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In this assay, a sandwich-type immunoassay on the surface of the fluorescent beads was established, as shown in Figure 9. The mouse anti-hMBP mAb “capture antibody” was covalently immobilized to the surface of one type of the fluorescent beads. The antibody- coupled beads were then incubated with samples containing the antigen “rhMBP”. For the detection of the captured antigen on the surface of the beads, a biotin-conjugated anti-His tag mAb “detection antibody” was employed. The well characterized interaction between the biotin label and the fluorescent marker streptavidin coupled R-phycoerythrin (SA-PE) was used to detect the antibody sandwich (Vignali, 2000).

Figure 9: Diagrammatic representation of the steps of the Bio-Plex immunoassay. A: anti-hMBP antibody - coupled beads, B: beads after incubation with samples containing the rhMBP, C: formation of the biotin-labeled antibody sandwich and D: antibody sandwich with the fluorescence label ready for detection by the Bio-Plex system.

2.3.7.1Coupling of anti-hMBP antibody to the fluorescent beads

Coupling of the anti-hMBP antibody to the surface of the fluorescent polystyrene beads was carried out according to the following in-house optimized protocols based on the manufacturer’s guidelines. A buffer exchange step for the antibody preparation was carried out using a phosphate buffered saline - pH 7.4 (PBS) in order to get rid of any amine additives. The final concentration of the antibody was determined by measuring its optical density at 280 nm. Suitable amounts of beads (20 µl ≈ 2.5 x 105 beads each) were transferred to three coupling reaction tubes. The beads were obtained by centrifugation and the bead pellet was re-suspended in 80 µl of the activation buffer. Aliquots of 2 µl of 50.0 mg/ml of freshly prepared solution of EDC closely followed by equivalent volumes of freshly prepared S-NHS solution of the same concentration were added to each tube in order to activate the surface of the beads. The bead suspension was incubated for 20 min at RT, away from light under constant rotation (25 rpm).

Activated beads were then washed with 2 x 150 µl PBS (pH 7.4) and then re-suspended in 100 µl PBS. In order to determine the optimal coupling conditions, the coupling reaction was carried out using three different amounts of the anti-rhMBP antibody (1.2, 1.8 and 2.4 µg). Suitable volumes of the antibody preparation equivalent to these

PBS then incubated with 250 µl of the blocking buffer for 30 min. Beads were then washed with 500 µl of the storage buffer then re-suspended in 50 µl of the same buffer. The concentration of coupled beads in each preparation was determined using a hemocytometer and the efficiency of the surface coverage in each case was evaluated.

2.3.7.2Validation of the coupling protocol

Aliquots from each bead preparation (≈ 10,000 beads each) were transferred into two sets of microfuge tubes which were labeled as “test” and “control”. The biotinylated anti-mouse antibody and the SA-PE solutions were prepared by diluting suitable amounts of each of them with the staining buffer to 2.0 µg/ml. Aliquots of 50 µl of the diluted antibody were transferred to the “test” tubes and equivalent volumes of the staining buffer were transferred to the “control” tubes. All tubes were incubated for 30 min at RT as described above then the supernatant was discarded. Aliquots of 50 µl of the diluted SA-PE were transferred to each of the “test” tubes and equivalent volumes of the staining buffer were transferred to the “control” tubes. All microfuge tubes were incubated for 10 min under the same conditions. The supernatant was then discarded and each of the pellets was re-suspended in 125 µl of the storage buffer then was transferred to a single well of a 96-well microtiter plate. The median fluorescence intensities (MFI) obtained from the “control” and “test” samples for each anti-hMBP antibody amount were then compared to identify the optimal coupling conditions. Coupling of the anti-hMBP antibodies to a larger number of beads was carried out using the optimized protocol. The concentration of the bead stock was determined as described above. Coupled beads were then stored away from light at 4 oC where they are stable for at least six months.

2.3.7.3Optimization of the detection step

In order to determine the optimum concentration of the detection antibody, three samples of the rhMBP reference standard over a wide range of concentration were prepared in 50 mM HEPES (pH 7.0). A similar procedure was followed to prepare an equivalent serial dilution of the WTcontrol sample. Aliquots with a volume equivalent to ≈ 10,000

assay buffer then washed with the same buffer. Aliquots of 50 µl of the prepared test samples and control samples were transferred to the filter plate (in duplicate) and were incubated with the coupled beads for 30 min. Samples were then incubated with different amounts of the biotin-labeled anti-His tag antibody (25 µl of 1:250, 1:500 and 1:1000) for another 30 min. Detection was carried out using the Bio-Plex system after an incubation step with 50 µl SA-PE (1:1000) for 10 min and the optimal dilution factor of the anti-His tag antibody was determined according to the MFI. All incubation steps were carried out at RT, away from light and on the surface of a plate shaker (300 rpm). A washing step was carried out after each incubation step using 3 x 100 µl of the assay buffer. Excess reagents and washing buffers were removed from the filter plate by vacuum filtration. The optimized conditions were used for all subsequent analysis of standard and test samples.

2.3.7.4Calibration, validation and application of the Bio-Plex method

A serial dilution of the rhMBP standard covering a wide range of concentration was prepared using 50 mM HEPES buffer (pH 7.0) in a 96-well microtiter plate. Blank and WTcontrol samples were included in the same plate. Suitable amounts of the coupled beads were transferred to the desired number of wells of a 96-well filter plate. Aliquots of 50 µl of the prepared samples were transferred from each well of the microtiter plate and added to the corresponding well in the filter plate. Samples were incubated with the coupled beads for 30 min followed by an incubation step with the biotin-labeled anti-His tag antibody (1:1000) for 30 min. The antibody sandwich was detected after another incubation step with the SA-PE system for 10 min. Standard, blank and WTcontrol samples were analyzed in triplicates and the fluorescent signal generated was detected using the Bio-Plex system. A calibration curve was obtained by plotting the MFI vs the rhMBP concentration. The same procedure was used to prepare and analyze all validation samples as well as samples containing unknown concentrations of the rhMBP. The MFI was used to predict the concentration of the rhMBP from the optimized calibration curve. The control and blank samples were included in every plate in order to detect any processing error.