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Oak Ridge Poster Sessions |
1
Response Biomedical Corp., Vancouver, British Columbia, Canada V5J 5J1;
2
Pathology and Laboratory Medicine and
3
Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5;
4
Datrend Systems Inc., Delta, British Columbia, Canada;
a address correspondence to this author at: Department of Pathology and Laboratory Medicine, University of British Columbia G131 ACU, 2211 Westbrook Mall, Vancouver BC, Canada V6T 2B5
The Rapid Analyte Measurement Platform (RAMPTM) is an enabling immunodiagnostic platform for quantitative analysis of a wide variety of analytes. Consisting of an immunochromatographic strip in a disposable cartridge and a scanning dual wavelength fluorescence reader, RAMP takes advantage of the inherent simplicity of lateral flow immunochromatography while providing a quantitative measurement of analyte concentration. Using 2–3 drops of whole blood, a test can be completed in 5 min by placing the blood on the cartridge and placing the cartridge in the fluorescence reader. No training or sample measurement is needed. The system has a low manufacturing cost and a short development time per assay. A patent was issued on the system in the US in May 1998 (1).
The criteria for point-of-care testing (2) are affordable cost, a disposable device, and/or minimal maintenance and minimal technical expertise required to perform tests. The sample should be whole blood, low volume, unmetered, and applied directly into the instrument or disposable. There needs to be a simple "goof-proof" strategy for recording collection time and result reporting, a simple strategy for calibration and quality control, data transfer capability compatible with the laboratory or hospital information system and a positive identification and specimen-tracking strategy that eliminates specimen identification error. Results should be rapid and in agreement with accepted "gold standard" tests. RAMP was designed with these requirements in mind.
The RAMP assay has much in common with qualitative immunochromatography assays (3)(4) in that it uses the accumulation of a particle population at a location on a membrane strip to indicate the presence of an analyte in the aqueous sample used to transport the particle population along the strip by capillary flow. In general, the particles carry adsorbed or coupled antibodies (Abs) against the analyte. The coated particles (colloidal gold or latex <1 µm in diameter) are dried into a carrier material or directly in the nitrocellulose membrane in the presence of a hydration agent to allow their easy mobilization when wetted by the sample. A few centimeters from the deposited particles, a second Ab against the analyte is dried onto the membrane, creating a "test line". Another Ab directed against the Ab on the latex is applied farther along the strip to form the "control line". The aqueous sample, which can be whole blood, serum, or urine, is contacted with the end of the strip and allowed to wick along it, hydrating the particle population and carrying it along in the capillary flow. As the particles move along the strip, Abs bind the antigen (Ag), with the amount bound depending on the Ag concentration. When the particles reach the test line, some fraction of the population is arrested by the membrane-associated Abs capturing particle-Ag complexes. When the remainder of the population reaches the control line, an additional fraction is arrested, regardless of the presence of bound Ag, indicating that the test was completed successfully. The visible presence of particles at one or both lines constitutes the transduction of the immune recognition reaction and indicates a positive or negative result.
RAMP assays differ from the qualitative assays in that they utilize two
classes of particles in every assay: the test particle and an internal
standard particle (see Fig. 1
A). Both particles are polystyrene from the same
manufacturing batch and thus have the same size and surface properties.
Each particle population is labeled internally with a distinct
fluorescent dye. The test particle population typically carries a
covalently coupled monoclonal antibody against the analyte,
whereas the internal standard particle carries an uninvolved covalently
coupled monoclonal antibody at the same surface concentration and of
the same isotype as the test Ab. In all respects except the actual
polypeptide sequence of the Ab, the two populations of particles are
identical.
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An obstacle to overcome in any immunochromatographic assay is the variability observed from strip to strip at the same analyte concentration attributable to variations in membrane properties, component aging, humidity effects, variable release of particles by the sample, or particle aggregation constraints. The internal standard particle population is used to overcome this variability. The two particle populations are mixed before being dried onto the membrane. When the sample is applied to the membrane strip, typically through a sample pad, which may, for example, allow separation of blood cells from plasma, the two particle populations are released equally and migrate down the strip with the sample. In a "sandwich" assay, a polyclonal antibody against the analyte is immobilized at the test line as described above for the qualitative assay. At a second, internal standard line, an anti-mouse IgG antibody arrests a fraction of both the remaining test and the internal standard particles. The RAMP reader scans the strip and measures the fluorescence intensity of each dye at the test line and internal standard line, from which it calculates the concentration of each particle at each location. The internal standard particles are subject to the same nonspecific interfering reactions as the test particles; therefore, the number of internal standard particles arrested at the internal standard line provides a measure of the fraction of the particles that have successfully reached the end of the strip and are able to participate in an immune reaction. Thus, the ratio of the concentrations of test to internal standard particle provides a measure of analyte concentration that is virtually independent of interfering factors. This ratio is therefore referred to a calibration curve to obtain the analyte concentration.
In a serum assay for myoglobin, the reader-based noise corresponds to a
concentration of ~0.3 pg/L. Typical test detection limits were
near 1.0 pg/L, and the CVs were 5–15% (range) for sera with added
myoglobin. Assay performance is independent of sample volume and
does not require a metered amount of sample. The data in Fig. 1B
show a
typical calibration curve for myoglobin. The error bars represent 1 SD
calculated on 4 separate days (over a period of 36 days) with five
points per concentration on each day. The mean of the CVs for the
calibrators was 13%.
Footnotes
fax 604-822-7635, e-mail don{at}pathology.ubc.ca
References
The following articles in journals at HighWire Press have cited this article:
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  S. Choi, E. Y. Choi, H. S. Kim, and S. W. Oh On-Site Quantification of Human Urinary Albumin by a Fluorescence Immunoassay Clin. Chem., June 1, 2004; 50(6): 1052 - 1055. [Full Text] [PDF]
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