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©KEN EWARD/BIOGRAFX 2001
Chromato-
hen a simple and selective method is required, there are few analytical techniques that compare with immunoassays. This group of methods uses antibodies or antibody-related molecules as reagents for chemical analysis (1, 2). Immunoassays are among the most specific of the analytical techniques, provide low detection limits, and can be used for a wide range of substances. These features, plus the fact that many samples need little or no pretreatment, have made immunoassays common in clinical testing, pharmaceutical analysis, environmental monitoring, and food safety testing. There are many ways in which immunoassays can be performed, but a recent approach based on LC, known as a chromatographic or flow immunoassay, has received particular attention (3–10). We examine this method’s basic principles, discuss various formats in which it can be performed, and describe the potential advantages, limitations, and applications.
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The variety of formats and labels, along with speed and Antibodies and their properties selectivity, makes these The key component in any immunoassay is the antibody. Antibodies, or immunoglobulins, are proteins produced by the imtechniquesmune system as a defense against foreign agents, such as bacte-
ria or viruses. The general structure of an antibody is shown in Figure 1, using immunoglobulin G (IgG) as an example. IgG is the most common type of antibody found in blood and contains four polypeptide chains—two identical long, or “heavy”, chains and two identical shorter, or “light”, chains—which are connected through disulfide bonds to produce a Y-shaped molecule. Other classes of antibodies, such as IgA and IgM, have similar structures but can contain several Y-shaped units linked together through an additional peptide chain. The bottom stem of an antibody is called the Fc region, and it contains essentially the same amino acid sequence from one antibody molecule to the next. The upper two arms of an antibody are known as the Fab regions. These consist of two identical binding sites, which can be removed from the rest of the David S. Hageantibody through digestion with the enzyme papain. The amino acid sequences in the F regions vary significantly among difMary Anne Nelsonferent antibodies. It is abthis feature that allows our bodies to produce up to 108 different types of antibodies, each with its University of Nebraska—Lincoln A P R I L 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
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in turn, led to interest in using these columns as a means of performing immunoassays. Binding site
Direct detection methods
In the earliest immunoassays, antibody binding diSample plug rectly generated a signal for analyte detection. An example is the determination of blood types, in which the presence of A or B antigens cause red Wash away undesired blood cells to clump together when they are mixed Fab region Regenerate solutes column with antibodies that bind to these factors (11). Direct detection was also used in the first chromatoElute analyte Fc region graphic immunoassays (Figure 1). In this approach, the sample is first injected onto an antibody column under mobile-phase conditions that allow any anaDetect or lyte present to strongly bind to the immobilized ancollect analyte Antibody structure tibodies on the column, while other sample com(IgG) ponents are washed through it. A second buffer is then passed through the column to dissociate the FIGURE 1. Basic structure of an antibody and its use in a chromatographic immunoretained analyte, which is collected for analysis or assay with direct analyte detection. The circles represent the test analyte, and the measured as it passes through an on-line detector. squares represent other unretained sample components. The initial application buffer is then reapplied to the column, and the immobilized antibodies are allowed to regenerate for the next sample. own unique binding affinity and specificity. Direct detection in chromatographic immunoassays has been Within an antibody’s binding sites, there is a well-defined used for determining various clinical analytes, such as anti-idiotypthree-dimensional arrangement of amino acids that can undergo ic antibodies, glucose-containing