Coupling purification and in situ immobilization process of monoclonal antibodies to clenbuterol for immunosensor application

Accepted Manuscript Coupling purification and in-situ immobilization process of monoclonal antibodies to clenbuterol for immunosensor application Hui Cao, Min Yuan, Li Li Wang, Jing Song Yu, Fei Xu PII: DOI: Reference:

S0003-2697(15)00045-7 http://dx.doi.org/10.1016/j.ab.2015.01.022 YABIO 11966

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

9 September 2014 26 January 2015 27 January 2015

Please cite this article as: H. Cao, M. Yuan, L.L. Wang, J.S. Yu, F. Xu, Coupling purification and in-situ immobilization process of monoclonal antibodies to clenbuterol for immunosensor application, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.01.022

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Coupling purification and in-situ immobilization process of monoclonal antibodies to clenbuterol for immunosensor application

Hui Cao#, Min Yuan#, Li Li Wang, Jing Song Yu, Fei Xu* School of Medical instrument and Food Engineering, University of Shanghai for Science and Technology, P.O. Box 454, No. 516, Jungong Road, Shanghai 200093, P.R China

# These authors contributed equally to this work. *Corresponding Author: Fei Xu, PhD Email: [email protected] Tel: +86-21-55271193 Fax: +86-21-55271193 Subject: Immunological procedure Short title: Immunosensor for detection of clenbuterol

1

Abstract Clenbuterol (CL), which promotes the growth of muscular tissue and reduction of body fat in pigs and cattle, has been confirmed to be a potential hazard to human health. In this study, a monoclonal antibody to clenbuterol (CL mAb) from a hybridoma culture supernatant was purified by an aqueous two-phase system (ATPS) at different polyethylene glycol (PEG) concentrations, PEG molecular weights, pH values and NaCl concentrations. Then the CL mAb was immobilized in-situ by directly adding polystyrene microspheres (PSMS) into a PEG phase containing CL mAb. Using the immobilized antibody, an immunosensor was constructed to detect the CL residues in pork samples. The results showed that using an ATPS composed of 15% (w/w) PEG6000, 15% (w/w) phosphate and 15% (w/w) NaCl at pH 8.0, the partition coefficient was 7.24, the activity recovery was 87.86% and the purification fold was 2.88. The PEG-CL mAb-PSMS retained approximately 98% of its initial activity after 30 ml PBS washings. After 30 days of storage, the CL mAb-PSMS lost almost 75% of its activity, while the PEG-CL mAb-PSMS retained as much as 95% of its initial activity. Furthermore, the constructed immunosensor obtained recoveries of 90.5% to 102.6% when applied to pork samples spiked with CL.

Key words: aqueous two-phase system; monoclonal antibody to clenbuterol; in-situ immobilization; immunosensor; clenbuterol detection

2

1. Introduction Clenbuterol (CL) is a beta-adrenergic drug usually employed as a bronchial dilating agent for the treatment of pulmonary diseases in humans and animals [1]. Due to its growth-promoting effect involved in increasing lean muscle mass and reducing fat deposition, CL is also commonly but illegally added at high doses to the livestock feed, especially for pigs and cattle, to improve the production of lean meat [2]. Intake of CL may result in human food poisoning, including muscular tremors, tachycardia, palpitation and dizziness, hence its use has been banned in livestock feed in most countries [3]. Although China has taken measures to restrict the use of CL in livestock feed, CL poisoning still occurs frequently. A number of analytical methods have been reported for the detection of CL. Immunoassay methods based on the specific recognition of antigens by antibodies have received greater attention, especially when used as immuosensors [4]. In the immunosensor method for detection of residues, antibodies are usually immobilized as probes onto different solid surfaces by physical adsorption or covalent coupling. Immobilization allows for increased antibody-antigen binding to improve detection sensitivity [5-7]. Although the immunosensor method has been employed as a rapid, efficient

