Ngoc Van

Thanh Nguyen

Claire Smadja
email: claire.smadja@universite-paris-saclay.fr

Myriam Taverna

Khoa Trong

Frédéric Mai

Thanh Halgand

Mai Duc

Trong Khoa Mai

Frédéric Halgand

Thanh Duc Mai
email: thanh-duc.mai@universite-paris-saclay.fr

Immuno-capture of biomolecules on functionalized magnetic beads: from characterization to fluorescent detection of a biomarker for Alzheimer's disease toward high performance biosensing

Keywords: magnetic beads, density and orientation, antibody anti-amyloid beta, size exclusive chromatogram, enzyme digestion, IdeZ

Introduction

Immuno-enrichment and immunoassays, relying on the capture of target peptides and proteins via specific antibodies for their subsequent preconcentration and fluorescent detection, are the gold techniques used in bioanalysis and biosensing. Among all solid supports for such purpose, magnetic beads have gained great attention thanks to the ease of manipulation via an external magnetic field and a variety of functionalities (commercially) available for magnetic particles [START_REF] Comanescu | Magnetic Nanoparticles: Current Advances in Nanomedicine, Drug Delivery and MRI[END_REF][START_REF] Piovesana | Magnetic Materials for the Selective Analysis of Peptide and Protein Biomarkers[END_REF]. The performance of immunocapture on magnetic beads nevertheless depends significantly on density and orientation of the grafted antibodies in order to allow their efficient and selective interaction with target molecules as well as avoid / minimize nonspecific adsorption [START_REF] Khizar | Magnetic nanoparticles in microfluidic and sensing: From transport to detection[END_REF][START_REF] Serra | The power of solid supports in multiphase and droplet-based microfluidics: towards clinical applications[END_REF][START_REF] Mai | Single-step immunoassays and microfluidic droplet operation: Towards a versatile approach for detection of amyloid-β peptide-based biomarkers of Alzheimer's disease[END_REF]. Control and characterization of on-beads immobilized antibodies are therefore of utmost importance to achieve the best immunocapture and immunoassay performance. Different characterization strategies have been developed for such purpose, notably spectrophotometric and colorimetric assays [START_REF] Moreira-Alvarez | Integrated analytical platforms for the comprehensive characterization of bioconjugated inorganic nanomaterials aiming at biological applications[END_REF][START_REF] Liu | Protein A-and Protein G-gold nanoparticle bioconjugates as nano-immunoaffinity platform for human IgG depletion in plasma and antibody extraction from cell culture supernatant[END_REF]. Gagey-Eilstein et al. communicated a chemiluminescent test to monitor the antibody grafting rate on streptavidinmagnetic beads [START_REF] Bouzas-Ramos | Controlling Ligand Surface Density on Streptavidin-Magnetic Particles by a Simple, Rapid, and Reliable Chemiluminescent Test[END_REF]. These methods nevertheless cannot provide any information on antibody orientation and/or can induce measurement biases mainly related to possible background interferences [START_REF] Kozlowski | Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates[END_REF][START_REF] Walkey | Nanoparticle Size and Surface Chemistry Determine Serum Protein Adsorption and Macrophage Uptake[END_REF][START_REF] Hinterwirth | Bioconjugation of trypsin onto gold nanoparticles: Effect of surface chemistry on bioactivity[END_REF]. Other methods such as surface plasmon resonance (SPR), dual polarization interferometry, spectroscopic ellipsometry and atomic force microscopy, can provide insight on antibody orientation mainly by measuring antibody dimensions/thickness on surfaces or shift angle [START_REF] Bae | Study on orientation of immunoglobulin G on protein G layer[END_REF][START_REF] Song | Comparative Study of Random and Oriented Antibody Immobilization as Measured by Dual Polarization Interferometry and Surface Plasmon Resonance Spectroscopy[END_REF][START_REF] Yoshimoto | Direct Observation of Adsorption-Induced Inactivation of Antibody Fragments Surrounded by Mixed-PEG Layer on a Gold Surface[END_REF][START_REF] Fujiwara | Measurement of antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy[END_REF][START_REF] Zhang | Direct quantification of surface coverage of antibody in IgG-Gold nanoparticles conjugates[END_REF][START_REF] Näreoja | Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay[END_REF]. Indirect methods for quantification of proteins on the surface of nanoparticles (NPs) were as well proposed, relying on hydrolysis of grafted proteins, followed by spectrophotometric or chromatographic quantification of primary amines, amino acids or peptides in solution [START_REF] Oliverio | Versatile and High-Throughput Strategy for the Quantification of Proteins Bound to Nanoparticles[END_REF][START_REF] Liu | Accurate and reliable quantification of the protein surface coverage on protein-functionalized nanoparticles[END_REF][START_REF] Shen | Restricted Proteolysis and LC-MS/MS To Evaluate the Orientation of Surface-Immobilized Antibodies[END_REF]. Our group recently proposed a new simple and rapid analytical approach to evaluate human antibody orientation and density on magnetic beads applied for immunocapture of a biomarker of inflammation (TNF-α) [START_REF] Laborie | Analytical methods of antibody surface coverage and orientation on bio-functionalized magnetic beads: application to immunocapture of TNF-α[END_REF]. This approach relies on the cleavage of immobilized antibodies by IdeS, a highly specific protease for human immunoglobulin G (hIgG). This approach using IdeS nevertheless is not adapted to mouse IgG, which represents the majority of antibodies commercially available for bioanalysis. Indeed, in the context of molecular diagnosis of Alzheimer's disease (AD), which is the most prevalent neurodegenerative disease worldwide causing a huge burden to the society and having no efficient AD treatment so far [START_REF] Van Thanh Nguyen | Recent Electrokinetic and Microfluidic Strategies for Detection of Amyloid Beta Peptide Biomarkers: Towards Molecular Diagnosis of Alzheimer's Disease[END_REF], mouse antibodies have been most widely used for immunoassays of amyloid (Aβ) beta peptides (notably Aβ 1-42 and Aβ 1-40 which are established biomarkers for AD). No human antibodies recognizing specifically these compounds have been available so far.