tetrasaccharide, granulocyte various noncovalent interactions, including dipole–dipole, ionic, colony stimulating factor, fibrinogen, human serum albumin, IgG, or nonpolar interactions, and hydrogen bonding (11). The com- IgE, interferon, interleukins, leukotriene, 2-microglobulin, transbination of these forces and the distinct shape of the binding ferrin, and tumor necrosis factor-␣ (2, 3,17 ). In many cases, results pocket allows antibodies to bind to other agents with equilib- can be obtained in only a few minutes with microliter volumes of rium constants in the range 105–1012 M–1. This strong and se- sample. These assays have been used with samples ranging from lective binding is what makes antibodies useful as reagents for serum, blood, and urine to cerebrospinal fluid and bone marrow determining chemicals in complex mixtures (1, 2). aspirates. The direct detection mode has also been used to examAlthough the chemical nature of antibodies was not known ine substances in aqueous solutions, cell cultures, and cell extracts. until the middle of the 20th century, they have been used as an- For example, such methods have been reported for detecting analytical reagents for almost 100 years. Antibodies were also used tithrombin III, bovine growth hormone, fungal carbohydrate antiin the 1920s by Landsteiner, who won the 1930 Nobel Prize in gens, glutamine synthetase, group A-active oligosaccharides, lymMedicine for his work with these reagents in developing the phocyte receptors, and tissue-type plasminogen activator (3). ABO system for identifying human blood types. More advanced Several techniques are available for directly monitoring anaassays appeared around 1960, when Berson and Yalow combined lytes as they elute from antibody columns. Proteins and pepradioactive labels with antibodies for identifying insulin in dia- tides are commonly detected by on-line absorbance measurebetic patients. This work, recognized by the 1977 Nobel Prize ments, whereas electrochemical detection has been used for in Medicine, gave rise to the methods of radioimmunoassays carbohydrates. Analytes with low concentrations can sometimes and competitive binding immunoassays, which are still common be monitored by derivatizing them with fluorescent tags or ratools in biomedical testing and research (11, 12). diolabels before injection. Another option is to collect fractions As antibodies gained popularity as analytical reagents, scientists of the analyte and later measure them by a traditional imbegan to consider them for chromatographic separations (13). As munoassay or an assay for biological activity (3). better support materials and immobilization methods appeared, The main advantages of a chromatographic immunoassay with antibodies were placed into columns and used as stationary phas- direct detection are its simplicity, speed, and ease-of-use. A major es. This approach, referred to as immunoaffinity chromatography, limitation is that the analyte must be present at high-enough levsoon became a popular tool for isolating biological substances els to allow direct detection. This problem can be overcome by (14, 15 ). Early work with this method used low-performance ma- using other formats that use labels for indirect analyte detection. terials such as agarose. By the end of the 1970s, more rigid and Another limitation is that direct detection works best when the efficient supports had been developed, which allowed immobi- analyte is eluted as a relatively sharp peak. This is accomplished by lized antibodies to be used with HPLC, giving rise to high-per- using a step gradient or fast gradient elution to rapidly dissociate formance immunoaffinity chromatography (HPIAC) (16). This, analytes from the antibody column. Although it is often possible 200 A
Binding site
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 , 2 0 0 1
to obtain hundreds of injections on a single antibody column with these gradient schemes, care must be taken to choose elution conditions that do not irreversibly damage the immobilized antibody or support. In some situations, it may be difficult to find an elution buffer that allows both fast analyte dissociation and long column lifetimes. As an alternative, relatively gentle conditions can be used to remove analytes from the antibody column, followed by capture and reconcentration by a second on-line column.
