microparticle-based immunoaggregation assay

Activities

1.1 Using a model biomarker (goat anti-rabbit IgG) for immunoaggregation assay demonstration 

To illustrate the immunoaggregation assay concept, goat anti-rabbit IgG (Ag) was firstly used to mimic a model biomarker (antigen). For preparation of microparticle-rabbit Ag aggregates, dynabeads M-280 Streptavidin (MP) was functionalized with biotinylated rabbit anti-goat IgG (rAb) through streptavidin and biotin binding. The rAb-MP conjugated solution was then placed on the magnet to separate the rAb-MP conjugate from the solution, and remove the unconjugated Ab supernatant. The rAb-MP conjugate was resuspended with PBS (PH7.4, 0.1% BSA) to a concentration of 53.40 µg/ml. Different concentrations of Alexa Fluor 488 labeled gAg ranging from 0.1 ng/ml to 320.0 ng/ml.

We used a fluorescent microscope to study the aggregation behavior of microparticles in  different solutions through bright field filters, i.e., Cy3 filter (552/575 nm) and GFP filter (494/518 nm). The number of Ag–rAb-MP aggregates for each biomarker concentration was counted. The numbers of MP, 2-rAb-MP aggregates, 3-rAb-MP aggregates and 4- rAb-MP aggregates were recorded separately. To ensure accuracy and repeatability, more than 1000 particles were counted for each sample and 3 samples were prepared and tested for each Ag concentration.  To confirm the result, MP-rabbit Ag aggregates were diluted to 100 ml PBS (pH7.4, BSA 0.1 mg/ml); the size and counts of the particle in the sample were measured using a particle counter.

1.2 Using a real biomarker (human ferritin) for immunoaggregation assay demonstration.

Human ferritin was used as an example to prove that microparticle-based immunoaggregation assay is valid for detection of real human biomarkers. The preparation procedure and characterization methods of microparticle-ferritin aggregates were similar to previous microparticle-rabbit Ag aggregates. M2.8-ferritin Ab conjugate was resuspended with PBS (PH7.4, 0.1% BSA) to two different concentrations of 53.40 µg/ml and 213.40 µg/ml to test different behaviour of microparticle-ferritin aggregates. Different concentration human ferritin samples ranging from 0.1ng/ml to 416 ng/ml were prepared by gradient dilution with PBS (PH7.4, 0.1% BSA). In a parallel study, 10% fetal bovine serum (FBS, Sigma-Aldrich, USA) was also used to dilute the human ferritin to different concentrations ranging from 0.1 ng/ml to 416.0 ng/ml to mimic the biomarker detection in complex media. 

1.3 Antifouling materials development.

Protein fouling on particle and sensor surfaces can dramatically reduce the detection sensitivity. Using antifouling materials can improve the detection sensitivity. In this work we developed a versatile and high performance zwitterionic polysaccharide platform and studied the structure-property-function relationships of zwitterionic polysaccharides, so that this platform can be readily adapted to the biomarker detection. As shown in Scheme 1, CB-Dex was synthesized via one pot reaction (see Scheme 1). The molecular weight and the degree of substitution were characterized by GPC and 1H NMR spectroscopy respectively. Zwitterionic CB side chains were introduced onto dextran backbone using the rational design approach. Three methacrylate (MA) modified dextran derivatives, with different degree of CB substituent, from 0 % (Dex-MA), 35 % (CB-L-Dex-MA), to 158 % (CB-H-Dex-MA) were employed in this study. All samples were kept at a similar MA ratio around 25 % (one MA unit per four glucose units). We used NMR to confirm the structure of the polymer. The antifouling properties of the materials were evaluated in the form of hydrogel.

Scheme 1 Synthetic route of Zwitterionic CB-Dex-MA

Findings

Figure 1 shows the microparticle (MP) aggregation behaviors in the bare streptavidin-MP solution (a), the rabbit Ab-MP (rAb-MP) solution (b) and the solution containing the goat antibody as a biomarker (Ag) and rAb-MP (c) measured by an optical particle sizer (accusizer). Figure 1 indicates that the addition of biomarker can trigger the aggregation of rAb-MP and the aggregation can be detected by the optical particles sizer (Accusizer). This study demonstrated that the immunoaggregation method is a versatile method and it can be combined with many particle sizing methods for biomarker detection.

Fig. 1 Accusizer measurement results for (a) the bare streptavidin-MP solution, (b) the rabbit Ab-MP (rAb-MP) solution, (c) the solution containing goat antigen (Ag) and rAb-MP.

To detect and determine the concentration of biomarkers, it is important to correlate biomarker concentration to aggregate size, concentration and volume. After the aggregates were formed for each Ag concentration, both single magnetic particles and aggregates were imaged and counted through microscope. For each sample, the volume fraction of aggregates (f), defined as volume ratio of aggregates to all counted particles, was calculated. The volume ratio of aggregates was plotted in Fig. 2 as a function of Ag concentration. As Figure 2 showed, the volume ratio of aggregates increased with the increase of Ag concentration in the range of 0.10 ng/ml to 40.00 ng/ml; the max volume fraction (65.7%) occurred at 40.00 ng/ml.  Above 40.00 ng/ml, aggregate volume fraction was reduced with the increase of rabbit antigen concentration. This is because the high concentration of rabbit antigen would saturate rabbit Ab on the surfaces of Ab-MP; hence the number of unreacted rabbit Ab on Ab-MP is too low to cause the aggregation. All the study showed that innovative microparticle-based immunoaggregation can be formed and biomarker concentration can be related to the volume fraction of aggregates.

Fig. 2 The relationship between the volume ratio of Ag-rAb-MP aggregates to all particles and the Ag concentration.