Use of Immunoassays in Neurological Diagnosis and Research

By Meriggioli, Matthew N

Objectives: To review the use of immunoassays in the diagnosis and research of disorders affecting the nervous system.

Methods: Systematic review of the English literature.

Results: Immunoassays have demonstrated utility for. (1) the detection of antigen (molecules, genes, gene products, peptides, hormones and drug metabolites) and (2) the detection of an immune response (antigen-antibody complexes and specific and non-specific populations of antibodies) in serum, cerebrospinal fluid, and central nervous system tissue.

Discussion: The specificity of the antibody-antigen interaction makes immunoassays an ideal diagnostic and research tool for the investigation of neurological disease. A number of immunoassays are available for this purpose, and the choice of a particular methodology generally depends upon whether one is detecting antigen, antibody or antigen-antibody complexes, and the nature of the biologic sample that is being tested. Ease of testing, sensitivity, specificity and cost are other important considerations. [Neurol Res 2005; 27: 734-740]

Keywords: Immunoassay; cerebrospinal fluid; antibody; antigen; central nervous system; ELISA; radioimmunoassay; immunofixation electrophoresis

INTRODUCTION

Immunoassays make use of antigen-antibody reactions, and are widely used in research and laboratory diagnosis of many neurological diseases. These assays may be categorized according to whether the goal of the assay is to detect antigen, antibody or antigen-antibody complexes, and according to the biological sample that is being tested (Table 1). Therefore, neurological immunoassays may be divided into assays that: (1) detect antigen in serum, cerebrospinal fluid (CSF), or the central nervous system (CNS) tissue; (2) detect antigenantibody complexes usually in serum or CSF and (3) detect and quantitate either non-specific or specific populations of antibodies in serum, CSF or CNS tissue.

NATURE OF ANTIGEN-ANTIBODY REACTIONS

The combination of antigen and specific antibody forms the basis for the immunoassays used in neurology and other areas of medicine1. Antibodies possess high specificity for a specific antigen. The part of a protein antigen that reacts with the antibody is called antigenic determinant. The bonds that hold the antigen in the antibody binding site are non-covalent, and by their nature reversible. Antibody affinity refers to the strength of the reaction between the antibody binding site and the antigenic determinant. Specificity is the ability of an individual antibody binding site to react with only one antigenic determinant. Cross reactivity occurs when an individual antibody binding site reacts with more than one antigenic determinant. This may occur when a cross-reacting antigen shares structural similarity with the immunizing antigen.

The higher the affinity of the antibody for the antigen, the more stable the interaction and the easier it is to detect in immunoassays. The ratio of antigen/antibody influences the size of the antigen-antibody complexes and affects their detectability. The physical form of the antigen (soluble or particulate) also influences how the reaction will be detected. The growth in the range, scope and clinical utility of immunoassays has largely arisen from the exquisite sensitivity and specificity that can be achieved using different types of antibodies. Antibody preparations used in immunoassays may be either polyclonal or monoclonal. Polyclonal antibodies are obtained from the serum of immunized animals, and are a mixture of antibodies with different affinities and specificities for the immunized material2. As would be expected, the response to immunization varies considerably from animal to animal, and standardization must be done with each preparation. Also, polyclonal antibodies may give unwanted cross reactions affecting the specificity of the assay.

Recently, polyclonal antibodies have been largely replaced in clinical immunoassays by monoclonal antibodies. Monoclonal antibodies are prepared in vitro by fusing plasma cells of an immunized animal with a cell line that grows continuously in culture (hybridoma)3. The monoclonal antibodies produced by these cells interact with a particular small region on an antigen called an epitope. Therefore, monoclonal antibodies are highly specific and, by and large, overcome the problems of unwanted cross reactions that occur with polyclonal antibodies. Another advantage is that the hybridoma cell line that produces monoclonal antibodies, can produce the same antibodies consistently and indefinitely.

Monoclonal antibodies possess high affinity and specificity for a specific antigen. It is this specific binding of antibody to antigen that allows detection of a particular molecule by a variety of immunoassay methods.