From this rationality, we report in this study the development of an analytical approach to simultaneously characterize the density and orientation of grafted mouse antibody on magnetic beads. This approach relies on selective enzymatic digestion of immobilized mouse antibodies at one specific site below the hinge region using FabRICATOR Z (IdeZ) to give F(ab')2 and Fc fragments from mouse antibodies, followed by separation and determination of these fragments with size exclusion chromatography (SEC) coupled to fluorescent detection (FLD). Optimization of the SEC-FLD method was made to allow separation of the F(ab')2 and Fc fragments from residual antibodies and the enzyme, allowing non-bias determination of the digestion efficiency via the F(ab')2 / Fc ratios. The developed approach was applied for three functionalized magnetic beads (i.e., Tosyl-activated, carboxylic, Pierce Protein G) grafting with immunoglobulin (IgG2a) from mouse. Following the antibodygrafting optimizations that can be monitored with our method, the activity of grafted antibodies was confirmed via on-bead immunoassays with fluorescent detection of amyloid beta 1-42 peptide (Aβ1-42) that is an established biomarker for AD molecular. The work was extended to three antibodies specific to different epitopes of Aβ1-42 (i.e., NAB228 for N-terminal, 12F4 for C-terminal, and 4G8 for 17-24 epitopes), serving for evaluation of the performance of bead-based immunoassays of Aβ1-42.

Experimental

Chemicals, reagents and samples

2-(Cyclohexylamino)ethanesulfonic acid (CHES), Tris(hydroxymethyl)aminomethane (TRIS), sodium dodecyl sulphate (SDS), sodium hydroxide (NaOH), potassium hydroxide (KCl), acid boric (H3BO3), sodium dihydrophosphate (NaH2PO4), disodium hydrophosphate (Na2HPO4), Tween 20, ammonium sulfate ((NH4)2SO4), EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride), S-NHS (N-Hydroxysulfosuccinimide sodium salt), mouse IgG2a (2 mg/ml) (mIgG), phosphate buffer saline 10X (PBS), Tris buffer saline 10X (TBS), ammonium hydroxide (NH4OH), human IgG from whole serum (hIgG at 10 mg/ml), as well as different anti-Alzheimer antibodies (1 mg/ml), including NAB228 and 6E10-HRP were all provided by Sigma Aldrich (St. Louis, MO, United States). Other antibodies 12F4-HRP, 12F4, and 4G8 were obtained from Biolegend (San Diego, California, United State).

