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Originally published July, 1998

RAMP: A quantitative immunoassay platform takes shape

Joanne Stephenson

The immunoassay technology developed during the 1960s relies on the natural function of human proteins called antibodies, which have the ability to recognize and bind to specific analytes called antigens. Simple immunoassays, such as dipstick pregnancy tests, indicate the presence of a target analyte by producing a color change that can be visually interpreted.

Such immunoassay devices are generally small, simple to use, and capable of delivering rapid results—advantages that have brought them considerable commercial success in both point-of-care and home-use applications. But a significant limitation of these tests is that their results are qualitative, and thus only beneficial in situations where a yes/no answer or detection of a threshold value is of use. However beneficial, such immunoassays have remained incapable of precise, highly sensitive, quantitative measurement.

The RAMP system by Response Biomedical promises quantitative results with immunoassay ease of use.

Response Biomedical Corp. (Vancouver) is seeking to eliminate these limitations with the development of its Rapid Analyte Measurement Platform (RAMP) technology. Since licensing the technology from the University of British Columbia in 1996, Response has developed RAMP assays for thrombospondin, microalbumin, myoglobin, and hepatitis B, and is now preparing to launch its products onto the international market.

RAMP is a platform technology which can be adapted to quantify virtually any immunologically active substance. As described below, each assay requires only a few drops of blood, serum, urine, or water. These samples are processed in an inexpensive immunochromatographic test device, with results provided in minutes by a low-cost, portable reader. Significantly, RAMP assays are being developed with the intention of obtaining waived status under the Clinical Laboratory Improvement Amendments (CLIA). The assays are simple, require no sample preparation or measurement, and are highly reliable. No technical training is required for operation of the system.

RAMP Components

In traditional immunoassays, colored latex particles have long been used as carriers for reagent antibodies. The results of such assays are determined by observing the accumulation of antibody-coated colored particles that have bonded to antigens in the patient sample. The RAMP technology adapts and improves this technique, enabling the system to detect and quantify concentrations of latex particles at sensitivities similar to enzyme-linked immunosorbant assays (ELISAs).

The RAMP system consists of two components: a disposable cartridge that houses an analyte-specific immunochromatographic strip, and a portable fluorescence reader that is used to quantify antibody-antigen complexes. The membrane strip component uses two populations of latex particles—each independently fluorescently labeled and tagged with monoclonal antibodies—to generate two independent signals during the assay reaction. One of these is the test reaction and the other is an independent internal standard.¹

The absolute concentration of complexed latex particles is determined by the RAMP reader. To perform this task, the reader calculates the ratio between the concentrations of latex particles in the test and internal standard reactions, refers to an analyte-specific calibration curve, and converts signal to concentration.

The proprietary chemical and optical methods used in the RAMP system are designed to reduce optical interference from the immunochromatographic membrane, thereby improving the signal obtained from the fluorescently dyed latex. The resulting improvement in sensitivity enables assays to be performed over the same range of analyte concentration as laboratory-based immunodiagnostic procedures such as ELISAs. A correlation between RAMP and ELISA assays at various concentrations of myoglobin is shown in Figure 1.

Figure 1. Correlation between RAMP and ELISA assays for various concentrations of myoglobin.The RAMP cartridge houses an analyte-specific test strip.

The manufactured cost of a disposable test cartridge and a reader is estimated to be less than $1 and $1000, respectively.

The RAMP Cartridge. The cartridge is a hollow casing, approximately 0.5 x 2.5 x 7.0 cm, molded of moisture-impervious solid material. Two openings in the casing provide access to the membrane for sample application at one end, and for viewing by the reader optics at the other.

Housed in the cartridge is a mylar-backed nitrocellulose strip on which the assay runs. Nitrocellulose was selected as the carrier material because of its well-known flow characteristics and its natural ability to bind proteins without requiring prior sensitization. The mylar backing provides increased handling strength and reduces permeation by the sample liquid. The internal standard that runs concurrently with every test assay enables the RAMP system to compensate for possible test-to-test variations in membrane properties.

The RAMP cartridge houses an analyte-specific test strip.

During the manufacturing process, reagents necessary to the assay are immobilized directly onto the membrane substrate. These reagents are applied by airbrush to large sheets of nitrocellulose, which are then slit into narrow strips to ensure identical, reproducible units. Reagents used on the strips include commercially available monoclonal and polyclonal antibodies.