and

convenient

detection

method

for

pollution

residues,

the

commercialization of immunosensor technology has achieved only limited success, mostly because of the generation of false signals caused by instability and poor loading of the immobilized antibody. To solve these problems, many processes have been proposed for antibody immobilization. Some of the most common methods such as entrapment or physical adsorption generally suffer from antibody leaching and/or diffusion limitations during operation. While on the other hand, immobilized antibodies prepared by covalent bonding are frequently inactivated during the immobilization processes. Thus, high stability and bioactivity of the immobilized antibodies with simple immobilization processes are typically required to reduce false signals in the immunosensor. To obtain increased antibody loading and activity, Guan et al. proposed that mouse ascites, hybridoma culture supernatants and genetic engineering products 3

containing monoclonal antibody (mAb) need to be purified before immobilization [8]. However, the traditional methods reported for mAb purification, mainly chromatography-based technologies, are too expensive and complex to scale up production for increasing mAb demands. Recently, some separating technologies with simplified process were introduced [9-12], showing ATPS as an ideal purification technique for the extraction and concentration of biomolecules because of its high productivity, simplicity, short processing time, cost-effectiveness, scalability and versatility. An ATPS is usually formed by combining either two incompatible polymers like PEG and dextran, or a polymer and a salt, commonly phosphate [12]. Compared with the PEG-dextran system, PEG-phosphate system can efficiently separate proteins with different hydrophobic properties because of the phase hydrophobicity and salting-out effects. Moreover, the PEG-phosphate system is easier to operate and scale up due to lower viscosity and lower cost of the salt phase [13]. ATPS technology has successfully been applied to the separation and purification of biological products, such as proteins, nucleic acids and viruses [14-16]. However, after purification by ATPS, some technical difficulties remain, such as recovery of antibodies dispersed in the PEG phase and recycling of the PEG. Schulze and Winter et al. reported that when native macromolecules were incubated in PEG, almost no loss of activity was found [8, 17]. Bradoo et al. recovered enzymes from the phosphate phase of an ATPS by immobilizing directly on solid carriers, thus allowing the recycling of the bottom salt phase [18]. Based on these reports, an alternative process that includes the purification of CL mAb by an ATPS and its in-situ immobilization directly in the PEG phase is hereby explored. Evidently, this novelty method could allow antibody recovery and immobilization in the PEG phase simultaneously, avoid the use of other complicated steps, and allow the recycling of the top PEG phase. In present work, the objective was to investigate an effective approach for the purification and in-situ immobilization of CL mAb in an ATPS system and apply the immobilized mAb in immunosensors for detecting CL in pork samples. The schematic diagram of the experimental procedure is shown in Fig. 1. 4

Fig. 1 2. Materials and methods 2.1 Materials Hybridoma cells secreting CL mAb were cultured, and the cultured supernatant containing CL mAb was centrifuged and collected for later purification. HRP-labelled CL was prepared using EDC/NHS crosslinking as described by Roda et al., with a coupling ratio of 1.8 [19]. PEG with molecular weights of 1000, 2000, 4000, 6000, 10000 and 20000 Da were purchased from Fluka (Buchs, Switzerland) and used without further purification. HRP-conjugated goat anti-mouse IgG was provided by Agrisera (Vännäs, Sweden). 3, 3’, 5, 5’-tetramethylbenzidine (TMB) was obtained from Sigma-Aldrich. Polystyrene microspheres (PSMS) of 3 mm diameter were produced by Janus materials CO., Ltd (Nanjing, China). All other chemicals were of reagent grade or higher. 2.2 ATPS extraction and characteristics of CL mAb 2.2.1 ATPS extraction of CL mAb Aqueous PEG/phosphate systems were prepared by weighing appropriate amounts of PEG stock solution (40% w/w), phosphate buffer stock solution (40% w/w), sodium chloride (NaCl) solution, hybridoma culture supernatant containing CL mAb and water, to a final weight of 10 g. 40 (w/w) phosphate buffer solutions with different pH values were prepared by using variable mass ratios of K2HPO4 to NaH2PO4. Slight adjustments to the final pH value were performed with a 40% (w/w) solution of K2HPO4. All system components were thoroughly mixed on a vortex shaker, equilibrated at 4°C for 60 min and then centrifuged at 1500 g for 10 min to ensure separation into two phases. The top and bottom phases were then carefully separated, and the volume of each phase was measured. Samples were taken from the hybridoma culture supernatant，the top phase and the bottom phase of the system for determining protein concentration and CL mAb activity. The partition of CL mAb was described by the partition coefficient (K), the activity recovery of CL mAb in the top phase (Y) and the purification fold (PF). The partition coefficient (K) of CL mAb was defined as the ratio of total activity 5

in the top phase ( At ) to that in the bottom phase ( A b ). It was calculated as follows: K=

