The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease

Abstract

Hydrogel biomarker capturing microparticles were evaluated as a biomaterial to amplify the sensitivity of urine testing for infectious disease proteins. Lyme disease is a bacterial infection transmitted by ticks. Early diagnosis and prompt treatment of Lyme disease reduces complications including arthritis and cardiac involvement. While a urine test is highly desirable for Lyme disease screening, this has been difficult to accomplish because the antigen is present at extremely low concentrations, below the detection limit of clinical immunoassays. N-isopropylacrylamide (NIPAm)-acrylic acid (AAc) microparticles were covalently functionalized with amine containing dyes via amidation of carboxylic groups present in the microparticles. The dyes act as affinity baits towards protein analytes in solution. NIPAm/AAc microparticles functionalized with acid black 48 (AB48) mixed with human urine, achieved close to one hundred percent capture and 100 percent extraction yield of the target antigen. In urine, microparticles sequestered and concentrated Lyme disease antigens 100 fold, compared to the absence of microparticles, achieving an immunoassay detection sensitivity of 700 pg/mL in 10 mL urine. Antigen present in a single infected tick could be readily detected following microparticle sequestration. Hydrogel microparticles functionalized with high affinity baits can dramatically increase the sensitivity of urinary antigen tests for infectious diseases such as Lyme disease. These findings justify controlled clinical studies evaluating the sensitivity and precision of Lyme antigen testing in urine.

Figure 1

a. Affinity baits (such as Acid Black 48) covalently incorporated in the microparticles bind proteins with high affinity. Due to the molecular size sieving property of the microparticles, only low molecular weight proteins may enter the microparticles. b. The process by which proteins are concentrated. The proteins are mixed with the microparticles. Low molecular weight proteins enter the microparticles. The solution is separated by centrifugation. The microparticles containing low molecular weight proteins form a pellet at the bottom of the test tube. The supernatant, which contains high molecular weight proteins, is removed.

Figure 2

Concentration effect of microparticle pre treatment. For an initial sample volume of 10 mL and a final collection volume of 50 μL the concentration factor is 200.

Figure 3

a. Correlation between temperature and average diameter of microparticles. Standard deviations represented by error bars. b. Correlation between pH and average diameter of microparticles. Standard deviations represented by error bars. c. and d. False color AFM images of the NIPAm-AB48 hydrogel microparticles. Diameter, measured on the AFM image of 20 microparticles, was 606.6 +/− 22 nm.

Figure 4

Silver stain SDS-PAGE demonstrating the ability of nine different types of dye-functionalized hydrogel microparticles (listed above each gel) to concentrate B. burgdorferi proteins in water. Acid Black 48 dye was determined to be the most effective at concentrating the total protein mix. OspA 31 kDa, OspB 34 kDa, IS = initial solution, S = supernatant, and P = microparticle content.

Figure 5

NIPAm-Acid Black 48 hydrogel microparticles sequester and concentrate B. burgdorferi proteins in synthetic urine in a pH dependent manner. SDS-PAGE silver stain of B. burgdorferi proteins OspA and OspB post incubation with NIPAm-AB48 microparticles in synthetic urine at three different pH values: 5, 6 and 7 (lanes 3, 5, and 6 respectively) showed optimal binding microparticle capacity at pH 5 (lane 3). Supernatant was completely depleted. IS = initial solution, S = supernatant, and P = microparticle content.

Figure 6

Western blot analysis demonstrating the presence of B. burgdorferi proteins captured from synthetic urine. a. OspA and b. OspB were detected in the protein mixture. NIPAm-AB48 microparticles completely depleted the supernatant and concentrated B. burgdorferi proteins present in solution. C= control, i.e. B. burgdorferi protein solution, S = supernatant, and P = microparticles. NIPAm/AB48 microparticles sequestered and concentrated OspA and OspB spiked in 100 μL of synthetic urine at a concentration of c. 7 μg/mL, 3.5 μg/mL, 0.7 μg/mL; d. 0.35 μg/mL, 0.07 μg/mL, and e. 0.07 μg/mL, 0.007 μg/mL, and 0.0007 μg/mL, respectively. This study indicates that the minimum detectable concentration of B. burgdorferi protein in 100 μL solution after microparticle concentration was 7 ng/mL, therefore a protein quantity of 700 pg. IS= initial solution, S = supernatant, and P = microparticles.

Figure 7

Western blot analysis showing the control mixture containing 7ng B. burgdorferi protein diluted in water, and the eluate from NIPAm-AB48 microparticles incubated with the tick proteins. The band pattern from the ticks was similar to that from the control protein mixture, indicating that the antibodies reacted with B. burgdorferi protein in the tick. C= commercial control, E= eluate from the microparticles.

Figure 8

Western blot showing the concentration of B. burgdorferi proteins from a. 1 mL and b. 10 mL human urine. U=plain human urine, IS= initial solution of B. burgdorferi proteins spiked in human urine, S = supernatant, and P = microparticles, E = eluate from the microparticles. c. Graph shows the concentration effect of the microparticles. The band intensity for the proteins OspA and OspB was greatly increased by the microparticles (more than 100 fold).

Figure 9

Microparticle concentration step was successfully coupled to reverse phase protein microarray (RPMA) calibrated immunoassay directed against outer surface protein A (OspA). After microparticle preprocessing, no OspA was detectable in the supernatant and the yield of the concentration process was higher than > 95%.