IMMUNOASSAYS DETECTING A SPECIFIC ANTIGEN IN SERUM OR CSF

Immunoassays of this type utilize one or more selected antibodies to detect the antigen(s) of interest. The measured antigens may be those that are normally present (such as thyroid hormone), expressed by a pathogen (such as a viral or tumor antigens), expressed in association with a specific disease process (such as myelin basic protein or 14-3-3 protein in the CSF) or that do not naturally occur in the body (such as a therapeutic drugs).

All immunoassays require the use of a tag that is a measure of the amount of antigen or antibody present. The tag is a molecule that reacts as part of the assay and produces a detectable change that can be measured in the blood, CSF or CNS tissue that is being tested. The most commonly used tags are: (1) radioactive compounds (radioimmunoassay); (2) enzymes that cause a change in color in a solution (enzyme immunoassay); (3) fluorescent compounds (immunofluorescent assay) and (4) molecules that emit light (chemiluminescent assay), Immunoassays may utilize either direct or indirect detection methods. In the direct technique, the tag is conjugated to the primary antibody that recognizes the targeted antigen (Figure 1A). The indirect technique uses a tagged secondary antibody that recognizes the non-tagged primary antibody for detection. The primary antibody binds to the antigen of interest. The secondary tagged antibody then binds to the constant portion of the primary antibody and thereby indirectly detects bound target antigen (Figure 1B). The indirect method is commonly used when no tagged primary antibody is available.

Table 1: Immunoassays in neurological disorders

Figure 1: Direct versus indirect immunoassays. (A) In direct immunoassays, labeled antibody (Ab) directly detects the presence of antigen (Ag). (B) In indirect immunoassays, a secondary antibody which recognizes the primary antigen-specific antibody has an attached label that identifies the presence of bound primary antibody

Immunoassays may also be competitive or noncompetitive. In competitive immunoassays, an unknown sample of antigen is mixed with a known sample of labeled antigen to compete for a limited number of binding sites on the specific antibodies (Figure 2A). In the non- competitive immunoassays, a labeled antibody directly binds to antigen in the sample (Figure 2B).

Radioimmunoassays

The first radioimmunoassay (RIA) was developed for detection and measurement of insulin in 1960 by Yalow and Berson4. The basic principle of RIAs is that antigenspecific antibodies are allowed to bind to a known quantity of radioactive antigen. The bound radioactivity is then measured. Patient serum or CSF containing an unknown amount of antigen is added to the antibody and radioactive antigen. The amount of radioactivity bound to the antibody will decrease as antigen in the sample competes for a fixed number of antibody binding sites. The amount of antigen in the specimen is determined by comparing the bound radioactivity with a standard curve. This is an example of a competitive immunoassay.

A second type of RIA, sometimes called immunoradiometric assay, uses radio-labeled antibodies in excess. Typically, specific antibodies are coated on beads or on the surface of plastic supports or wells. The solution containing the test antigen is added and the antigen is allowed to bind to the fixed antibodies. After washing to remove unbound antigen, an excess of radiolabelled antibody specific to a different epitope on the test antigen is added. After washing to remove unbound radiolabelled antibody, the remaining radioactivity is measured.

Figure 2: Competitive versus non-competitive immunoassays. (a) In the competitive immunoassay, unknown antigen in the sample is combined with a known quantity of labeled antigen and specific antibody. The antigen in the sample will compete for binding to the antibody, and the quantity of labeled antigen will be reduced. Therefore, the lower the signal generated by the labeled antigen, the higher the concentration of antigen in the tested sample. (b) In the non-competitive immunoassay, labeled antibody binds to antigen in the sample and the intensity of the signal is directly proportional to the amount of antigen present in the sample

Radioimmunoassays have been widely utilized to detect and quantify hormones in drug metabolites. RIAs are commercially available for the detection of myelin basic protein in CSF samples. The most specific diagnostic test for autoimmune myasthenia gravis is an RIA which utilizes ^sup 125^Iα-Bungarotoxin, a radiolabelled snake venom toxin5. Immun\oradiometric assays are widely used in the field of neuroendocrinology. RIA is an extremely sensitive and precise technique for determining very small amounts of antigen. The main disadvantage of this technique is the health hazard associated with working with radioactive substances. For this reason, enzyme immunoassays have largely supplanted RIA in many laboratories today.