Competitive binding immunoassays For low-concentration substances that cannot be directly measured, there are several immunoassay formats that combine antibodies with labeled agents for indirect detection. An example is the competitive binding immunoassay originally developed by Berson and Yalow for insulin. They combined patient samples or insulin standards with a fixed amount of radiolabeled insulin and a limited amount of anti-insulin antibodies. After these components had been mixed and allowed to interact, the normal and labeled insulin bound to antibodies were separated from that which remained free in solution. Next, the amount of labeled insulin in the bound fraction was measured. As the amount of unlabeled insulin increased, there was a decrease in the amount of labeled insulin that was detected in the antibody-bound fraction. This inverse relationship was then used to quantitate the insulin from the standard or sample (11). A competitive binding immunoassay can also be performed on a chromatographic system (Figure 2a). The simplest approach is to mix the sample with a fixed amount of a labeled analogue of the analyte. This mixture is then injected onto a column that contains antibodies that bind both the analyte and its labeled analogue. This format is known as the simultaneous injection method. The analogue that is used can be tagged with different labels, as shown in the box on this page (1–3). The amount of this analogue that binds to or passes through the column is determined as it is injected in the presence of standards that contain known concentrations of the analyte. This information is used to prepare a calibration curve, like the one shown for human serum albumin in Figure 2b. Clinical, pharmaceutical, and environmental agents have been measured with this technique, including adrenocorticotropic hormone, human chorionic gonadotropin, IgG, insulin, testosterone, theophylline, thyroid-stimulating hormone, thyroxine, transferrin, and isoproturon (3, 17 ). A closely related technique is one in which the sample and labeled analogue are applied separately to the antibody column. This approach, sequential injection, has been used for quantitating ␣-amylase, human serum albumin, and triazine herbicides (3). Like simultaneous injection, an indirect measure of the analyte is obtained by measuring the amount of labeled analogue that elutes in either the unretained or retained fractions. An advantage of this approach is that even an unlabeled preparation of the analyte can potentially be used as the “labeled analogue”, provided that this produces a sufficient signal for detection. Another advantage over simultaneous injection is that there are no interferences from the sample when the labeled analogue is detected in its
unretained fraction. One possible disadvantage of this format is that it requires two steps for applying the sample and analogue to the antibody column. A third way of performing a competitive binding immunoassay by chromatography is competitive displacement. The compounds 2,4-dinitrophenyl lysine, cortisol, thyroxine, and trinitrotoluene have all been detected by this method (18–21). First, a labeled analogue is applied to an antibody column to occupy some or all of the column’s binding sites (Figure 2c). When a sample is later passed through this column, the analyte binds to sites that are momentarily unoccupied by the labeled analogue as it undergoes local dissociation and reassociation. The net result of this competition is a displacement of the labeled analogue from the column, with the size of its displaced peak being proportional to the amount of analyte in the injected sample. A typical calibration curve for this approach is a straight line. A big advantage of this method is that its response has a direct, rather than inverse, relationship to analyte concentration. Another advantage is that a single application of the labeled analogue to the antibody column can often be used for many sample injections, which helps to increase sample throughput. However, the technique is highly dependent on flow rate and the labeled analogue’s rate of dissociation. When using high-affinity antibodies and slowly dissociating analogues, this approach tends to produce broad peaks (3, 18–20).
Immunometric assays Another type of immunoassay that has been conducted in chromatographic systems uses labeled antibodies or antibody fragments for analyte detection. One example is the sandwich immunoassay. This format is also known as a two-site immunometric assay because it uses two types of antibodies that bind to separate regions on the chemical of interest. The first of these antibodies is attached to a solid-phase support and is used to extract the analyte from samples. The second antibody contains an easily measured tag and is combined with the analyte either before or after sample extraction; the purpose of this antibody is to provide a signal that can later be used for analyte detection and measurement. The combination of all these reagents results in a “sandwich” complex on the column, in which the analyte is located between these two types of
Labels used in chromatographic immunoassays. Detection method Absorbance Chemiluminescence Electrochemical activity Fluorescence Radioactivity Thermal measurements 1
Labels Horseradish peroxidase1, human serum albumin, transferrin Acridinium ester, horseradish peroxidase1 Alkaline phosphatase1, glucose oxidase1, horseradish peroxidase1 Alkaline phosphatase1, fluorescein, horseradish peroxidase1, liposomes2, Lucifer Yellow, Texas Red Iodine-125 Alkaline phosphatase1
Enzymatic labels used to generate suitable products for detection. Liposomes used to encapsulate large amounts of internal marker compounds (e.g., carboxyfluorescein) that are later released for detection.