HPLC grade isopropanol (IPA), methanol and acetonitrile were purchased from VWR (Pennsylvania, US). FabRICATOR enzyme (Ides) and FabRICATOR Z enzyme (Idez) were obtained from Genovis (Lund, Sweden). Amyloid beta peptide Ab 1-42 (Aβ1-42) was purchased from Eurogentec (Seraing, Belgium). FP-488 NHS ester solution was purchased from Interchim (Montluçon, France). Pierce Protein G (ProG), Tosyl-activated (Ts) and Carboxylated (COOH) magnetic beads, QuantaRed Enhanced Chemifluorescent HRP kit were provided by Thermo Fisher (Massachusetts, United States). All buffers were prepared with deionized water and were filtered through a 0.22 µm membranes (Pall Corporation, New York, USA) prior to use. For mass spectrometry experiments, acetonitrile and formic acid were purchase from Sigma Aldrich.

-6 -

Apparatus and Material

Deionized water used in all experiments was purified using a Direct-Q3 UV purification system (Millipore, Milford, MA, USA). Conductivity and pH values of buffer solutions and samples were measured by a Seven Compact pH meter (Mettler Toledo, Schwerzenbach, Switzerland). SEC-FID experiments were carried out using a 1260 Agilent UPLC, and a 1260 fluorescent detector (λexcitation: 280 nm, λemission: 345 nm) (Agilent Technologies, Santa Clara, CA, USA).

Two columns, including Agilent AdvanceBio SEC (2.7μm particle size, 130A° pore size, i.d 7.8 , length 300 mm) and BioSEC-3 (3μm particle size, 300A° pore size, i.d 4,6 mm, length 300 mm) were employed for SEC-FLD at temperature of 25 °C. Fluorescence detection with the excitation and emission wavelengths of 530 nm and 582 nm respectively were carried out with a FP-750 Spectrofluorometer from Jasco (Lisses, France) using a 40 μL quartz cuvette (Starna Scientific, Essex, England). Mass spectrometry measurements were performed on a QToF Synapt G2 Si (Waters, Manchester) equipped with ion mobility. Parameters were optimized to obtain the best signal. In brief, capillary voltage was set to 3.5 kV, source temperature to 40°C, the cone voltage and the offset were fixed at 50 V, désolvatation temperature was 450 °C, and nebulizer gas flow to 500 L/hr. Calibration was performed on the 500 to 5000 m/z range using TFANa. Error on the measurement was 0.2 ppm.

Methods

Antibody digestion in solution

IdeS FabRICATOR enzyme 5000 U and IdeZ FabRICATOR Z enzyme 2000 U were dissolved in 100 μL and 50 μL miliQ water, respectively, then aliquoted to 2 μL and stored at -20 o C. For IgG digestion, 2 μL IdeS enzyme (100UI) was mixed with 50 μL of 100 mM phosphate buffer (pH 7.0) whereas 2 μL IdeZ enzyme (80UI) was added to 50 μL of 10mM phosphate buffer and 10 mM sodium chloride (pH 6.5). hIgG and 6E10 were diluted in PBS to 50 μL at concentration of 0,1 mg/ml and then digested with IdeS at 37 0 C on a thermomixer at 650 rounds per minute (rpm) for 90 minutes. NAB228 and 6E10 (0,1 mg/ml) were prepared and digested with IdeZ under the same conditions for 120 minutes.

Antibody grafting on magnetic beads

Protein G magnetic bead 50 µL of ProG magnetic beads (10 mg/mL) was added to 2 mL LoBind Eppendorf vials and gently vortexed with 500 µL of Tris solution containing 0.05 % v/v of Tween 20. The supernatant was removed, and the beads were washed one more time with 1 mL of Tris-Tween 20 (0.05%). A magnet was employed to retain magnetic beads during removal or addition of a suspension solution. After removal of the supernatant, a predefined volume (0-5-15-22,5-45-60-75-90 μL) of NAB228 at 2 mg/mL was subsequently added to beads. The Tris-Tween 20 (0.05%) solution was then added to 500 μL. The mixtures were then incubated at 25°C, 650 rpm for 1 hour on a Thermomixer. The protocol was also carried out with 100 µL ProG (10 mg/mL) and 4 batches of NAB228 (0-10-55-120 μL at 2 mg/mL). After incubation, the supernatants were collected. The beads were then washed twice with 500 µL of Tris-Tween 20 (0.05%) solution. The washing solutions were collected for subsequent analyses.