At Ab

The activity recovery (Y) of CL mAb in the top phase was determined as the ratio of total activity in the top phase to that in the culture supernatant of hybridoma cells before partition, and expressed as percentage. In general, high Y values indicated that most of CL mAb were partitioned to the top phase with increasing partition coefficient. Y was calculated as follows:

Y (%) =

At K = Ab + At 1 + K

The purification fold (PF) of CL mAb was calculated as the ratio of the specific activity observed in the top phase ( At Ct ) to the initial activity found in the culture supernatant of hybridoma cells before partition ( A0 C0 ).The PF value is dependent on the K values of CL mAb and contaminated proteins in the top phase and bottom phase. PF was calculated as follows: PF =

At Ct A0 / C 0

Where Ct is total protein mass in the top phase. A0 and C 0 are the total activity and total protein mass of CL mAb in the culture supernatant of hybridoma cells before partition, respectively. 2.2.2 CL mAb activity assay The activity of CL mAb was determined by an immunoassay method. Polystyrene microtiter plates (Thermo Fisher Scientific, MA, USA) were coated using 100 µl/well of CL mAb and incubated at 4°C for overnight, after which the plates were thoroughly washed with PBS-T (0.01 M phosphate buffer at pH 7.2 containing 0.1% v/v Tween-20). The plates were then sealed with 3% (w/v) gelatin at 37°C for 2 h. After 2h, the plates were washed with PBS-T and incubated with HRP-conjugated goat anti-mouse IgG for 1 h at 37°C. At the end of the incubation period, plates were washed three times and incubated with 50 µl/well of TMB substrate solution at room temperature. The colour reaction was stopped by adding 50 µl/well of 2 M sulphuric acid. The absorbance at 450 nm representing the CL mAb activity was measured 6

using a microplate reader (Thermo Fisher Scientific, MA, USA). 2.2.3 Quantitative analysis of protein Protein mass was determined by the Bradford method using a Coomassie assay reagent [20]. To avoid interference from other components within each phase, samples were analysed against blanks containing the same phase composition but without adding hybridoma culture supernatant. Bovine serum albumin (BSA) was used as a protein standard. Absorbance was monitored at 595 nm in a microplate reader (Sunnyvale, CA, USA). 2.2.4 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed with 12% separating gel to evaluate the purity of samples from the top and bottom phases of the ATPS. Samples were diluted in 62.5 mM Tris-HCl buffer at pH 6.8, containing 4% (w/v) SDS, 0.01% (w/v) bromophenol blue and 10% (w/v) glycerol, and then, the samples were denatured at 100°C for 5 min. The electrophoresis was run at 110 V and 36 mA for 75 min. The gel was stained with a buffer solution consisting of 0.05% (v/v) Coomassie Brilliant Blue G-250, 30% (v/v) methanol and 10% (v/v) acetic acid. 2.3 In-situ immobilization and characteristics of immobilized CL mAb 2.3.1 In-situ immobilization of CL mAb CL mAb was immobilized by directly adding PSMS carriers into the top PEG phase of the ATPS or the culture supernatant of hybridoma cells. After simple pre-treatment, appropriate amounts of the PSMS carriers were immersed into 20 ml of CL mAb-enriched PEG phase or the culture supernatant and shaken at a constant speed for at least 12 h at 4°C to adsorb CL mAb. The two types of immobilized CL mAb obtained were washed with distilled water and then stored at 4°C for later analysis. The immobilized CL mAb prepared from CL mAb-enriched PEG phase was sealed with 3% (w/v) gelatin at 37°C for 2 h, and then packed into Tygon columns (3 mm×50 mm). Both ends of the column were sealed with rubber plugs attached to filter cloth of certain pore diameters. The packed columns were stored at 4°C for evaluation in an immunobiosensor. 7