Figure 3: Sandwich ELISA. In sandwich ELISA, antibody specific for a particular antigen is bound to a micro-plate. The sample containing antigen is added, and a second labeled antibody specific for a second distinct epitope on the antigen is added, then the resulting signal is measured. This technique is used for detecting larger antigens

Enzyme immunoassays

In the early 1970s, the quest for a simple but sensitive method for the quantitative detection of antigen or antibody led to the development of enzyme-coupled reagent assays or enzyme immunoassays (EIAs)6. Enzyme immunoassay is the general term for an assay that uses an enzyme-bound antibody to detect antigen. The enzyme catalyzes a color reaction when exposed to substrate. The enzyme- linked immunosorbent assay (ELISA) is a widely used EIA for quantitation of antigen or antibody. In this technique, enzyme- labeled antibody (or antigen) is bound to a solid support (e.g. tubes, beads, microtiter plate well, etc.), and patient specimen containing antigen (or antibody) of interest is added. A color change indicating the presence of the product of enzyme substrate reaction is the endpoint used to detect the presence of antigen. ELISA has become one of the most utilized methods for quantification of antigens in samples because of their sensitivity and specificity and being simpler and less costly than other analyses.

Types of EL ISAs

There are essentially two main types of ELISA: (1) sandwich ELISA and (2) competitive ELISA. Sandwich ELISA is used for detection of larger antigens containing at least two epitopes. A micro-plate is coated with antibody specific for one epitope of the antigen of interest. Then the sample containing the antigen is incubated on the coated plate. Finally, an enzymeconjugated antibody specific for a second epitope on the antigen of interest is added, the presence of enzyme conjugate bound to the plate is detected using an appropriate substrate, the resulting signal is measured with a micro-plate reader7. A linear relationship exists between the signal and the concentration of antigen, and this relationship is standardized using known concentrations of antigen (Figure 3).

In competitive ELISA, the micro-plate is coated with antibody specific for a single epitope on the antigen of interest. Next, sample containing antigen and labeled antigen bound to a detection enzyme are added and incubated on the coated plate. The quantity of enzymeantigen conjugate bound to the plate is detected with an appropriate substrate as for the sandwich ELISA. In this case, however, there is an inverse relationship between the signal obtained and the concentration of antigen in the sample.

The use of ELISA technique is extensive in both clinical and research arenas, and allows detection and quantification of substances such as peptides, proteins, antibodies and hormones. The advantage of ELISA testing is that it allows for sensitive screening of a large number of samples, and testing is relatively simple to perform. However, routine intrassay and interassay variability is significant, and false-positives are not uncommon owing to non- specific antibody binding. For this reason, a positive ELISA assay may require follow-up testing with a confirmatory immunoassay such as Western blotting (see below).

In neurological disease, ELISA has been used to detect Barrelia burgdorferi antigens8 and mycobacterium tuberculosis antigen9’10 in CSF. Research applications include the detection of phosphorylated tau and amyloid precursor protein11, as well as neuron-specific enolase12. ELISA is probably best recognized as the primary method for testing blood for HIV seroconversion.

Immunoflorescence assays

In 1942, Albert Coons first demonstrated that antibodies could be labeled with molecules that fluoresce13. These fluorescent compounds are referred to as fluorochromes. In immunofluorescence assays (IFA), antigen-specific antibody is tagged with a suitable fluorochrome (i.e. fluorescein isothiocyanate or FITC) and applied to the sample of interest. Unbound conjugate is rinsed off and the sample is examined under a fluorescence microscope. The presence of a specific antigen will cause the appearance of localized color (usually green) against a dark background. This is called a direct IFA because the antibody with the fluorescent tag is added directly to antigen in the sample. Direct IFA is best suited for the detection of antigen. An indirect IFA may also be performed in which antigen-specific antibody is detected by a second anti- immunoglobulin antibody labeled with a fluorescent tag. This analysis is better suited for the detection of antibodies (see below).

Direct IFA is used for the detection of varicella-zoster virus in swabs from cutaneous lesions of patients suspected of having herpes zoster14. The analysis of IFA assays is subjective and requires experienced personnel as well as an appropriately equipped fluorescence microscope.