Tosyl-activated magnetic bead 10 µL of Dynabeads MyOne Tosylactivated (100 mg/mL) was added to 2mL LoBind Eppendorf vials and washed with 500µL of 0.1 M sodium borate buffer (pH 9.5) using a Thermomixer for 15 min C at 25°C and 650 rpm. Pre-defined volumes (0-10-55-90-120 μL) of NAB228 solution (2 mg/mL) were added to beads. 60 µL of 3M ammonium sulphate buffer (pH 9.5) and 20 µL of 0.1M sodium borate buffer were then added. The mixtures were then incubated at 37 °C on a Thermomixer (650 rpm) for 16h. The supernatants were collected with the help of a magnet. The beads were washed twice with 500 µL of PBS-Tween 20 (0.05%) and once with 500 µL of PBS. The washing solutions were also collected for subsequent analyses by SEC-FLD.

Carboxylated magnetic bead

100 μL of a Dynabeads MyOne carboxylic acid suspension (10 mg / mL) were rinsed twice with 1 mL PBS after removing the suspending solution. 500 μL of EDC solution (10 mg / mL inPBS) and 500 μL of S-NHS solution (10 mg / mL in PBS 1X) were then added into the washed beads, followed by the addition of NAB228 2 mg/ml (10 μL or 55 μL). The mixture was incubated for 3 hours under 650 rpm at room temperature (RT). The supernatant was collected. The antibody-bound magnetic beads were subsequently washed twice with 1 mL of PBS, followed by an incubation at RT with 1mL of 50 mM Tris-HCl (pH 7.4) for 15 min in order to quench the non-reacted activated carboxylic acid groups. The antibody-bound magnetic beads were then washed 3 times with 1 mL of PBS -0.1 % Tween-20. A magnet was employed to retain magnetic beads during removal or addition of a suspension solution.

Digestion of NAB228 grafted on magnetic beads by IdeZ

100 µL of enzyme Idez (3 UI) was used for 1 μg NAB228. The enzyme solutions were added directly to grafted and washed magnetic beads and incubated for 3h at 37 o C on a Thermomixer at 650 rpm. Further optimization of the enzymatic digestion procedure can be referred to section 3.1.

SEC-FLD conditions

The SEC-FLD analyses of digested samples were conducted on two Agilent columns: BioSEC-3 (3μm particle size, 300A°, i.d 4,6 mm, length 300 mm) and AdvanceBio SEC (2.7μm particle size, 130A° pore size, i.d 7.8, length 300 mm). Both columns were equilibrated with the mobile phase for 5 column volumes prior to analysis; and flushed with a mixture of miliQ water (80%) and MeOH (20%) for 15 column volumes after each analysis.

The flow rate was fixed at 0,3 ml/min for both columns. For each analysis, 2 µL of sample was injected, and analyzed with the phosphate buffer 50 mM that contains 10 % IPA and 150 mM KCl (pH 6,8).

Calibration curves were made using native NAB228 prepared in different matrices and under different conditions that were used for on-bead antibody grafting protocols. The calibration curve served for the study on Tosyl-activated beads was made with NAB228 (at six points from 0,05 to 0.3 μg) diluted in ammonium sulphate 3 M and sodium borate buffer 0,1 M at pH 9.5 and incubated on Thermomixer at 37 o C during 16h. The one used for the study on proG beads was done with NAB228 (6 concentration points) prepared in Tris-Tween 20 (0.05%) and incubated at 25 o C for 1h on Thermomixer. The non-grafted condition was used as the reference with six points of native mouse IgG2a prepared in PBS. Three SEC-FLD analyses were implemented for each concentration. The respective calibration curve for each bead type was used to determine the non-grafted NAB228 amount in supernatant and washing solutions. The successfully grafted NAB228 was estimated from the difference between the initial antibody concentration and the recovered ones in the supernatant and washing solutions.

Immunoassay of Aβ 1-42 peptide

-10 -A volume of 50 µL Aβ 1-42 was incubated with 200 µg magnetic beads coated with the desired antibodies (12F4, 4G8 or NAB228) and 5 µL 6E10-HRP or 12F4-HRP antibodies at concentration of 0.04 µg/ml in PBS 1X on a mixer at RT for 1 hour. The beads were then washed 3 times (10 min each time on a mixer) with 400 µL PBS 1X/ 0.1 % BSA (m/v)/ 0.1 % Tween-20 (v/v). Then, 100 µL QuantaRed solution was added to the washed beads. The incubation was carried out over 7 min on shaking, followed by addition of 10 µL QuantaRed Stop solution. The colour intensity of the achieved solution was measured with the excitation and emission wavelengths of 530 nm and 582 nm, respectively.