2.3.2 Activity assay for immobilized CL mAb The activity of the immobilized CL mAb was determined by ELISA method. 100 mg of immobilized CL mAb was placed into test tubes and fully immersed in 3% (w/v) of gelatin for 2 h. After washing with PBS-T, the immobilized CL mAb was incubated with the HPR-labelled goat anti-mouse IgG solution for 1 h at 37oC. At the end of the incubation period, the immobilized CL mAb was washed three times and incubated with 100 µl of TMB substrate solution at room temperature. The colour reaction was stopped by adding 100 µl of 2 M sulphuric acid. Microspheres of immobilized CL mAb were removed from test tubes, and the absorbance of the remaining reaction solution was measured at 450 nm using a microplate reader. 2.3.3 Surface characteristics of immobilized CL mAb and PSMS carrier The surface morphology and roughness of the immobilized CL mAb and PSMS carrier

were

observed

by

atomic

force

microscopy

(AFM,

Shimadzu

HD-WET-SPM-9500J3, Kyoto, Japan). The AFM images were obtained in contact mode with a triangular cantilever (force constant of 0.05 N/m) supporting an integrated Si3N4 pyramidal tip. Second-order flattening was applied to all images to remove the background slope. The average roughness (RMS value) was calculated from the AFM image data. 2.3.4 Storage stability and operational stability of the immobilized CL mAb The two types of immobilized CL mAb freshly prepared by the procedure described in Section 2.3.1 were stored in phosphate buffer (50 mM, pH 6.0) at 4°C for 30 days. Their residual activities were tested every 5 days to evaluate storage stability. Immobilized CL mAbs were packed into Tygon columns, and their operational stabilities were measured. PBS was continuously pumped through the packed column at a flow rate of 1 ml/min using a peristaltic pump (THOMAS, Germany) for 30 min, and the residual activities of the antibodies were determined to evaluate their operational stability. 2.4 Construction of an immunosensor system An immunosensor system based on flow-injection analysis (FIA) consisted of a peristaltic pump (Shimadzu LC-10Ai, Kyoto, Japan), a six-port valve (Rheodyne 8

7725, Cotati, California, USA), an immuno-reactor containing one immobilized antibody column and one reference column, and a spectrophotometric detecting unit. Using the immunosensor system, the CL content in samples could be determined by the method of direct competitive ELISA. This method has only one incubation step, while indirect ELISA usually involved two separate immunochemical reactions as well as the use of a labelled secondary antibody [21]. In immunosensor method, 20 µl of the sample solution were mixed with 20 µl of HRP-labelled CL in a 40-fold dilution, and then, the mixture was injected into the immunosensor system. As the mixture flowed through the immuno-reactor, the CL present in the sample solution and the HRP-labelled CL competitively reacted with immobilized CL mAb based on the principle of direct competitive ELISA. After washing away the unbound CL, the substrate solution (TMB) was injected into the system and hydrolysed by the HRP bound on the immobilized antibody column. The absorption detected by the spectrophotometric unit represented the CL contents in samples. The reference column and the immobilized antibody column were identical, except that the reference column was full of carrier without antibody and was used to eliminate unspecific interferences which were not produced by the antibody reaction. 2.5 Sample preparation Non-contaminated pork samples were purchased from the local supermarket and finely ground in a household meat grinder. Samples of ground pork weighing 15 g were spiked with 150 µl of CL solution at concentrations ranging from 0.05 µg/ml to 0.8 µg/ml. The spiked pork samples were fully mixed using a homogenizer for 2 min and extracted with 50 ml of 0.10 mol/l HCl by shaking in an incubator for 30 min at 80oC. The samples were centrifuged for 10 min at 6000 g, and the resulting supernatants were collected for recovery studies. The relative standard deviation (RSD) was used to evaluate the precision of the assay. 2.6 Statistical analysis Data were expressed as the mean ± SD. Statistical significance was determined by one-way analysis of variance with Dunnett’s post-hoc test using SPSS 13.0 software. P-values less than 0.05 were considered to be significant. 9