2
antibodies. After any excess labeled antibodies have been washed from the column, those that remain will provide a signal that is directly proportional to the amount of extracted analyte. Figure 3a shows one scheme for performing a sandwich immunoassay, in which the sample and labeled antibodies are allowed to bind before being injected onto a column that contains immobilized antibodies for extraction. Alternatively, the sample and labeled antibodies can be injected sequentially onto the extraction column, which eliminates the preincubation step but tends to produce worse detection limits and requires more labeled antibodies than simultaneous injection. Sandwich immunoassays can potentially be performed with any of the labels listed in the box on p 201 A, including enzymes, fluorescent markers, and chemiluminescent tags. This format has been used to measure several analytes of clinical interest, such as ferritin, parathyroid hormone, IgG, IgM, and antibovine IgG antibod-
(a) Step 1: Sample injection + label + Step 2: Elution of retained analyte and label Elution buffer (b)
Relative amount of bound analogue
1 0.8 0.6 0.4 0.2 0
0
1 2 3 Human serum albumin (nmol)
4
(c) Step 1: Injection of label
Step 2: Sample injection
Step 3: Elution of retained analyte and label Elution buffer FIGURE 2. (a) General scheme for simultaneous injection. (b) Calibration curve for human serum albumin by simultaneous injection based on data generated from Ref. 43. (c) General scheme for competitive displacement. Green circles are the analyte, blue circles are the label.
ies, and has also been used to quantitate human serum albumin and insulin in fermentation broths and cell cultures (2, 3, 17 ). A significant benefit of sandwich immunoassays is that they produce a signal directly proportional to the amount of injected analyte (Figure 3b). The fact that two types of antibodies are used gives this technique much higher selectivity than competitive binding immunoassays, which use only one antibody preparation per analyte. In addition, the greater selectivity and lower background signal of sandwich immunoassays help provide lower detection limits than can be obtained by most competitive binding methods. The main disadvantage is that they can only be used for substances that are large enough to allow simultaneous binding to multiple antibodies. This is not a problem with competitive binding immunoassays, which work equally well for small or large analytes. The one-site immunometric assay is another type of chromatographic-based immunoassay. In this technique (Figure 3c), the sample is first incubated with a known excess of labeled antibodies or Fab fragments that are specific for the analyte of interest. After binding between the analyte and antibodies has occurred, this mixture is applied to a column containing an immobilized analogue of the analyte. This column serves to extract any excess antibodies or Fab fragments that are not bound to the original analyte. Those antibodies or Fab fragments that are bound to the analyte will pass through this column as a unretained peak. Detection can then be performed by looking at the size of this unretained fraction, which gives a signal that is directly proportional to the analyte’s initial concentration. Applications of the one-site immunometric assay in chromatographic systems have included detecting ␣-(difluoromethyl)ornithine, thyroxine, and 17-estradiol (22–24). One benefit of this approach over the sandwich immunoassay is that it can be used to measure both small and large solutes. However, like a sandwich immunoassay, it also gives a signal that is directly proportional to the amount of analyte in a sample. The fact that the column contains an immobilized analogue of the analyte can also be an advantage because a wider range of elution conditions can be used with antibody columns. A disadvantage of this technique is that a different analogue column must be produced for each analyte; this is a more demanding task than generating multiple types of antibody columns because each analogue may require its own unique method for immobilization. Another limitation is that pure and highly active labeled antibodies or Fab fragments are ideally required to provide a low background signal for analyte detection. This same requirement is desirable, but not as crucial, in sandwich immunoassays.
Homogeneous immunoassays All of the formats discussed so far have been heterogeneous immunoassays, in which an immobilized antibody or analogue is used for the analysis of solution-phase samples. But it is also possible to use chromatography to perform a homogeneous immunoassay in which the analyte, antibodies, and any labeled components remain in solution during the measurement process. This can be accomplished by using a column with restricted-access media for solution-phase separation of
small analytes from their antibody-bound fractions. For instance, this approach has been used in a competitive binding immunoassay for atrazine and related compounds (2). In this technique, samples are combined with a fluorescein-labeled analogue of atrazine and anti-atrazine antibodies and then injected onto a reversed-phase restricted-access column. This column retains the analytes and labeled analogues that were free in solution, but not their larger antibody-bound forms. The result is a separation of these two fractions in