The latex particles are prepared in the absence of surfactant, eliminating the potential for solubilization of antibodies and proteins and improving coupling capacity. The particles are matched to the membrane pore size such that they are transported along the membrane by capillary action of the fluid sample. The raw latex particles are dyed with proprietary fluorescent dyes, then coated with appropriate commercially available antibodies.

The RAMP Reader. Immunoassays based on fluorescence have substantially greater sensitivity and dynamic range than those based on earlier-generation detection techniques. Nevertheless, the development of such assays has been limited by the need for stable dyes that do not cause sample interference, and the fact that the instrumentation required was both complex and expensive. The RAMP technology overcomes these limitations by using proprietary dyes that are both stable and free of interference, and a simple detection system built with commercially available off-the-shelf components.

The reader incorporates two detectors, one for each wavelength of dye used to label the latex particles. When the chemical reaction in the cartridge is complete, the reader emits ultraviolet light, causing the dyes to fluoresce at two wavelengths that can be detected by the reader. The reader uses proprietary software on programmable, read-only memory chips which are plugged into each detector. These chips enable the reader to perform multiple types of assays and automatically determine which test is being run. The chips are designed to automatically calibrate the reader on a lot-specific basis.

System Operations

A RAMP assay can be designed for one of two traditional immunoassay modes depending on the target analyte. Sandwich assays are used for analytes with a high molecular weight (such as cardiac markers), while inhibition assays are used for analytes with a low molecular weight (such as hormones and therapeutic drugs).

To perform a test, the operator places an unmetered fluid sample containing a soluble analyte into the well of a test cartridge specific to the analyte of interest, and inserts the cartridge into the reader. The reader automatically prompts the operator to enter a patient identification number; once this number has been entered, no further intervention by the operator is required. A bar code on the cartridge contains test-specific information, including the identity of the analyte being tested, lot number, and calibration details.

Once inserted into the cartridge, the solution suspected of containing the immunogenic analyte contacts the nitrocellulose membrane strip; moves through the contact, detection, and internal standard zones; and is finally quantified by the reader. The operational sequence of a RAMP sandwich assay is as follows.

Contact Zone. The liquid sample is drawn by capillary action along the membrane through the contact zone (Figure 2). There, it comes into contact with the two populations of fluorescently labeled antibody-coated latex particles embedded during manufacture. Both particle populations are coated with the same surface concentration of antibody, but are labeled with different dyes and different antibodies. The test particles are labeled with an antibody against the analyte of interest. The internal standard particles are labeled with an "anti-rat" or "anti-mouse" antibody directed against a control reagent that does not react with the antibody directed against the analyte.

Figure 2. A liquid sample is added to the application zone at one end of the RAMP cartridge and migrates along the membrane by capillary action, passing through the contact zone.

When the liquid migrates through the contact zone, the colored latex particles are mobilized and carried along the membrane strip (Figure 3). If present in the sample, the target analyte binds covalently to the antibody on the test latex particles. The fluid sample, along with bound latex particles, unbound particles, and internal standard particles, is then transported by capillary action through the strip to the detection and internal standard zones.

Figure 3. When the liquid sample migrates through the contact zone, colored latex particles are mobilized and carried along the membrane strip by capillary flow. If present in the sample, the target analyte binds covalently to the antibodies on the latex.

Detection Zone. Further along the membrane strip, the detection zone is embedded with a second immobilized antibody specific to the target analyte. If the fluid sample contains the target analyte, it will be bound by the antibody in the detection zone, which arrests the migration of the attached latex particles. If no target analyte is present, the latex particles will migrate past the detection zone. The quantity of latex particles immobilized in the detection zone is directly related to the concentration of target analyte in the sample (Figure 4).

Figure 4. The quantity of latex particles immobilized in the detection zone is directly related to the concentration of target analyte in the sample.

Internal Standard Zone. Past the detection zone is the internal standard zone, which is embedded with an immobilized mouse or rat immunoglobulin. The internal standard particles bind with this antigen and are arrested at this line.

Results. The reader's two detectors separately measure the amount of fluorescence emitted by the complexes bound on the detection and internal standard zones. To establish a quantitative reading, the reader calculates the ratio between the two reactions.

The internal standard corrects for membrane variability and provides an internal validation of the assay. Since the internal standard should always detect a consistent amount of fluorescence, any variation in this measurement should also be reflected in variation of the test measurement. For instance, membrane variations that cause a lower-than-normal reading on the internal standard should have the same effect on the test measurement. By calculating the final test result as a ratio between the two measurements, the RAMP system automatically accounts for such variations. No reading from the internal standard indicates that the test was not successfully completed, and acts as an automatic quality control.