Chemiluminescent immunoassays

Chemiluminescence refers to the emission of light caused by a chemical reaction15. A large number of molecules are capable of chemiluminescence and may be conjugated to either antigen or antibody. These labels may then be used in reactions that are similar to those described for RIAs and EIAs. Measurement of light from a chemical reaction is highly useful because the concentration of an unknown can be inferred from the rate at which light is emitted. The rate of light output is directly related to the amount of light emitted and proportional to the concentration of the luminescent material present. Chemiluminescent assays have become more widely used in recent years because of their excellent sensitivity and the reagents are stable and nontoxic15. Chemiluminescence assays are used in the determination of serum B12 levels16.

Western blot

Western blotting, sometimes called immunoblotting, is a reliable method for checking any sample for the presence of a specific antigen17. The detection is based on the molecular weight of an antigen and the interaction of the antigen with a specific primary antibody. The procedure involves using a denaturing gel (containing sodium dodecyl sulphate and polyacrylamide) to separate the proteins in a particular sample. These proteins are then transferred and irreversibly bound to a nitrocellulose membrane support. Nonspecific binding sites on the membrane are then blocked and primary antibodies recognizing the antigen of interest are added. A secondary antibody that recognizes the constant region of the primary antibody is usually used. The secondary antibody is usually linked to an enzyme that gives a colored product such as alkaline phosphatase, horseradish peroxidase, or pnitrophenyl phosphatase. Radioactive, fluorescent and chemiluminescent labels may also be used.

Western blotting is highly sensitive, does not require high- affinity antibodies, and is useful in identifying specific antigens recognized by either polyclonal or monoclonal antibodies. In addition, it may be used to confirm the specificity of antibody binding to protein antigens detected by other methods (i.e. ELISA). However, Western blotting is more time consuming and expensive than ELISA.

IMMUNOLOGICAL TECHNIQUES TO DETECT ANTIGENS IN TISSUE secTIONS AND CELLS (IMMUNOFLUORESCENCE AND IMMUNOHISTOCHEMISTRY)

Some antibodies to proteins only recognize the surface features of the native, folded protein, and do not effectively recognize the same protein when denatured. Therefore, the three-dimensional structure of the protein of interest needs to be preserved as much as possible. If the protein is present in serum or CSF, this is not a problem, but detection of tissue proteins often requires the use of frozen tissue sections which are fixed only after the antibody- antigen interaction has occurred, Immunofluorescence microscopy is an excellent technique for the localization of target molecules in tissue sections. As described above, the fluorescent dye may be attached directly to the specific antibody, or the bound antibody may be detected by a fluorescent anti-immunoglobulin antibody (indirect immunofluorescence). By attaching different dyes to different antibodies, the localization of two or more molecules may be determined in a single tissue section. Recent development of the confocal fluorescent microscope, which utilizes computer-aided techniques to produce ultrathin sections of tissues, has further enhanced the resolution of fluorescent microscopy.

An alternative to immunofluorescence microscopy for the detection of antigens in tissue sections is immunohistochemistry. In this technique, a specific antibody is coupled to an enzyme that causes a color reaction product in situ. The localization of colored product may then be determined by light microscopy. Immunohistochemistry has provided major insights regarding phenotypic brain tumor markers that have been regularly utilized in clinical brain tumor diagnosis18.

The expression of cell surface molecules or even the intracellular expression of molecules may be assessed by staining the cell with fluorescently labeled probes (antibodies) that are specific for those molecules. A flow cytometer can then be used to measure the fluorescence on individual cells in a suspension and thereby determine the number of cells expressing the molecule of interest. A fluorescence-activated cell sorter (FACS) is a flow cytometer that allows determination of separate cell populations according to which and how much fluorescent probe they specifically bind19. FACS analysis is freque\ntly used to determine the relative numbers of different subpopulations of lymphocytes. FACS strategies are currently being developed to isolate neural stem cells from other lineage-restricted progenitor cells20.

Use of antibodies in identification of genes and their products

An first step in isolating the gene that encodes for a protein utilizes antibodies specific for the protein of interest to isolate it from cells. Small amounts of the amino acid sequence of the protein can then be used to construct a set of synthetic oligonucleotides corresponding to possible DNA sequences, which are then used as probes to isolate the gene encoding the protein. Antibodies may also be used to isolate the protein product of a known gene. The amino acid sequence of the known gene can be deduced from the nucleotide sequence of the gene, and synthetic peptides representing parts of this sequence are made. Antibodies are raised against these peptides and purified. Labeled antibody is then used to stain tissue to determine the amount and distribution of the normal gene product. This is the procedure used to detect the presence of dystrophin in muscle tissue from patients suspected of having Duchenne’s muscular dystrophy21.

