Report. Comparison of magnetic beads coating protocols for immunoaffinity capillary electrophoresis. Natalia Gasilova, Hubert H.

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1 Report Comparison of magnetic beads coating protocols for immunoaffinity capillary electrophoresis Natalia Gasilova, Hubert H. Girault Laboratoire d Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland The immunoaffinity capillary electrophoresis (IACE) analytical system with UV-detection developed previously in the laboratory (Gasilova, N., Gassner, A.-L., Girault, H.H.. Electrophoresis 2012, 33, ) for bovine β-lactoglobulin B detection was used for testing coating protocol for tosyl-activated Mbs (1.29 µm diameter, Estapor R01-24, Lot N 8369/2, Merk, France). The Estapor coating protocol was compared with the one proposed for Dynabeads (MyOne Tosylactivated, Invitrogen, Oslo, Norway). Experimental section Mbs coating: 1. Estapor protocol 4 µl of rinsed tosyl-activated MBs were mixed with 280 µl of coating buffer, 166 µl of 3 M ammonium sulfate in coating buffer and 54 µl of antibodies (1.48 mg/ml). The mixture was incubated overnight (~12h) at room temperature under continuous moderate stirring to avoid MBs sedimentation. After the beads were incubated with blocking buffer during 1h at room temperature under continuous moderate stirring. Then the beads were rinsed with washing buffer and stored in PBS buffer, containing 0.025% of Tween 20 and 0.02% of sodium azide. 2. Dynabeads protocol 2 µl of rinsed tosyl-activated MBs were mixed with 8 µl of coating buffer (100 mm sodium borate, ph 9.5), 8 µl of 3 M ammonium sulfate in coating buffer and 8 µl of antibodies (5 mg/ml). The mixture was incubated 24 h at 37ºC under continuous moderate stirring to avoid MBs sedimentation. After incubation, beads were rinsed with 10 mm PBS buffer (8 mm Na 2 HPO 4, 2 mm NaH 2 PO 4, 150 mm NaCl, ph 7.4) and stored in same PBS buffer, but also containing 0.025% of Tween 20 and 0.02% of sodium azide.

2 BCA test: To roughly estimate the amount of antibodies bound to the Mbs surface, a BCA (bicinchoninic acid) protein test (BCA kit, Pierce, Rockford, USA) was carried out following the standard manufacturer protocol. Detected amount of antibodies bound per 1 mg of Mbs is the following: 42 µg for Estapor coating protocol and 24 µg for Dynabeads coating protocol. Estapor coating protocol allowed reaching two times better efficiency of the antibodies binding than Dynabeads protocol. IACE experiment: IACE experiments were carried out with a 7000 CE apparatus (Agilent, Waldbronn, Germany) equipped with a UV diode-array detector. Fused silica capillaries (50 µm i.d, 375 µm o.d, 41.5/50cm effective/total length, BGB analytik AG, Böckten, Switzerland) coated with 5% HPC solution were used for separation. For the MBs capture, two permanent cylindrical magnets (Nd-Fe-B, 4 mm diameter, 12 mm length, Supermagnete, Zürich, Switzerland) were placed directly around the capillary at a distance of 14.5 cm from the inlet using a homemade Plexiglas holder. Throughout the IACE experiments the following buffers were used: leading electrolyte and washing buffer (50 mm ammonium acetate, ph=8), sample buffer (10 mm PBS with 0.1% of Tween 20), elution buffer (10% acetic acid with 0.1% TFA, ph=1.5), separation buffer (10% acetic acid, ph=2). Working concentration of Mbs was 0.5 mg/ml, β-lactoglobulin B working concentration was 5 µg/ml. The scheme of the performed IACE experiments is presented below (Figure 1): 1. Magnetic beads trapping 2. Sample injection and capture 3. Direct elution UV DAD 4. t-itp/ce separation Figure 1. Schematic representation of IACE-UV experiment with tosyl-activated Mbs.

3 UV / mau - MBs injection during 300s at 39 mbar and trapping by permanent magnets inside the separation capillary; - Washing with washing buffer, 250s at 39 mbar; - Sample injection during 600s at 39 mbar; - Washing during 650s; - Elution via direct injection of elution buffer during 200 s at 39 mbar; - Separation of the sample via application of 24 kv; - Detection by UV (at 200 nm); - MBs removal and capillary washing with high pressure, at 1000 mbar. Each experiment was repeated in tree replicates, n=3. Results of IACE experiments Electropherograms obtained during IACE analysis of β-lactoglobulin B using Mbs coated using either Estapor or Dynabeads coating protocols are presented in Figure IACE with Tosyl-activated Mbs Estapor coating protocol Dynabeads coating protocol lactoglobulin B ,0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 10,6 10,8 11,0 Time / min Figure 2. IACE analysis of 5 µg/ml solution of β-lactoglobulin B with Mbs coated by Estapor (black line) or Dynabeads (blue line) coating protocol. Mbs concentration is 0.5 mg/ml.

4 UV / mau As can be concluded from the displayed results the areas of β-lactoglobulin B peak obtained in case of Mbs coated using Estapor coating protocol are larger than in case of Dynabeads coating protocol: area of (±0.009) units vs area of (±0.006), respectively. The same conditions of IACE analysis were utilized in both cases including the level of preconcentration introduced by t- ITP step. Therefore, Mbs coated with Estapor protocol showed better analytical performance. In order to decrease the time of coating procedure the Mbs incubation with antibodies was performed during 6h at 37ºC instead of overnight reaction (~12h) at room temperature. The other steps of coating protocol were remained the same. The electropherograms obtained using these Mbs are presented in Figure 3. For these experiments the solution of 50 µg/ml β-lactoglobulin B was analysed IACE with Mbs coated by Estapor protocol lactoglobulin B 37 o C room temperature ,0 10,2 10,4 10,6 10,8 11,0 11,2 11,4 11,6 11,8 12,0 12,2 Time / min Figure 3. IACE analysis of 50 µg/ml solution of β-lactoglobulin B with Mbs coated by Estapor coating protocol at 37ºC (black line) or room temperature (blue line). Mbs concentration is 0.5 mg/ml. The areas obtained for β-lactoglobulin B peaks in case of coating at 37ºC and at room temperature were the following: 2.14(±0.05) units vs area of 2.28 (±0.06) units, respectively. Coating of Mbs with Estapor procedure at 37ºC provided similar efficiency of antibodies binding to Mbs surface as the same procedure performed at room temperature, but within less time.

5 Conclusions The results of performed IACE analysis of β-lactoglobulin B indicate that Estapor coating protocol provides higher efficiency of Mbs coating with antibodies than the Dynabeads protocol. Moreover, the Estapor coating protocol performed at 37ºC ensure similar coating efficiency as the same protocol performed at room temperature but within less experimental time.