2 Materials
2.1 Bead Coupling
1. Beads: MagPlex
® magnetic microspheres.
2. Plates: 96-well half-area flat-bottom polystyrene plates.
3. Plate shaker.
4. Plate magnet (see Note 1).
5. Activation buffer (1Â): 100 mM monobasic sodium phosphate, pH 6.2; store at 4
C for up to 1 month and at
À20
C for long term.
6. EDC solution: Prepare aliquots of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in screw-capped tubes
and store at À20
C. Dissolve in activation buffer to 50 mg/
mL directly prior usage (see Note 2).
7. S-NHS solution: Prepare sulfo-N-hydroxysuccinimide aliquots
in screw-capped tubes and store at 4
C. Dissolve in activation
buffer to 50 mg/mL directly prior to usage (see Note 2).
8. Coupling buffer: 100 mM 2-(N-morpholino) ethanesulfonic
acid (MES), pH 5.0, and store at 4
C for up to 1 month and at
À20
C for long term.
9. Wash buffer: 0.05% (v/v) Tween 20 in 1Â PBS, pH 7.4
(PBST).
10. Solution for antibody coupling: R-Phycoerythrin-conjugated
anti-species antibodies diluted to 0.25μg/mL in PBST (see
Note 3).
11. Storage buffer: Blocking reagent for ELISA diluted to 2.7 mg/
mL in PBS and supplemented with 0.1% (v/v) ProClin.
2.2 Sample Labeling
1. Sample dilution buffer: 1Â PBS, pH 7.4.
2. Labeling solution: 10 mg/mL sulfo-N-hydroxysuccinimidepolyethylene oxide biotin (NHS-PEO4-Biotin, Pierce), dissolved in dimethyl sulfoxide (DMSO) directly before use.
3. Stop solution: 1 M Tris–HCl (pH 8.0), stored at +4
C.
2.3 Assay Procedure
1. Assay buffer (1Â): 0.1% (w/v) casein, 0.5% (w/v) polyvinylalcohol, and 0.8% (w/v) polyvinylpyrrolidone, prepared in PBST
and stored at +4
C for up to 3 months and at À20
C for the
long term. Supplement before use with 40μg of purified HBR6
(Heterophilic Reagent 6, Scantibodies) per sample (see Note 4).
2. Plates: 96- or 384-well half-area flat-bottom polystyrene plates.
3. Crosslinking solution (10Â): Prepare 4% paraformaldehyde
(PFA) solution and store at +4
C. Dilute 1:10 in PBS prior
to usage.
4. Detection solution: Dilute R-phycoerythrin modified streptavidin to 0.5μg/mL in PBST directly before use and protect
from light exposure (see Note 3).
Minimizing Rheumatoid Factor Interference
145
2.1 Bead Coupling
1. Beads: MagPlex
® magnetic microspheres.
2. Plates: 96-well half-area flat-bottom polystyrene plates.
3. Plate shaker.
4. Plate magnet (see Note 1).
5. Activation buffer (1Â): 100 mM monobasic sodium phosphate, pH 6.2; store at 4
C for up to 1 month and at
À20
C for long term.
6. EDC solution: Prepare aliquots of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in screw-capped tubes
and store at À20
C. Dissolve in activation buffer to 50 mg/
mL directly prior usage (see Note 2).
7. S-NHS solution: Prepare sulfo-N-hydroxysuccinimide aliquots
in screw-capped tubes and store at 4
C. Dissolve in activation
buffer to 50 mg/mL directly prior to usage (see Note 2).
8. Coupling buffer: 100 mM 2-(N-morpholino) ethanesulfonic
acid (MES), pH 5.0, and store at 4
C for up to 1 month and at
À20
C for long term.
9. Wash buffer: 0.05% (v/v) Tween 20 in 1Â PBS, pH 7.4
(PBST).
10. Solution for antibody coupling: R-Phycoerythrin-conjugated
anti-species antibodies diluted to 0.25μg/mL in PBST (see
Note 3).
11. Storage buffer: Blocking reagent for ELISA diluted to 2.7 mg/
mL in PBS and supplemented with 0.1% (v/v) ProClin.
2.2 Sample Labeling
1. Sample dilution buffer: 1Â PBS, pH 7.4.
2. Labeling solution: 10 mg/mL sulfo-N-hydroxysuccinimidepolyethylene oxide biotin (NHS-PEO4-Biotin, Pierce), dissolved in dimethyl sulfoxide (DMSO) directly before use.
3. Stop solution: 1 M Tris–HCl (pH 8.0), stored at +4
C.
2.3 Assay Procedure
1. Assay buffer (1Â): 0.1% (w/v) casein, 0.5% (w/v) polyvinylalcohol, and 0.8% (w/v) polyvinylpyrrolidone, prepared in PBST
and stored at +4
C for up to 3 months and at À20
C for the
long term. Supplement before use with 40μg of purified HBR6
(Heterophilic Reagent 6, Scantibodies) per sample (see Note 4).
2. Plates: 96- or 384-well half-area flat-bottom polystyrene plates.
3. Crosslinking solution (10Â): Prepare 4% paraformaldehyde
(PFA) solution and store at +4
C. Dilute 1:10 in PBS prior
to usage.
4. Detection solution: Dilute R-phycoerythrin modified streptavidin to 0.5μg/mL in PBST directly before use and protect
from light exposure (see Note 3).
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145