Abstract
The high surface area of microsphere makes it a good tool for carrying the antibody or antigen in the immunoassays. Using a commercial streptavidin coated microsphere, the modification of antibody on the microsphere is simple and fast. Compare to normal ELISA, the antibody coated microsphere can lower the detection limit and improve the performance of the normal immunoassay. In this chapter, we will try to modify the antibodies on the commercial available microspheres and study the performances of the modified microspheres.
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4.1 Modification of biotinylated antibody on the streptavidin coated microspheres
Introduction
Compare to the planar immunoassays, nanoparticle or microsphere based suspension arrays are a more powerful detection methods. The advantages of the suspension array are low sample requirement, short operation time, low cost, and easy to use.1,2 The important role in the suspension array is microsphere.
The high surface area of microsphere can allow it to carry extensive biomolecules, such as antibodies, antigens, oligonucleotides, or DNA strands.3-5 The large loading capacity of microsphere makes the suspension array more sensitive and selective. In recent years, many research groups have noticed the potential of microspheres and many immunoassays have been developed using microspheres.6-10
Baba et al. presented an immuno-pillar chip using microsphere as an antibody carrier to detect the target in the immunoassay.6 In brief, the microchip substrate was a cyclic olefin copolymer and had rectangular microchannels. The anti-human CRP coated beads was mixed within the hydrogel pillars. The assay procedure was simple. The sample solution was injected into the microchannels and incubated for a short time. After incubation, the channels would be washed by PBS three times. The fluorescent-labeled secondary antibody solution was added in the microchannels and incubated for a short time. After incubation, the channels were washed with PBS three times. Finally, using an inverted
fluorescence microscope detected the fluorescence signal.
The CRP sandwich immunoassay was performed as a model sample. The incubation time was set differently: 1 min, 3 min, and 5 min, which corresponded to total assay time of 4 min, 8 min, and 12 min. The fluorescence signals rose as CRP concentration increased. The limit of detection (LOD) for all different incubation times was ~100 pg mL-1. Because the pillars consisted of pores, antigen and antibody were diffused into the pillars. By using FITC-labeled antibody, they estimated the diffusion rate of antibody was 0.4 μm s-1. Also, the assay was carried out with AFP, PSA, and multiplex samples. The results were similar. This developed immuno-pillar chip was easy-to-use and only needed a trace amount of sample.
In this section, the detection antibody was coated on the microsphere using streptavidin-biotin interaction. The commercial microsphere was modified with streptavidin which can react with biotin on the detection antibody. The S-HRP was used to generate the color signals from the detection antibody coated microspheres. The blue color signals can be used to quantify the amount of detection antibody which was bound on the SμS.
Experimental section
100 μL of SμS suspension solution (the original concentration of SμS suspension was 12.5 mg beads / mL stored buffer) was taken and centrifuged at 10,000 g for 5 min to remove the stored buffer. 300 μL of PBS2 was used to wash the microsphere three times. The microsphere was resuspended in 300 μL
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of PBS2 and the concentration of the microsphere suspension was 5 mg mL-1. The saturated volume of biotinylated antibody on a SμS was 2.4 μg / mg beads.11 180 μL of 60 μg mL-1 IL-7 DetA was added into the microsphere suspension and incubated for 8 h at 4°C. After 8 h incubation, the microsphere suspension was washed three times with 300 μL of PBS2 by centrifuge at 10,000 g. The waste buffer will be removed after every wash step and the microsphere was re-suspended in a fresh buffer. 100 μL of 1.5 x 10-4 mg mL-1 S-HRP was added into the microsphere suspension and incubated for 20 min at rt on a shaker (v = 160 rmp). After 20 min, the microsphere was washed three times with 300 μL of PBS2. Finally, 100 μL of ready-to-use TMB was added per wells and reacted for 15 min in dark at rt. This experiment was the initial trial to assure the biotinylated antibody can actually bind to the SμS.
A control experiment was also performed to assure the color generated from TMB was not because the non-specific binding of S-HRP in the eppendorf. 100 μL of SμS suspension solution was taken and centrifuged at 10,000 g for 5 min to remove the stored buffer. 100 μL of PBS2 was used to wash the microsphere three times. The microsphere was resuspended in 300 μL of PBS2 and the concentration of the microsphere suspension was 5 mg mL-1. No antibody was added in this microsphere suspension. 100 μL of 1.5 x 10-4 mg mL-1 S-HRP was added into the microsphere suspension and incubated for 20 min at rt on a shaker (v = 160 rmp). After 20 min, the microsphere was washed three times with 300 μL of PBS2. Finally, 100 μL of ready-to-use TMB was added per wells and reacted for 15 min in dark at rt.
Additionally, the concentration of DetA on the microsphere was changed to gradient in the following experiment. The gradient concentrations of DetA can allow us to understand the optimized condition of the surface saturation on a SμS. 20 μL of SμS suspension solution was added in 6 clean eppendorfs and centrifuged at 10,000 g for 5 min to remove the stored buffer. The microspheres were washed three times with 100 μL of PBS2. The microsphere was resuspended in 50 μL of PBS2 and the concentration of the microsphere suspension was 5 mg mL-1. The microspheres were incubated with 100 μL of various concentrations of DetA for 8 h at 4°C (see Table 2). After 8 h, the microspheres were washed three times with 100 μL of PBS2. 100 μL of 1.5x10-4 mg mL-1 of S-HRP was added to the microsphere suspensions and allowed it to shake for 20 min at rt. After 20 min, the microspheres were washed three times with 200 μL of PBS4. Finally, 100 μL of TMB was added into the microspheres and allowed it to sit for 15 min at rt in the dark.
Table 2. The concentrations of IL-7 DetA that were used for the quantification of the surface saturation on SμSs.
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