3. Results and discussion

3.1 Optimization of PEG/ phosphate ATPS extraction Partition of a target compound in the PEG/ phosphate system depends on its intrinsic and extrinsic properties. Intrinsic properties include size, electrochemical properties,

surface

hydrophobicity

and

hydrophilicity,

and

conformational

characteristics of target compound. Extrinsic properties include type; molecular weight and concentration of components forming the ATPS; ionic strength and pH of the ATPS; and extraction temperature, among other properties [22]. By manipulating these extrinsic properties, it was possible to change the partition behaviour of the target compound. Thus, we investigated the effect of the pH, NaCl concentrations, PEG molecular weight and PEG concentrations on the partition efficiency of CL mAb in the PEG/ phosphate ATPS. 3.1.1 Effect of the pH of phosphate phase on the CL mAb partition efficiency It is well known that biomolecule partition in an ATPS is influenced by the pH of the system. The pH of the system generally affects the partitioning behaviour of proteins by changing the electrical charge of the target protein itself. Thus, in order to improve the extraction of CL mAb to the top PEG phase, systems containing 10% (w/w) PEG 1000, 15% (w/w) phosphate and 10% NaCl (w/w) with variable pH (6.0-10.0) were investigated. As shown in Fig. 2, the partition parameters of CL mAb increased with increasing pH from 6.0 to 9.0, while above pH 9.0, the partition parameters decreased. At pH 8.0, the partition parameters reached their highest values, with purification fold of 1.54, partition coefficient of 2.91 and activity recovery of 74.42%. Accordingly, pH 8.0 was selected for further study. Fig. 2 The results obtained could be explained by the fact that the iso-electric point of the CL mAb was near 8.0 [23]. Far away from the isoelectric point, the CL mAb carried greater net positive or negative charge, which favoured the partitioning of the antibody to the bottom phase due to electrostatic interactions between the antibody and the potassium-phosphate phase. Near the isoelectric point, CL mAb had a smaller net charge, resulting in the target antibody being attracted to the PEG-rich 10

top phase by hydrophobic interaction. 3.1.2 Effect of NaCl concentrations on the CL mAb partition efficiency Previous studies have shown that the neutral salt concentration had the strongest effect of all of the extraction performance parameters of an ATPS [24]. Therefore, the partition behaviour of CL mAb in an ATPS was investigated with variable NaCl concentrations at pH 8.0. The partition efficiency of CL mAb increased with increasing NaCl concentrations (Fig. 3). When the NaCl concentration was increased to 15% (w/w), the partition coefficient (4.27) and purification fold (1.91) were improved significantly (P<0.05), and > 80% of the total CL mAb was able to be recovered. Maximum partition efficiency was achieved at 20% (w/w) NaCl, which gave a partition coefficient of 4.88, a purification fold of 2.02 and an activity recovery of 83.01%. This behaviour is in agreement with the findings of Andrews et al. in 1996, whose study showed that the addition of NaCl to a polymer/salt ATPS could shift the preferential partitioning of IgG from the salt-rich bottom phase to the PEG-rich top phase [25]. Fig. 3 Hydrophobic interactions between the CL mAb and the phase-forming components were probably responsible for the selective partition of the antibody to the top phase. When NaCl was added to the system, there was a decrease in the total mass of free water [26]. In fact, as the NaCl concentration increased to 15%, the volume of the top phase decreased by 48%, while the volume of the bottom phase decreased by only 6%, making the PEG-rich phase more concentrated and hence more hydrophobic. Therefore, less free water would be available for antibody solvation, resulting in the exposure of hydrophobic patches on the antibody surface, in turn promoting the antibody’s hydrophobic interactions with PEG [24, 27]. In addition, the partitioning of CL mAb to the phosphate-rich phase might no longer be thermodynamically favoured due to the addition of NaCl, and partition to the PEG-rich phase might be more favourable. The purification fold and activity recovery was not significantly different between 15% and 20% NaCl concentrations (P>0.05), hence 15% NaCl was selected for the next study. 11

