstaining

    Immunohistochemical staining using chromogens offers benefits from having a counterstain applied that enhances the contrast and facilitates the observation of histological features. The most common type of counterstain used for FFPE samples is hematoxylin that stains cellular cytoplasm with a pale bluish color, and stain cell nuclei in a darker bluish nuance. Fluorescent stainings are usually not counterstained with hematoxylin, since the detection method is not based on light microscopy. Instead, the most common way to obtain counterstaining for fluorescence is to label cell nuclei by adding fluorescent dyes that bind nucleic acids.. After the actual immunohistochemical reaction, the only remaining steps are to coverslip and seal the sample for protection and long term storage. The most common way is to "glue" the coverslip to the sample using commercially available purpose-made resins.

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    Figure 3.27 Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubles are marked green by an antibody bound to FITC and actin filaments are labelled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial cells

    Image from NIH ImageJ-Programmpaket

    Specific examples

    IHC is widely used in both research and clinical practice. The Human Protein Atlas (HPA) project is a prime example of how high-throughput IHC is used to achieve large-scale mapping of the human proteome in a multitude of tissues, cancers and cells. In the HPA project, a streamlined in-house large scale antibody production chain facilitates the generation of specific antibodies, which after passing basic characterization and validation regimes, are used to systematically stain tissue microarrays containing hundreds of tissue cores within a single experiment. The system for IHC employed by HPA relies heavily on standardization of protocols and automatisation using machines, but the evaluation of the optimal titration for each antibody is performed manually before the antibody is approved for staining on the full set of tissues. Each stained tissue core is annotated with respect to immunohistochemical staining in tissues and cell types, and thereafter published as a high resolution image on the web portal to be freely viewed by anyone.

    In clinical practice, IHC is mainly used within pathology to aid physicians to evaluate tissue specimens with respect to healthy and or diseased states, to set diagnoses, and to define the molecular subtype of different types of cancer. A specific example where IHC is used diagnostically is when pathologists are presented with a metastatic tumor sample and the tissue origin of the primary tumor is unknown. In these cases, pathologists use a panel of different antibodies that target tissue specific proteins, such as prostate-specific antigen for prostate cancer, or estrogen receptor for gynecological cancers, or cytokeratin 20 for gastrointestinal cancers (Gremel et al., 2014). Once a broad classification is made, additional tissue specific antibodies are used to further pinpoint the origin of the primary tumor. This information is useful for choosing the best or most appropriate strategy for drug therapy and/or to locate the primary tumor for radiation therapy and/or surgery.

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