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Lateral flow test strips are immunochromatographic assays used to test for an array of target analytes. Creating a functional and sensitive lateral flow assay draws on principles from biology, chemistry, physics and engineering. These are explored in Rapid Lateral Flow Test Strips: Considerations for Product Development.
While several types of detector particles can be used in the manufacture of lateral flow assays, latex microspheres are the most versatile of the particles currently used. In addition to being available in a wide range of sizes, they can be detected by colorimetry, fluorometry, or paramagnetism, depending on the specific particle composition. They are also available with a range of coupling chemistries for covalent attachment of antibodies and other types of molecules.
In lateral flow assays, the detector particles are required to move through the porous structure of the membrane. The size of the particles will affect their mobility due to physical interactions with the pore walls. Thus, small particles will move faster than larger particles. Assuming that the particle population is monodisperse with uniform size and shape, differences in assay sensitivity and specificity may be expected. The inherent flow rate of the membrane also affects the rate of particle movement. The net result is that an assay manufacturer must determine the appropriate microsphere size and membrane flow rate to achieve the desired levels of sensitivity and specificity.
In this article, we examine the relationship between microsphere size, membrane flow rate, and assay sensitivity, using blue Estapor® carboxyl-modified dyed microspheres and Hi-Flow™ Plus membranes.
Methods
Lateral flow test strips for the detection of hCG were assembled using blue Estapor® carboxyl–modified dyed microspheres (Table 1), Hi-Flow™ Plus lateral flow membranes (Table 2) and anti-hCG antibodies (Fitzgerald Industries [Table 3]).
Microspheres were conjugated to test antibodies using a two-step EDC/Sulfo-NHS covalent coupling procedure. Microspheres were washed twice with 50 mm MES buffer (pH 6.0) before activation with EDC and Sulfo-NHS. Following activation, the microspheres were washed with 50 mm MES buffer (pH 6.0), coated with 60 mg* hCG beta antibody #10-C25E (Fitzgerald) per gram of microspheres, and mixed on a rotary wheel for 2 hours at room temperature. Following antibody coating, the microspheres were quenched with 30 μL of ethanolamine and mixed on a rotary wheel at room temperature for 30 minutes. The microspheres were washed twice and blocked in 50 mm Tris (pH. 8.0), 0.5% casein for 2 hours by mixing at room temperature on a rotary wheel. Finally, the microspheres were resuspended to a final concentration of 1% (w/v) in 50 mm Tris (pH. 8.0), 0.5% casein. For conjugate pad application, the hCG conjugates were diluted to 0.065% (w/v) in 50 mm Tris (pH 8.0) containing 0.5% casein, 2.5% trehalose, 10% sucrose, and 0.5% polyvinylpyrrolidone. An aliquot of 1 mL microsphere suspension was applied to each glass fiber diagnostic pad (0.5 x 30 cm, SureWick® G041 Glass Fiber Pad, Merck Millipore) to yield about 10.8 μg of microspheres per test. Conjugate pads were dried overnight at room temperature and stored in a sealed foil pouch with desiccant sachets until used for test strip assembly.
Hi-Flow™ Plus membranes were striped with capture reagent solutions using a Kinematic Matrix 1600 Reagent Dispensing Module under these conditions:
- Relative humidity: 40% to 60%
- Temperature: 18–22 °C
- Bed speed: 10 cm/sec
- Reagent dispense rate: 10 μL/sec
The test line solution contained hCG beta antibody #10-C25D (Fitzgerald) at a concentration of 1 mg/mL in 50 mm MES (pH 6.0). The control line solution contained a polyclonal goat IgG, raised against mouse IgG, at a concentration of 1 mg/mL in Milli-Q® water. Membranes were dried at 37 °C for 2 hours and then assembled together with conjugate pads, sample pads (SureWick® C083 Cellulose Fiber Pad treated with 10 mm Tris [pH 8.2], 1% Tween 20, 0.75% BSA) and absorbent pads (SureWick® C083 Cellulose Fiber Pad) into lateral flow test strips (Figure 1).
The functionality and sensitivity of the lateral flow test strips were evaluated with a range of hCG (BBI Solutions) concentrations prepared in human urine (Biochemical Diagnostics Inc). Microsphere mobility and nonspecific binding were evaluated visually. Signal intensity at the test lines was evaluated visually and also quantitatively using an ESEQuant Lateral Flow Immunoassay Reader (Qiagen). Photographs of selected test strip series have been included where appropriate.
Results
After constructing and running test strips with hCG samples of known concentration, the signal intensities at the test lines were evaluated colorimetrically. For each combination of microsphere size and membrane speed, the data were graphed and are presented below.
On Hi-Flow™ Plus 75 (Figure 2a) and Hi-Flow™ Plus 90 (Figure 2b), the fastest flowing membranes, the highest signal intensities from the quantitative analysis were obtained with the 0.478 μm diameter microspheres; and the lowest signal intensities were obtained with the 0.185 μm diameter microspheres. Interestingly, the microspheres of intermediate diameters (0.288, 0.413, and 0.422 μm) produced similar signal intensities.