MS measurements of Aβ 1-42 peptide

Five lots of Aβ 1-42 peptide stock solution at 2 mg/ml were prepared by balancing and dissolving in corresponding volume of DMSO. The Eppendorf vials were vortexed gently with hand. Then, the stock solutions were diluted in NH4OH 0.16% to obtain the samples at a concentration of 5 μM. These samples were injected directly into MS to verify the quality of Aβ 1-42. For MS conditions see the section 2.2 above.

Results and Discussion

Enzymatic digestion of grafted antibodies and analysis of released fragments

Our approach (see Fig. 1) to characterize the performance of on-bead antibody grafting relies on the selective enzymatic digestion of the grafted antibody below the hinge position to release (Fab')2 and Fc fragments. The analysis of the remaining antibody as well as these released fragments, which are typical for an antibody, can then provide insight in the density and the orientation of the grafted antibodies thanks to the ratio of F(ab')2 's signal to Fc one.

Unlike chemically mediated or trypsin-based methods, our approach is softer and more selective, and can provide both information on antibody density and orientation. The challenges here were to find an appropriate enzyme that works on the target mouse antibodies and develop the subsequent separation strategy for determination of the resulting fragments.

In our case, SEC which is frequently used for separation of proteins and peptides was chosen for analysis of F(ab')2 and Fc fragments. The obtained F(ab')2 and Fc fragments and residue IgG possess respective molecular weights of about 100, 50 and 150 kDa. Phase mobile optimization was first carried out to separate these target fragments from the residual antibodies in the solution (Fig. 2). Among two salts commonly used in SEC buffers, KCl offered more symmetric and less tailing peaks than NaCl (Fig 2 A-B vs Fig 2 C-D). Indeed, KCl was found stronger than NaCl in suppression of the secondary interaction between target molecules and the stationary phase, probably due to the larger size and higher interactivity of K + cation [START_REF] Duivelshof | A generic workflow for the characterization of therapeutic monoclonal antibodies-application to daratumumab[END_REF]. It was observed from Fig 2 C vs Fig. 2 D that the column of a smaller pore size (130A°) provides better resolution for separation of F(ab')2 and Fc. Moreover, thanks to these optimized conditions, the peak of IdeZ enzyme was well separated from the Fc fragment (see Figs. 3 and4), which was not the case in the previous work using IdeS [START_REF] Laborie | Analytical methods of antibody surface coverage and orientation on bio-functionalized magnetic beads: application to immunocapture of TNF-α[END_REF]. This would help avoid the bias in quantification of the digested fragments.

The optimized SEC-FLD conditions were then used to monitor the IgG digestion optimization. FabRICATOR IdeS, which was found to offer fast reaction on hIgG, and was found to work well in our previous work on hIgG [START_REF] Laborie | Analytical methods of antibody surface coverage and orientation on bio-functionalized magnetic beads: application to immunocapture of TNF-α[END_REF] was tested as the reference. By comparing the peak of IgG before and after digestion in Figs. 3A-B, one can see that almost 80% of hIgG was digested with IdeS without optimization, whereas only 10% of mouse IgG1 could be digested under the same conditions. Indeed, IdeS can digest only antibodies having CPPCPAPELLG/GPSVF sequence at hinge position that is typical for human, rabbit and sheep IgG. This enzyme nevertheless is less favorable for mouse IgG, which represents the majority of antibodies commercially available for bioanalysis and is the most widely used one for immunoassays of Aβ peptides. To overcome this problem, FabRICATOR Z IdeZ was selected for mouse IgG. This kind of enzyme shows its specificity on mouse IgG2a or IgG3, having CPAPNLLG/ GPSVF sequence at the hinge site. As can be seen in (iii) temperature (low 30 o C and high 44 o C). The response to be evaluated was the digestion efficiency estimated via F(ab')2 : Fc peak-area-ratio. A Pareto chart was used to determine the optimal conditions for complete digestion of IgG while using the lowest enzyme unit as possible. Among the investigated factors, the one with the highest impact was found for the added enzyme unit. As can be seen from Fig. 4, with 3 U of enzyme IdeZ for 1 µg IgG (in an incubation over 3 hours at RT), quasi-complete digestion (98%) of IgG in solution, calculated according to peak areas of initial IgG and residual one after digestion, could be achieved.

