Advances in detection technologies have enabled new, efficient diagnostics, but only if manufacturers thoroughly understand the assay design process.
The West Nile Detect IgM Capture ELISA by InBios International Inc. (Seattle) is a sandwich-type competitive assay. (Photos Courtesy InBios International Inc.)
Immunological approaches to diagnostic assays have come a long way since the concept was introduced for insulin in the 1960s, and for thyroxine in the 1970s.1,2
Immunoassays use antigens or antibodies as analytical tools and are based on the observation that in such a system, the distribution of free, as opposed to bound, analyte forms are quantitatively proportional to the analyte concentration in the system. Occasionally, assays are based on a specific binding protein in combination with antibody detection. Today, immunoassays are performed routinely, with or without sophisticated instrumentation. They range from simple point-of-care tests for use in the field or doctor's office to advanced clinical assays being run in high-throughput instruments with advanced robotics in clinical reference laboratories and blood banks. In addition to the integral role of diagnosing infectious disease, the immunoassay has revolutionized endocrinological testing, and is playing a growing role in the detection of cancer and various autoimmune diseases.3–6
With the boom of scientific technology and global monitoring of disease, immunoassay design has become a rapidly growing field. This article will focus on the immunodiagnostic approaches of InBios International Inc. (Seattle) to global infectious diseases, primarily in humans. It will also examine how small companies deal with regulatory and marketing issues, using as examples the development of an FDA-approved enzyme-linked immunosorbent assay (ELISA) for West Nile virus and a lateral-flow rapid assay to detect visceral leishmaniasis.
The Immunoassay Concept
Because immunodiagnostics use the antigen-antibody reaction as their primary means of detection, it is necessary to review some basic principles of this reaction before discussing test design.
Antibodies are host proteins produced in response to the presence of foreign molecules (antigens) in the body. They bond to antigens to form an immune complex that signals uptake and degradation by host phagocytic immune cells. Antibodies are synthesized primarily by plasma cells, terminally differentiated cells of the B-lymphocyte lineage. They are a large family of glycoproteins that share key structural and functional features. Functionally, they can be characterized by their ability to bind both antigens and specialized cells or proteins of the immune system.
Structurally, antibodies are composed of one or more copies of a characteristic unit that can be visualized as forming a “Y”. Each Y contains four polypeptides—two identical copies of a polypeptide called the “heavy chain” and two identical copies of a polypeptide called the “light chain.” Antibodies are divided into five immunoglobulin (Ig) classes: IgG, IgM, IgA, IgE, and IgD. This classification is based on the number of Y-like units and the type of heavy-chain polypeptide they contain. Each Ig class plays a specific role in the immune response, with IgM production being the initial antibody response for most primary infections.
The region of an antigen that interacts with an antibody is known as an “epitope.” An epitope is defined only by its interaction with the binding site of an antibody, and as such is not an intrinsic property of any particular structure. Because antibodies can recognize relatively small regions of antigens, occasionally they can find similar epitopes on other molecules. This forms the molecular basis of a cross-reaction. However, the presence of similar epitopes does not necessarily imply a functional relationship. The binding of the antibody to the antigen depends entirely on noncovalent interactions, and the antigen-antibody complex is in equilibrium with the components in the free state.
Simply stated, the immunoassay concept is to use one analyte—either antigen or antibody—to sequester and detect the presence of the other. For example, the antigen from an infectious agent can be used in an external assay to detect the presence of antibodies produced in response to infection by that agent.
Selecting the Matrix
Figure 1. (click to enlarge) Major steps and considerations in the immunoassay development process.
A number of considerations go into making a commercial immunoassay (see Figure 1). Once a target disease has been decided on, the first development step is to define the most effective source of analyte in body fluids or tissues. Depending on the availability of analytes, assay systems have been developed using plasma, serum, whole blood, cerebrospinal fluid (CSF), sputum, urine, or solid-tissue extracts. The nature of the matrix chosen is important both for the clinical sensitivity and specificity of the assay and the nature of the disease.
Analytes may be present in one or more matrices, but at considerably different concentrations. For example, distinct assays have been designed to incorporate different matrices in the following circumstances:
• Early pregnancy testing that detects human chorionic gonadotropin using urine and blood.
• HIV antibody analysis in both serum and saliva.
• The indication of West Nile virus infection through serum testing and the diagnosis of nervous system involvement through the detection of analyte in CSF.
• The assaying of Mycobacterium tuberculosis in sputum, and of antibodies to the organism or related proteins in serum and urine.
