I can't remember seeing this article before, can you?
RAMP®: High Accuracy from Immunochromatographic Assays by the Use of Internal Control Ratios
Lynn P. Cloneya, Linda J. Spiller1, Whalley K. Fong1, Joanne E. Harris1 and Paul C. Harris1
1 (Product Development, Response Biomedical Corporation, 8855 Northbrook Court, Burnaby, BC, V5J 5J1 Canada)
aauthor for correspondence: fax 604-412-9830, e-mail lcloney@responsebio.com
RAMP® (Rapid Analyte Measurement Platform) is a platform technology that can be adapted to quantify immunologically active substances. The system consists of two components: a disposable test cartridge that houses an analyte-specific immunochromatographic nitrocellulose membrane strip and a portable scanning fluorescence reader that quantifies antibody-antigen complexes. The unique feature of the RAMP technology is that an internal control is run and measured concurrently in every assay, allowing the system to compensate for inherent test-to-test and reader-to-reader variations (1).
In the RAMP System, the test sample is delivered into the sample well of a RAMP test cartridge and the cartridge is inserted into the reader. As the sample migrates along the strip, fluorescently dyed latex particles conjugated to analyte-specific antibodies bind to antigen, if present in the sample. Antigen-bound particles are immobilized at the detection zone by specific antibodies, and some of the excess particles are immobilized at the internal control zone by anti-immunoglobulin antibodies. On completion of the test, the reader measures fluorescence (F-units) emitted by the complexes bound at the detection and internal control zones and calculates a ratio between these measurements.
Immunochromatographic assays are dynamic in nature. In these tests the sample is in contact with the immobilized capture antibodies for only a short time; therefore, the degree of antibody-antigen binding is subject to variability. For any fixed amount of free antigen, the captured signal will increase with longer contact time. This contact time is affected by such factors as capillary speed, sample viscosity, humidity, and temperature (2)(3)(4). Nitrocellulose membranes differ in structure, wetting characteristics, and surface quality. These variations can occur both within and between lots and can cause large discrepancies in the test results (5). In the RAMP System, the detection and internal control zones are positioned in close proximity to each other on the nitrocellulose strip; therefore, factors that affect the binding of particles at the detection zone similarly affect binding at the internal control zone. A ratio of the measurement at these two zones, the RAMP ratio, then corrects for variations in binding.
Fluorescence-based assays are generally difficult to operate quantitatively because of a lack of stable fluorescent standards for instrument standardization (6)(7). In addition, the detection of fluorescent signal is affected by differences in instrument optical configurations, filtering, and excitation light wavelength and intensity. The RAMP ratio factors out the absolute units of fluorescence through the ratio calculation itself; the ratio is a relative value of the measurement of signal at the detection zone divided by the sum of the detection zone and the internal control zone. The signal intensity can vary within the linear range of the instrument and not change the ratio. With the ratio, the readers do not require routine recalibration; this is an important feature for point-of-care testing devices (8).
The RAMP reader and the RAMP myoglobin assay have been cleared for sale by the US Food and Drug Administration and the Canadian Therapeutic Products Directorate. Additional RAMP tests have been developed for use in both clinical and environmental applications. To demonstrate the broad application of this technology with effective correction for test-to-test variation of binding at the detection zone, we prepared samples containing different amounts of each of six different analytes over their respective diagnostically relevant ranges. Serial dilutions of solutions or suspensions of each of the analytes, including three environmental agents (Bacillus anthracis, botulinum toxin, and ricin) and three cardiac markers [myoglobin, creatine kinase-MB (CK-MB), and troponin I] were prepared, and aliquots were tested using the corresponding RAMP test. Triplicates of each dilution were tested for the first three, and replicates of five were tested for each dilution of cardiac markers. A 70-µL aliquot of each sample was appropriately diluted and delivered into the sample well of a RAMP test cartridge, and the cartridge was inserted into the reader. The mean, SD, and CV were calculated for the binding of each dilution at each of the two zones. The corresponding RAMP ratio was calculated for each dilution, and the CV of these ratios, as well as the mean CV across the analyte range, are reported in Table 1 .
View this table:
[in this window]
[in a new window]
Table 1. Reproducibility of analyte test results.
