Scheme of experiment setup in macro- and micro-scales. (a) Prepared magnetic anti-Aβ immunosorbent is inserted in PDMS microchip using syringe pump where they are self-assembled into affinity microcolumn. (b) Sample containing Aβ peptides is applied into the chip and (c) the specific immunocapturing is performed. After proper washing (d) the specifically captured peptides are eluted using compatible eluting reagent according to subsequent method (e).
Scheme of immobilization techniques employed for IgG on magnetic beads with various site-groups
Binding efficiency (%) of SiMAG-active Chemicell microbeads (1 mg) with different site-groups for human serum IgG (100 µg) immobilization as determined by SDS-PAGE (3 replicates)
16.5% urea/tricine/tris SDS-PAGE of Aβ peptides (1-38, 1-40, 1-42) eluted by the various elution reagents: 0.05% trifluoroacetic acid (TFA; pH 2.3), 0.25% formic acid (FA; pH 2.35), 0.16% ammonium hydroxide (OH; pH 11.0), and 100 mM ammonium bicarbonate (NH4HCO3; pH 8.0); molecular weight standard 1.4–26.6 kDa (MW, BioRad).
(a) Master for PDMS molding, (b) scheme of PDMS microfluidic device, and (c),(d) detail of anti-Aβ plug/affinity microcolumn in microfluidic channel (1 mm × 0.25 mm × 20 mm) formed by magnetic microbeads self-organized using 2permanent magnets.
Monitoring of on-chip Aβ 1-42 immunocapture on anti-Aβ IS (pAb) by dot blot on nitrocellulose membrane. The process consists of Aβ 1-42 capture (0–100 μl; flow rate 50 μl/h), washing (110–260 μl; flow rate 200 μl/h), and elution of captured Aβ 1-42 (270–370 μl; flow rate 200 μl/h) by 0.05% TFA.
CE-UV of Aβ mixture of three synthetic peptides (1-38, 1-40, 1-42) from IP performed on anti-Aβ IS (pAb): original sample (OS; sample before IP), binding fraction (BF; sample with non-IP Aβ peptides), and three successive elution fractions (E1–3; fractions with IP Aβ peptides eluted by 0.16% NH4OH). E1–3 show preferential capture of Aβ 1-42. Analytical conditions were according to Verpillot et al. (2008).
(a) Electropherogram from MCE of fluorescently labeled Aβ 1-37 and 1-42 from μIP on anti-Aβ IS (mAb); FP = FluoProbe. (b) Comparison of fluorescence intensity (FI) ratio of Aβ 1-37/1-42 peptides in samples obtained from μIPs on anti-Aβ IS (pAb) and anti-Aβ IS (mAb) (averages of 3 repetitions). OS = original sample (before IP).
Aβ peptides separated by Aβ-SDS-PAGE/immunoblot on polyvinylidene fluoride membrane (according to Wiltfang et al. (2002)). (a) Batchwise IP of CSF sample on anti-Aβ IS (pAb) and anti-Aβ IS (mAb). CSF = CSF sample before IP; IP (pAb) = batchwise IP on anti-Aβ IS (pAb); IP (mAb) = batchwise IP on anti-Aβ IS (mAb). (b) μIP of CSF sample on anti-Aβ IS (mAb). Aβ ST = Aβ standard peptides mixture corresponding to levels in CSF; μIP = μIP on anti-Aβ IS (mAb).
Quantification of amyloid β (Aβ) isoforms in human CSF before and after μIP.
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