Thus, selecting the matrix or matrices best suited to the source of analytes and presentation of the disease is an important first step of building a diagnostic assay.
The Detection Method
Table I. (click to enlarge) Clinical study data for the West Nile Detect IgM Capture ELISA by InBios International Inc. Serological sensitivity = 99.4%, with a 95% confidence interval of 96.8–99.9%. Serological specificity = 100%, with a 95% confidence interval of 97.1–100%.
Since the use of radioactivity in the design of the radioimmunoassay (RIA) was first described 36 years ago, many alternate detection systems have been championed. The use of radioactive labels such as tritium (3H) and iodine (125I) and gamma or scintillation counters has largely been replaced by newer, nonradioactive methods. There are, however, some assays that require exquisite sensitivity and still use isotopes. Generally, though, the new detection agents are safer and more environmentally friendly, and have led to the development of more-advanced and high-throughput instrumentation.
Detection systems can be divided into two types, based on the test format. For rapid-type assays, analyte specific visual detection agents, such as reagents conjugated to colloidal gold or selenium or colored latex-type particles, are used.7 These particles are sensitive, but usually not as sensitive as an enzyme-linked immunoassay. As expected, the visual and inexpensive, one-step elements of detection are essential for point-of-use test design.
For instrument-compatible assays such as ELISAs, a solid- or liquid-phase system is most desirable. In this format, various enzymes, including horseradish peroxidase, alkaline phosphatase, and beta-galactosidase can be conjugated to analyte specific reagents. Following the analyte reaction, certain enzyme activities are studied using a sensitive substrate. This can include either chromogenic-, fluorogenic-, or chemiluminescence-generating substrates, which are typically detected by light-absorbance measurements (optical density). In addition, analyte specific reagents can be coupled directly to a fluorescent, chemiluminescent, or bioluminescent tag.
This new generation of detection agents provides extremely sensitive detection systems that can be used with high-throughput instrumentation and are helpful in developing simultaneous multianalyte assays.
Selecting the Format and Method
Deciding whether to test for the presence of antigen or antibody depends largely on the target diagnosis. In screening drugs of abuse, for example, inhibition immunoassays are used. For diagnosing malarial and hepatitis B viral infections, as well as several bacterial pathogens that cause acute illness, testing for antigen in blood, rather than for antibody, is crucial. Antibody detection in blood has also been critical for visceral leishmaniasis, hepatitis C, and syphilis infection. With some diseases that elicit prolonged immunity responses, discerning between primary and secondary infections can be challenging. Cell-based assays, such as those that use T-cells for cytokine or interferon-gamma induction to diagnose latent infection, are becoming popular for diseases such as tuberculosis.8
Table II. (click to enlarge) Clinical study data for the Kalazar Detect rapid test for visceral leishmaniasis by InBios International Inc. Serological sensitivity = 100%, with a 95% confidence interval of 97.9–100%. Serological specificity = 93%, with a 95% confidence interval of 88.5–96%.
The ELISA and the rapid, or dipstick, test currently are the two predominant formats in immunodiagnostics. ELISAs are created by coating the antigen or antibody on a suitable plastic. To complete the reaction, an enzymatic detection method with a color-forming substrate is required.
ELISAs can be either competitive or noncompetitive. The noncompetitive, or sandwich type, of ELISA is a standard tool for quantifying the antibody or antigen in a serum. One supplied analyte “captures” the complementary antigen or antibody in the serum. Then, a second labeled antibody coupled to a detection method completes the sandwich.
The West Nile Detect IgM Capture ELISA by InBios International is an example of this type of assay. To conduct the test, serum samples are applied to microtiter wells that have been coated with antihuman IgM. A West Nile–specific recombinant antigen is then applied to bind any West Nile IgM antibody complex. Finally, a West Nile antigen-specific antibody, coupled to a detection method, is used to “sandwich” the antigen and produce quantitative results.
In the competitive ELISA, a known amount of labeled analyte directly competes with the same analyte in the patient's serum. A well-designed ELISA is a prerequisite, even if the design goal is a rapid test. ELISAs help identify the correct sera (both positive control and negative control) used to optimize the subsequent lateral-flow or flow-through dipstick assay. However, while the ELISA format affords a sensitive and detailed look at analyte-sera interactions, it typically relies on large instrumentation and long reaction times, and therefore cannot truly be called a rapid test.
Figure 2. A workstation by BioDot Inc. (Irvine, CA) for continuous in-line dispensing, coating, and impregnation of lateral-flow membranes.