A comparison of the reproducibility of test results for each analyte was made between the binding of particles at the detection zone only and the RAMP ratio. In all cases there was an improvement in the mean CV for test results across the analyte ranges tested when the RAMP ratio was used. The CV of Bacillus anthracis test results was improved twofold, from 7.5%, using detection-zone-binding only, to 3.8% when internal control zone binding was used to calculate the RAMP ratio. Likewise, the mean CV for botulinum toxin and ricin test results were improved 2.2-fold and 1.6-fold, respectively. Similarly, for the cardiac markers the mean CV for myoglobin, CK-MB, and troponin I test results were improved 5.7-fold, 2.3-fold, and 2.2-fold, respectively.
The RAMP ratio was shown to correct for even large variability in binding of latex particles at the detection zone. For example, data from five replicates of 50 µg/L myoglobin showed extreme differences in binding at the detection zone (CV = 35%). The replicates with the highest fluorescence measured at the detection zone also showed the highest fluorescence measured at the internal control zone. The RAMP ratio calculated with measurements at the two zones effectively corrected for these test-to-test variations, giving a CV of 3.8%. Without this correction, the variability in the results that would be reported using absolute fluorescence measured at the detection zone alone would render the test information inaccurate.
Results from the RAMP environmental tests are reported in the field as positive or negative by comparing the calculated ratio to a defined threshold. The cardiac tests are quantitative, and test results are interpolated and reported from a lot-specific calibration curve that is uploaded and stored in the memory of the reader. To compare the reproducibility of quantitative test results reported using either the detection zone data only or the RAMP ratio, troponin I calibration curves were plotted using either detection zone fluorescence or the RAMP ratio over the reportable range of 0–64 µg/L. The concentration of troponin I was interpolated from the curves for concentrations (n = 5) ranging from 0.5 to 32 µg/L. The mean CV for troponin I results reported across this range was 14% using the detection zone only. The CV of these results was improved to 5.6% when interpolated from the ratio calibration curve. For example, replicates of the 1 µg/L solution were reported as 0.78–1.04 µg/L (range) from the detection zone calibration curve and as 0.98–1.06 µg/L from the RAMP ratio calibration curve.
The results clearly show that if the binding at the detection zone alone is used to quantify the analyte, the variability would be substantial and would give less accurate information. The results also demonstrate that the RAMP ratio can effectively correct for inherent variability in fluorescent particle binding at the detection zone by standardizing the signals by use of the internal control zone. Improved precision is achieved by use of the RAMP ratio rather than simple absolute measurements of fluorescence at the detection zone. Results from six different analytes demonstrate that the reproducibility of RAMP System results is improved in all cases by use of the RAMP ratio. The RAMP technology can be used for rapid and reproducible quantification of a broad range of analytes for both clinical and environmental applications. Use of the internal control effectively corrects for test-to-test and reader-to-reader variability.
References
Stephenson J. RAMP: a quantitative immunoassay platform takes shape. IVD Technol 1998;4:51-56.
Jones KD. Troubleshooting protein binding in nitrocellulose membranes. Part 1: principles. IVD Technol 1999;5:32-41.
Jones KD. Troubleshooting protein binding in nitrocellulose membranes. Part 2: common problems. IVD Technol 1999;5:26-35.
Fisher AS, Alcox DA, . Millipore Corporation, Bedford, MA, USA. Structure-function relationships of immunochromatographic substrates. Polym Mater Sci Eng 1997;76:465-466.
Beer HH, Jallerat E, Pflanz K, Klewitz TM. Quantification of cellulose nitrate membranes for lateral-flow assays. IVD Technol 2002;8:35-42.
Holtom GR, Thrall BD, Chin B, Wiley HS, Colson SD. Achieving molecular selectivity in imaging using multiphoton Raman spectroscopy techniques. Traffic 2001;2:781-788.[CrossRef][ISI][Medline] [Order article via Infotrieve]
Smith TC. The use of fluorescent dyes to measure membrane potentials: a response. J Cell Physiol 1982;112:302-305.[ISI][Medline] [Order article via Infotrieve]
Wilding P, Ciaverelli . Hand-held sensor systems. Price CP Hicks JM eds. Point-of-care testing 1999:41-66 AACC Press Washington. .
clinchem.org |