Test strips run with the 0.478 μm microspheres exhibited two artifacts. First, a quantifiable signal was detected on the negative control strip (i.e., 0 mIU hCG/mL) on Hi-Flow™ Plus 90. Second, faint lines were visible above and below the primary control line (Figure 2c). These represent the edges of the liquid line when the control line reagent was initially applied to the membrane. Another feature that can be seen is a decrease in the intensity of the control line as the intensity of the test line increases. This is expected, given that the control line is capturing only the microspheres that travel beyond the test line.
When the signal intensities were evaluated on Hi-Flow™ Plus 120 (Figure 3a) and Hi-Flow™ Plus 135 (Figure 3b), similar trends were observed. The highest signal intensities were obtained with the 0.478 μm diameter microspheres; and the lowest signal intensities were obtained with the 0.185 μm diameter microspheres. The microspheres of intermediate diameters (0.288, 0.413, and 0.422 μm) also produced similar signal intensities. As with Hi-Flow™ Plus 90, a quantifiable signal was detected in the negative control with the 0.478 μm microspheres (Figure 3c, 3d). Smaller microspheres did not produce a nonspecific signal on the negative control strips (Figure 3c, 3d).
On Hi-Flow™ Plus 180, the slowest flowing membrane, the trends were generally the same (Figure 4a). The highest signal intensities were obtained with the 0.478 μm diameter microspheres; and the lowest signal intensities were obtained with the 0.185 μm diameter microspheres. The microspheres of intermediate diameters (0.288, 0.413, and 0.422 μm) produced similar signal intensities. One significant difference occurred with 0.478 μm microspheres. Above 160 mIU hCG/mL, the signal intensity leveled off well below the values observed on the faster flowing membranes. A quantifiable signal was also measured on the negative control.
On all Hi-Flow™ Plus membranes, the results obtained with the 0.413 μm and 0.422 μm Estapor® microspheres indicate that the difference in COOH charge density (21 vs 284 μEq/g) did not affect their performance.
Optimization of 0.478 μm Estapor® Microspheres
The measurable signals observed with the 0.478 μm microspheres on the hCG-negative controls indicated that nonspecific binding was occurring at the test line. Attempts to eliminate nonspecific binding by reducing the amount of microspheres in the assay were not fully successful. Thus, we investigated modifications to the conjugation procedure. The most effective approaches were optimizing the quenching and blocking steps. Specifically, the amount of ethanolamine used to quench the microspheres was reduced by 50% and the blocking agent was changed from 0.5% (w/v) casein to 1% (w/v) fish skin gelatin.
The optimized 0.478 μm microspheres were re-evaluated in the hCG assay on Hi-Flow™ Plus 75, Hi-Flow™ Plus 135, and Hi-Flow™ Plus 180 membranes. On Hi-Flow™ Plus 75, the signal intensity was reduced at all concentrations compared to the standard microspheres (Figure 5a). On the control lines, the faint lines above and below the primary line, which were observed with standard microspheres (Figure 2c), were no longer present (Figure 5b).
Conclusion
In this series of experiments using hCG detection as a model system, we have demonstrated the efficacy of Estapor® microspheres in lateral flow test strips manufactured on Hi-Flow™ Plus membranes. The optimal microsphere diameter for any given membrane has to take into account the sensitivity and specificity required in the final test strip design. Artifacts also need to be minimized so that interpretation of results is unambiguous.
Estapor® microspheres of 0.185 μm diameter were found to be the poorest performing regardless of membrane flow rate. Signals were undetectable at 25 mIU hCG/mL at all flow rates, and signal intensity at the control lines was uniformly less intense compared to larger microspheres. It is believed that these particles are flowing too rapidly to be efficiently captured at the test and control lines and that the color density of the particles is too low for them to be detected at the same efficiency as larger microspheres.
For all but Hi-Flow™ Plus 180, Estapor® microspheres with a diameter of 0.478 μm produced the highest signals. While this suggests that these microspheres are the best choice for Hi-Flow™ Plus 75, 90, 120, and 135, the artifacts that accompanied these higher signals require consideration of other aspects of the performance of the test strips. Optimization of conjugation to the 0.478 μm microspheres eliminated the nonspecific signals at 0 mIU hCG/mL observed on all membranes but also reduced the signal intensity for hCG-positive samples. Thus, there appears to be no performance advantage.
For Hi-Flow™ Plus 180, Estapor® microspheres from 0.288 to 0.422 μm diameter were found to give comparable performance up to a concentration of 16,000 mIU hCG/mL. Signal intensity did not level off with increasing hCG concentration as was observed with the 0.478 μm diameter microspheres. Thus, these microspheres appear to be interchangeable.
In conclusion, Estapor® microspheres ranging from 0.288 to 0.478 μm diameter were found to be effective as detector particles in lateral flow assays configured on Hi-Flow™ Plus membranes. While we have provided general guidelines on pairing microspheres with the membranes, test strip manufacturers should optimize each combination within the specifications for sensitivity and specificity that are required for the assay to be acceptable in the marketplace.
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