In rapid diagnostic tests, similar to those that InBios has developed for leishmaniasis and Chagas (T. cruzi) diseases, the antigen-antibody reaction occurs on a membrane support. These assays are conventionally called “antibody-capture assays.” In this process, the antigen is attached to a solid support, and labeled antibody is allowed to bind (see Figure 2). After the excess antibody flows upward (in a lateral-flow assay), the test is either quantitated by measuring the intensity of the test line, or yes/no information is extracted by visually confirming the test line. The latter method is suited for rapid screening and point-of-use applications.
Recent technological advances have created multiplex formats and instrumentation that enable concomitant sampling of multiple analytes (see Figure 3). When applied to 96-well ELISAs, the multiplex format allows up to 100 distinct target immunoassays within a single assay well. In one particular multiplex system, target analytes are bound to a microsphere distinguished by a precise internal dye ratio. The appropriate instrumentation reads both a fluorescence reporter bound to the captured analyte and the microsphere itself to distinctly analyze each miniimmunoassay within a single well suspension.9 This technology seems likely both to improve specificity and broaden the spectrum of immunodiagnostics.
Monoclonal or Polyclonal Antibodies?
Figure 3. Sample prototype of a multiplex rapid-test cassette, by PTG Global (Santa Ana, CA).
If a test design involves antigen capture, the next step is to produce antibody. Antibodies primarily of the IgG class are used in developing immunoassays for specific target antigens, and can be either polyclonal or monoclonal. Typically, polyclonal antibodies are developed in animal species such as sheep or goats, where large quantities can be produced. In general, polyclonal antibodies, when used to develop an immunoassay, are affinity-purified against the antigen to render them monospecific.
Monoclonal antibodies are typically developed in mice using well-established hybridoma techniques, but other species, such as rats and rabbits, have also been used. Monoclonal antibodies can be designed to hit different epitopes on the same antigen molecule, making them the antibodies of choice for use in sandwich immunoassays. They can also be designed with high affinity and specificity. In contrast, affinity-purified polyclonal antibodies would hit multiple epitopes on the antigen of interest unless the polyclonal was either developed to a specific peptide epitope or was rendered monospecific by affinity-purifying against that peptide epitope.
In the case of indirect immunoassays, where, for instance, host antibodies to a particular antigen are detected, an antibody against the specific immunoglobulin would be used in conjunction with a labeling system, thus also requiring antibody production. Again, considerations for choosing the target immunoglobulin would include analyzing the most effective and encompassing tool for diagnosis.
Choosing the Antigen
Whole-cell lysate, purified protein, and recombinant protein are all sources for antigen when an antibody capture test is being developed. Sometimes, sufficient specificity is attained using lysates or partially purified proteins. Cross-reactivity and high background levels may become a factor when using multiple analytes, and therefore the effort of creating recombinant proteins is generally rewarding, especially for future regulatory submission purposes. In addition, the cloning of particularly strong epitopes will often reduce background and increase sensitivity.
The buffer in which the antigen will remain dissolved also merits attention. Typical buffers used in rapid-test development include 10 mmol phosphate, phosphate buffer saline (PBS), borate, borate with dissolved salt, sodium acetate, and others. The selection of a buffer's pH is crucial for optimal antigenic activity and is influenced by the isoelectric point (pI) of the antigen. For some buffers, a peculiar problem arises: Newly made tests perform well and provide good sensitivity and specificity; but as they age, performance worsens.
Moreover, buffers are antigen dependent. One antigen may perform well in PBS (pH = 7.4) and may provide the desired shelf life. However, the same buffer and pH may give disastrous results with a different antigen. There is no general rule for the selection of buffer. Experimental results are the only indicators.
When an antigen is dialyzed in a given buffer and the buffer is not compatible, the antigen might precipitate out of solution. This is an early indication of an adverse antigen-buffer interaction that might produce poor antigenic activity.
Knowledge of the antigen's pI can also help researchers select the appropriate buffer. The antigen to be detected can be a circulating antibody to a specific antigen, which is the case in the Kalazar Detect rapid test for visceral leishmaniasis by InBios. In this antibody capture assay, a recombinant protein, rK39, with repeating epitopes, is immobilized on a solid phase (membrane or 96-well plate) and incubated with serum from visceral leishmaniasis patients. This is followed by incubation with goat antihuman IgG-HRP (ELISA) or labeling with colloidal gold for lateral flow.