Within 8 to 15 minutes, depending on which assay is being run, the test result appears on an LED readout. The result can also be stored, printed, or downloaded to a laboratory or hospital information system.

Applications Development

So far, four assays have been developed using the RAMP platform. The initial three, thrombospondin, microalbumin, and myoglobin were chosen for the purpose of defining the system's intellectual property position. Development of these assays demonstrated the suitability of the RAMP system for performing a sandwich assay with the same sensitivity as an ELISA using the same antibody pair (see Figure 1). With the assay for microalbumin, it was shown that the RAMP system is also suitable for inhibition assays.

In general, the maximum sensitivity of a RAMP assay is determined by the binding constant of the antibody involved, as in any other immunoassay. The useful concentration typically ranges from 1/10 of the dissociation constant of the monoclonal antibody to a factor of 10 above this value.

In the thrombospondin assay, the system uses a fluid sample of whole blood. The amount of thrombospondin measured in the sample is directly proportional to the number of platelets in the blood sample. Platelet count is an important parameter of normal hemostasis; low platelet counts may be associated with clinical bleeding. In clinical use, platelet counts are commonly monitored for patients with platelet-destructive disorders or abnormalities of platelet production, including those undergoing chemotherapy. The RAMP assay for thrombospondin measures platelet counts over the clinically relevant range of 5–800 x 10¹¹/L.

The microalbumin assay uses a fluid sample of urine, and does not require dilution of the sample. Urinary albumin is a measure of proteinuria caused by kidney damage. Repeated quantitative measurement of urinary albumin levels enables clinicians to assess the degree of a patient's renal dysfunction and its progress over time. This assay is sensitive to albumin concentrations ranging from tens to hundreds of µg/ml, while using a monoclonal antibody with a much lower dissociation constant.

The myoglobin assay uses a fluid sample of whole blood or serum. The concentration of myoglobin and its time dependence is of diagnostic importance in the early assessment of cardiac damage in suspected myocardial infarction. Clinical trials of the RAMP myoglobin assay are expected to begin in the third quarter of 1998.

The most recently developed application for the RAMP platform is an assay for hepatitis B surface antigen. This assay is intended for use in identifying disease and monitoring a patient's disease state during the course of treatment.

RAMP provides a platform for the development of a broad range of assays to detect immunogenic compounds, infectious and toxic agents, and therapeutic molecules. Entities detectable using RAMP assays include proteins, hormones or enzymes, glycoproteins, peptides, small molecules, polysaccharides, antibodies, nucleic acids, drugs, toxins, viruses or virus particles, portions of a cell wall, and other compounds.

Assuming the ready availability of highly specific, commercially available antibodies, new RAMP applications can be developed and ready for clinical trials in as little as three months. Such a rapid development phase is made possible by the use of a mathematical model that can predict the behavior of a RAMP assay, including the amount of antigen that will bind to the latex particles and the optical signal that will be produced by the latex arrested in the detection zone. This model provides guidelines for the initial selection of the assay's parameters, as well as for further development and refinement of the finished assay.

The rapid development cycle for RAMP assays is making it possible to develop a number of new products quickly and simultaneously. The next set of RAMP tests to be developed will include assays for coagulation, cancer detection, infectious disease detection, and therapeutic drug monitoring. Codevelopment of additional assays can be started at any time and will depend upon corporate partnerships.

Conclusion

Response Biomedical's business strategy is to maintain research and development of RAMP assays in-house; to retain control of manufacturing; and to form strategic alliances for the sales, marketing, and distribution of the assays around the world. Marketing partners will be responsible for obtaining regulatory approval for the assays in their geographic area. Product launch of the RAMP system is expected to take place early in 1999, when the myoglobin assay will be released to the international market. That assay is expected to be released in the United States at the end of 1999.

The RAMP platform technology will enable researchers to develop numerous assays that can provide quantitative results comparable to large, laboratory systems while retaining the inherent low cost, simplicity, and portability of dipstick technology. The system potentially offers diagnosticians a means of quickly obtaining quantitative results at the point of care—characteristics that could eventually prove more cost-effective than traditional laboratory testing.

Reference

1. Brooks DE, and Devine D, Quantitative immunochromatographic assays, U.S. Pat. 05,753,517, May 19, 1998. Joanne Stephenson is director of business development for Response Biomedical Corp. (Vancouver).