Compared to the digestion performance obtained with the commercial protocol for IdeZ (see Figs. 2 C-D where there was much residual IgG after the digestion), that achieved with our optimized conditions is superior with almost no trace of IgG left (Fig. 4).

Optimization of the density and orientation of antibodies grafted on magnetic beads

The optimized digestion conditions (i.e., incubation with 3 UI IdeZ over 3h at 37 °C) and the optimized SEC-FLD ones (i.e., column of 130 A° pore-size, phosphate buffer containing 150 mM KCl) were employed to release and determine the F(ab')2 and Fc fragments from onbead grafted NAB228 antibody (mouse IgG). Quantification of grafted NAB228 was made with following the formula (1), respectively: mgrafted = minitial -(msupernatant + mwashing) [START_REF] Comanescu | Magnetic Nanoparticles: Current Advances in Nanomedicine, Drug Delivery and MRI[END_REF] where minitial, mgrafted, msupernatnant and mwashing are the initial quantity of antibodies, the quantity of antibodies grafted on 1000 µg beads, and those of remaining antibodies in the supernatant and washing solutions, respectively.

The peak ratios for F(ab')2/Fc fragments released from NAB228 antibodies grafted on Tosylactivated, ProG and carboxylated magnetic beads were determined and compared (Fig. 5).

With the same quantity of antibodies available for grafting, proG and tosyl-activated beads allowed higher amount of grafted NAB228, whereas very little trace of grafted antibodies could be found with carboxylated ones. As shown in Fig 5A andB, proG and tosyl-activated beads would require different initial antibody amount to achieve the best F(ab')2/ Fc ratio.

The best grafted antibody density and orientation was achieved with 110 µg antibodies per 1 mg bead for tosyl-activated beads (at F(ab') / Fc of 2.9), whereas for proG beads the best F(ab')2 / Fc ratio of 2.5 was achieved at a higher quantity of 240 µg antibodies per 1 mg beads (Fig. 5C). This can be explained by the fact that tosyl-activated beads with a covalent group (H3CC6H4SO2) has more affinity to Fc fragment. Note that further addition of antibodies led to less grafting performance on tosyl-activated beads (Fig. 5C), probably due the abundant presence of PBS used for antibody suspension, which is the blocking solution and not favorable for tosylated groups on magnetic beads, according to the instructions in the commercial protocol. In the case of proG, at low IgG quantity (< 50 µg), the non-covalent linkage is more effective towards F(ab')2, leading to less-oriented antibody grafting (with decreased F(ab')2 / Fc peak ratio). At higher IgG quantity, the tendency was reversed, leading to an improved in F(ab')2/Fc peak ratio for ProG beads. This important observation indicates that different magnetic beads would require different antibody quantities to reach the maximum density and best orientation. Under our best grafting conditions, 74.4 % of antibodies grafted on tosyl-activated beads were at the right orientation, calculated from the (F(ab')2 / (Fc + F(ab')2) peak ratio. This was 71.9 % for the case of ProG beads. Using the formula (1), the maximum grafted quantity of NAB228 antibodies on ProG and tosylactivated beads were estimated to be 43 μg and 35 μg per 1000 µg beads, respectively. Note that different conditions (i.e., the matrices, the temperatures and incubation times) were used for grafting antibodies on different beads. To avoid bias in the quantification of grafted antibodies, different calibration curves were made for different bead types, using NAB228 prepared in respective grafting matrices (see Fig. S1 in the supporting information ESI). Note also that the peak of IdeZ was observed together with those of F(ab')2 and Fc fragments after the digestion (Fig. 5). The peaks of IdeZ in the supernatants after incubation with different beads were lower than that of IdeZ in the initial one, which was due to some absorption of IdeZ on to ProG and tosyl-activated beads during the incubation for digestion of grafted IgG, as demonstrated in Fig. S2 in the ESI. The peak of residual IdeZ, if not well separated from those of target F(ab')2 and Fc, could induce some interference to these target peaks, and therefore confirm again the need to have an improved SEC-FLD method in our case to separate IdeZ from the target fragments.