During the development process, it is necessary to create a prototype that incorporates all of the required assay features. At this stage, sensitivity and specificity are assessed and the assay reagents fine-tuned. The resulting prototype is field tested before being committed to clinical trials.
Figure 4. Kit reagent lots are tested as a part of the assay quality control process.
To carry out thorough prototype testing, confirmed positive samples as well as negative controls are used to establish sensitivity and specificity data. (Inadequate specificity, for example, might necessitate looking at alternate antigens or adding epitopes.) Interlab, interassay, and intraassay reproducibility data must also be assessed to establish the robustness of the assay. The stability of kit reagents is tested at a variety of temperature and environmental conditions (e.g., humidity), and dilution ELISAs are used to accurately pinpoint antigen and conjugate concentrations (see Figure 4). Both real-time and accelerated stability are studied to determine kit shelf life. In the end, a design goal of the assay is to incorporate optimum sensitivity and shelf life without losing specificity.
Field and Clinical Studies
Clinical trials are needed to validate an assay and determine its performance characteristics in preparation for regulatory submission. Typically, trials are performed at three or more clinical sites. The locations of these sites should be pertinent to disease prevalence and the locations where the tests will be used. The purpose is to show that the test's results are reproducible at different sites and that its performance characteristics are confirmed independent of the manufacturer. Studies of the assay's specificity, sensitivity, interfering substances, and cross-reactivity are performed, as are comparisons to established reference methods. For example, among West Nile virus diagnostics, the plaque reduction neutralization test (PRNT) serves as the standard method for comparison.
Tables I and II show data from clinical studies for the West Nile Detect IgM Capture ELISA and Kalazar Detect rapid test. Each data set represents one of the several sites that are included in the field trials. Data such as these are an important part of the regulatory submission procedure and help direct future work (e.g., the need to modify test sensitivity). It is not unusual to see differences in test sensitivity and specificity among different geographical regions, which influences antigen and antibody selection.
To be sold commercially in the United States, immunoassay kits must obtain regulatory approval. For assays that involve human testing, this means approval through FDA. Veterinary applications require approval from the U.S. Department of Agriculture. With regard to FDA, most of the IVDs discussed in this article would require either a premarket approval or a 510(k) clearance.
Once a test is submitted to FDA, the clinical trial results are reviewed and additional studies performed as necessary to answer specific questions. This may take anywhere from three to six months before final approval is received. As the process unfolds, marketing can continue; however, all kits and reagents must be labeled “for research use only.” As part of a regulatory approval, manufacturers are open to inspection and scrutiny by FDA or other regulatory bodies to ensure product quality.
To market an assay in the European Union or European Economic Area, a similar approval process is required, resulting in the mandatory CE marking of the product. The practice of using one or more analyte specific reagents to develop a diagnostic product for use in-house is referred to as “home-brew” manufacturing and is common in less-developed parts of the world, where healthcare expenditures prohibit the widespread use of commercial kits. The quality of these tests is often quite good and they can be customized for specific needs. However, overall, they may lack the standardization and quality control assurances that accompany approved kits.
Once regulatory approval has been obtained, marketing of the diagnostic can begin. Marketing for products from large companies is typically performed through the companies' own marketing and sales divisions. However, for small companies, such departments are typically not feasible until sales reach a certain level. Instead, small companies must rely on other methods for promoting their products. These include providing information at scientific symposia through an exhibitor's booth and presenting scientific abstracts or talks describing the performance of their products. A growing number of small diagnostics companies are also using Web sites to promote and sell their products. When marketing IVDs to foreign countries, it may be prudent to set up distributorships in the countries of interest. These distributors help speed both the importation of approved test kits and their sale to laboratories and testing sites. They can also help deal with the foreign FDA-equivalent authorities, if necessary.
There is an urgent need to develop reliable, user-friendly, and inexpensive diagnostic tools and vaccines for countering infectious diseases worldwide. Monitoring potential pandemics has become increasingly difficult with today's global migration patterns, and effective isolation of a disease requires quick diagnosis. Furthermore, the specificity and sensitivity of a test must be of the highest quality to be fully adaptable. The considerations for immunoassay design presented here represent an overview of the multifaceted development process.
(R to L) Bonnie J. Stewart, PhD, is project manager, Raymond L. Houghton, PhD, is director of new product development, WJW Morrow, PhD, is director of new technology acquisition, and Syamal Raychaudhuri, PhD, is chief scientific officer at InBios International Inc. (Seattle). The authors can be reached at firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, and email@example.com, respectively.
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