IMMUNOASSAYS THAT CHARACTERIZE AN IMMUNE RESPONSE

In neuroimmunology, immunoassays are important tools used to diagnose the presence of an immune response in the CNS, or other nervous system tissues. The detection and quantification of monoclonal antibodies, antigen-antibody complexes and antigenspecific antibodies (to infectious, tumor or self antigens) are very useful in the evaluation of infectious, inflammatory or autoimmune neurological disorders, and may also yield useful information regarding disease activity and response to treatment.

Detection of monoclonal or oligoclonal immunoglubulins

Electrophoresis is a technique for separation of ionic molecules (usually proteins) by their differential migration through a gel when exposed to an electrical field. Smaller molecules with more negative charge will travel faster and move farther toward the anode of an electrophoretic strip. Similar molecules will tend to group together on the gel where they may be visualized by staining procedures. In immunofixation electrophoresis22, proteins are first separated by electrophoresis on a support (agarose) according to their size and charge. The medium is then overlaid with monospecific antisera, usually with activity against the three major immunoglobulin classes (IgG, IgM and IgA). lmmunoglobulins are precipitated by the antisera, and after a few hours, the gels are washed to remove unpreciptitated proteins. The gels are stained, and if a monoclonal protein is present a characteristic band will be formed.

The presence of distinct IgG (oligoclonal) bands in CSF, but not serum, is indicative of local immunoglobulin synthesis and occurs most commonly in multiple sclerosis23. Immunofixation electrophoresis and a variation of this technique called isoelectric focusing are two methods used to detect these bands. A patient’s CSF and serum are run side-by-side using either of these two techniques. Following the separation step, a protein stain is applied to both specimens, and the banding patterns of the proteins in CSF and serum are compared with one another. The presence of two or more IgG bands in CSF that are not present in serum is a positive test for oligoclonal banding. Greater than 90% of MS patients show oligoclonal banding in their CSF24. The finding of oligoclonal bands is not specific because they may also be found in Guillain-Barr syndrome, CNS infections (including neurosyphilis and HIV infection) and even post-stroke25.

Detection of antigen-antibodu complexes

One way of determining it an antigen-antibody reaction has occurred is to detect the presence of the complexes formed between antigen and antibody. The amount of antibody present by changes it induces may be determined in the physical state of the antigen, such as precipitation of soluble antigens or clumping of particulate antigens. These are called secondary reactions, compared with primary reactions in which the direct binding of antibody to its antigen is measured. The ease of detection will depend upon the affinity of the antibody for the antigen, the stability of the reactions, the antigen/antibody ratio and the physical form of the antigen.

Precipitation reactions

Antigen-antibody reactions that occur in serum or CSF may lead to large crosslinked antigen-antibody complexes that precipitate out of solution. When the antigen is soluble, the precipitation of the antigen is being generally looked for after the production of large insoluble antigen/antibody complexes. Immunoprecipitation is a technique in which an antibody specific for one protein antigen in a mixture is used to precipitate-and thereby isolate-that antigen from the mixture. Precipitation reactions may also be used to determine the presence of antibody in serum samples. When 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 liter.

Agglutination

If the antigen is particulate, agglutination reactions are used. Direct agglutination is a general term used for techniques that utilize the macroscopic clumping (agglutination) of particulate reagents as an indicator of the presence of an antigen-antibody reaction. Examples include hemagglutination, latex agglutination and coagglutination. When the antigen is an erythrocyte, the term hemagglutination is used, and this type of testing forms the basis for the determination of a person’s blood type. In latex agglutination, antigencoated latex particles may be used to detect antibodies in a sample. Alternatively, antibodies may be absorbed to the latex particles for determining the presence of a particular antigen. Coagglutination applies a similar principle for the detection of antigen without the use of latex particles. Latex agglutination and coagglutination have been adopted for rapid and direct detection of soluble bacterial and fungal antigens in the CSF of patients with suspected meningitis26. Rapid antigen detection using these techniques may provide diagnostic confirmation in cases in which cultures and Gram stain are negative in patients who have received antimicrobial therapy.