Immunoassays with fluorescent detection of Aβ1-42 on magnetic beads

To validate the aforementioned optimization, the tosyl-activated and proG beads grafted with NAB228 (an anti Aβ1-42 antibody specific at N-terminal of this peptide) at their optimal concentrations were employed to carry out immunoassays of Aβ1-42 (Fig. 6). Since Aβ1-42 peptide is prone to aggregation [START_REF] Van Thanh Nguyen | Recent Electrokinetic and Microfluidic Strategies for Detection of Amyloid Beta Peptide Biomarkers: Towards Molecular Diagnosis of Alzheimer's Disease[END_REF], the good quality of each batch was first confirmed with MS (see Fig. S3 in the ESI). Aβ1-42 solutions were prepared freshly from the confirmed batch just before each immunoassay series. Detection in this case was made with another anti

  Fig 3C, more than 60% of NAB228 antibody (mouse IgG2a type) was digested in the first trial, which provided encouraging results. IdeZ enzyme was therefore chosen for further optimization of mouse IgG2a digestion. A two-level-three-factors (3 2 ) ½ fraction experiment was designed to optimize the digestion protocol using Minitab statistical software 17. The three factors covered (i) enzyme unit for 1μg IgG digestion (1 -3 UI), (ii) incubation time (2 to 4 hour) and

Figure captions:Fig. 1 .F(ab') 2 F(ab') 2 - 18 -Fig. 2 .F(ab') 2 F(ab') 2 Fig. 3 . 2 FFig. 4 . 3 F(ab') 2 - 21 -Fig. 5 . 3 FFig. 6 .

 122182223243221536 Figure captions: Fig. 1. Proposed strategy for characterization of density and orientation of antibodies

Acknowledgement

This work has been financially supported by the 'bourse d'excellence de l'ambassade de France' (for PhD scholarship of N.V.T. Nguyen).

Aβ1-42 antibody specific at C-terminal of this peptide, which is bound with HRP to trigger subsequently the enzymatic reaction with Quanta Red substrate followed by fluorescent detection (Fig. 6A). NAB228 grafted on ProG showed higher fluorescent signals than those obtained with tosyl-activated beads (Fig. 6B). Further results on the immunoassays of Aβ1-42 can be seen in Fig. S4 in the ESI. This observation was in good accordance with the results indicating that more antibodies were grafted on ProG beads than on tosyl-activated ones, with equivalent antibody grafting orientation (see section 3.2 above).

Employing the antibody / bead ratios optimized for NAB228, the test on immunocapture of Aβ1-42 was also extended to other antibodies specific to this peptide, i.e., 12F4 specific to Nterminal and 4G8 specific to 17-24 epitopes (Fig. S5 in the ESI). An antibody specific to Nterminal of Aβ1-42 (6E10-HRP) was employed in this case for detection of the captured peptide. Among three capture antibodies, NAB228 exhibited the best on-bead capture performance, regardless of the bead type used (tosyl-activated or ProG). The signals obtained with 12F4 and 4G8 were relatively low for both types of beads. The limit of detection was 10 nM nfor NAB228 and 20 nM for other antibodies. This implies that for each antibody, reoptimization of the antibody : bead ratio would be needed. And the optimal ratio achieved for 1 specific antibody (NAB228 in our case) cannot be generic to other ones. This remark is important, because so far one tends to follow the same antibody : bead ratio according to the established grafting protocol for different antibodies, which may lead to non-optimal immunocapture performance.

Conclusions

We developed successfully an enzyme-based approach for evaluation at same time the density and orientation of mouse IgG2a immobilized on three different magnetic beads. Using this -16approach to follow the optimization of antibody grafting conditions, more than 70 % of antibodies could be grafted on tosyl-activated and protein G beads in the right orientation. The developed method was applied for monitoring the density and orientation of anti-Aβ 1-12 antibodies on different magnetic bead supports, serving for immunoassays and fluorescent detection of this amyloid-beta peptide. The antibody NAB228 grafted on ProG beads exhibited the best performance, with the detection limit of 10 nM achieved for Aβ 1-12. The strategy using IdeZ enzyme to check the density and orientation of mouse antibodies on magnetic beads provides us a powerful tool for optimization and improvement of the performance of immunoassays and biosensing of target biomolecules on magnetic beads.

Other biosensing applications targeting different biomarkers using this enzyme-based method are envisaged to further demonstrate the significance of our work.