DETECTION OF SPECIFIC ANTIBODIES IN SERUM OR CSF

Detection and quantification of specific antibodies are important in the diagnosis of many infectious and autoimmune neurological conditions. The main categories of assays used in this setting are enzyme immunoassays, Western blotting, radioimmunoassays, immunofluorescence and immunohistochemistry. All of these techniques have been described above.

In most cases, ELISAs are useful for testing large numbers of serum or CSF, particularly when the target antigen is available in purified form. In the ELISA technique for determining antibody concentration, target antigen is bound to a solid surface. Patient serum or CSF is added, which allows the binding of any specific serum antibodies to the antigen. A second enzymatically tagged anti- human IgG antibody is added, and the amount of antibody binding correlates with the level of color that develops. As noted above, ELISAs are sensitive and relatively easy and inexpensive to perform. However, minor methodological differences can greatly alter reproducibility, sensitivity and specificity of the assay, and each individual laboratory must provide its own standards for a positive or negative test. Patient serum may contain polyspecific antibodies which bind non-specifically to a variety of substrates, including the plastic wells of the ELISA plate. Therefore, it is often necessary to confirm a positive ELISA test with Western blot analysis. Examples of antibody tests in which ELISA is typically utilized in the field of neurology include tests for anti-GM1 antibodies27, anti-interferon-β28, anti-myelin associated glycoprotein (anti-MAG)27, and for the detection of IgM antibodies to West Nile virus29.

Western blotting is useful for the detection of antibodies directed against proteins of known molecular weight. The method of Western blotting for detecting protein antigens is described above. For detecting specific antibodies, target tissue proteins are separated by electrophoresis and transferred to a membrane support. The proteins are then exposed to patient serum or CSF, which allows the binding of any serum antibodies that are present to the specific antigen on the membrane support. A second anti-human immunoglobulin antibody, with an attached fluorescent or enzymatic marker, allows for the detection of antibody binding. This method is used for the detection of many of the paraneoplastic antibodies including anti- Hu and anti-Ro antibodies30.

Finally, radioimmunoassays are still used to diagnose the two major disorders affecting the neuromuscular junction, myasthenia gravis and Lambert-Eaton myasthenie syndrome5 . Their use, however, is becoming less frequent due to the inherent complexities involved in using radioactive compounds.

DETECTION OF ANTIBODY BINDING TO CNS TISSUE

Immunofluorescence and immunohistochemistry are used mainly to detect antibody binding to tissue or cells. In these techniques, tissue is first exposed to patient serum or other tissue fluids and allowed to bind to specific antigen on the tissue slice. Antibody binding is detected by anti-human immunoglobulins that are either enzymatically or fluorescently labeled. If antibodies to a specific tissue protein are present, they will bind in an anatomically appropriate pattern.

FUTURE ADVANCES

Advances in immunological reagents and laboratory technology will continue to improve the sensitivity and specificity of immunoassays in the future. However, it is important to remember that the most critical component of any immunoassay is the antibody itself. \Improvements in the affinity, specificity and mass production of antibodies will probably be the most important advances dictating the future of immunoassay technology and its continued applicability to diagnosis and research in CNS disease. Furthermore, it is becoming apparent that inflammatory responses are key elements in the pathogenesis of various types of CNS disorders32. These disorders include not only multiple sclerosis, but stroke, traumatic brain injury and neurodegenerative disorders. Therefore, the identification of important inflammatory mediators using immunoassays may improve our understanding of the pathogenesis of these disorders, and may open up new avenues for their treatment.

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Matthew N. Meriggioli

University of Illinois at Chicago Department of Neurology and Rehabilitation Medicine, 912 S. Wood Street. 855 N, M/C 796 Chicago, IL 60612, USA

Correspondence and reprint requests to: Matthew N. Meriggioli, MD, University of Illinois at Chicago Department of Neurology and Rehabilitation Medicine, 912 S. Wood Street. 855 N. M/C 796 Chicago, IL 60612. USA. [mmerig@uic.edu] Accepted for publication July 2005.

Copyright Maney Publishing Oct 2005