3.1.3 Effect of PEG molecular weight (MW) on the CL mAb partition efficiency To study the effect of PEG MW on the partitioning efficiency of the CL mAb, partition experiments were performed in different aqueous two-phase systems by varying the PEG MW (1000, 2000, 4000, 6000, 10000 and 20000 Da) and maintaining the phase compositions (PEG/phosphate/NaCl) in constant proportion (10/15/15 %) at pH 8.0. As shown in Fig. 4, as the PEG Mw increased from 6000 to 20000, the PF, Y and K values decreased significantly (P<0.05). This observation is similar to that reported by Almedia et al., which showed that increasing PEG MW led to a decrease in the free volume available in the top phase and an increase in the exclusion effect. The polymer thus adopted a more compact conformation with intramolecular hydrophobic bonds and hindered the partition of target antibodies into the top phase [28-30]. No significant difference was noticed in the PF, Y and K (P>0.05) at PEG MW of 1000 to 6000. This result might be explained by the fact that target antibody partition is mainly dependent on the PEG-protein interaction in low Mw PEG [26]. Therefore, the PEG MW was kept at 6000 for the next experiments. Fig. 4 3.1.4 Effect of the concentration of PEG 6000 on the CL mAb partition efficiency The partitioning behaviour of CL mAb in the ATPS was studied within the 10-15% (w/w) of PEG concentrations. The phosphate concentration, NaCl concentration and pH were maintained at 15%, 15% and 8.0. As shown in Fig.5, the PEG concentration had a significant influence on the partitioning of CL mAb in the ATPS (P<0.05). At 15% PEG 6000, the partition coefficient reached its highest value of 7.24, with a purification fold of 2.88 and an activity recovery of 87.86%. When a lower PEG concentration was used, the partition coefficient, activity recovery and purification fold decreased accordingly. Fig5 The explanation for this observation could be that the hydrophobicity differences between the top and bottom phases would be enhanced with increasing PEG concentration, and more CL mAb would be partitioned to the top phase [31]. However, a further increase of the PEG concentration might result in increased viscosity and 12

increased interfacial tension between the two phases of the ATPS. Therefore, resistance to the partitioning of CL mAb to the top PEG phase would be increased. The PEG concentration was kept at 15% for the next experiments [32]. 3.2 SDS-PAGE of proteins partitioned by the ATPS Using the optimized extraction conditions, proteins in the hybridoma culture supernatant were partitioned into the top and bottom phases of the ATPS, and the SDS-PAGE of the proteins is shown in Fig. 6. CL mAb was enriched in the top phase, shown by the two distinct bands approximately 50 and 30 kDa, corresponding to the heavy and light chains of the antibody [33]. Most of the contaminant proteins were partitioned to the bottom phase. This result showed that ATPS extraction was a rapid and efficient method for separating CL mAb from the hybridoma culture supernatant. Fig. 6 3.3 Comparison of two types of immobilized CL mAb in operational and storage stabilities The operational stabilities of the two types of immobilized CL mAb were compared, and the results are shown in Fig. 7. The CL mAb-PSMS lost more than 50% of its initial activity after 30 ml PBS washings. However, at the same washing condition, the PEG-CL mAb-PSMS retained approximately 98% of its initial activity, indicating that these antibodies were fixed permanently in the matrix. The storage stability of PEG-CL mAb-PSMS and CL mAb-PSMS was also investigated. As shown in Fig. 8, after 30 days of storage, the CL mAb-PSMS lost almost 75% its activity, while the PEG-CL mAb-PSMS still retained as much as 95% of its initial activity. In addition, the initial activity of the PEG-CL mAb-PSMS was significantly higher than that of the CL mAb-PSMS (P<0.05). These results suggested that the stability and loading of immobilized CL mAb were improved obviously due to the introduction of PEG during the in-situ immobilization. Fig. 7 and Fig. 8 AFM was applied to investigate the surface characteristics of PEG-CL mAbPSMS，CL mAb-PSMS and PSMS. The two immobilized CL mAb surfaces were significantly rougher than the PSMS surface (P<0.05), and most of the peaks of the 13

immobilized CL mAb surfaces were irregular and much higher than those on the PSMS surface (Fig. 9a-c). This result demonstrated that the CL mAb immobilization onto the PSMS carriers made the surface morphology look different. AFM also showed that the PEG-CL mAb-PSMS surface displayed a clear network micelle structure (Fig. 9d), and the surface roughness was significantly lower than that of the CL mAb-PSMS surface but higher than that of the PSMS surface (P<0.05). These observations suggested that the CL mAb molecules of the PEG-CL mAb-PSMS were densely packed into the network micelle formed by the PEG, therefore, their activity was maintained during storage and operation by preventing the antibodies from dispersing. This retention of activity is of great importance to the sensitivity and the reproducibility of biosensors. Furthermore, other researchers found that the protective mechanism of PEG is probably due to a positive effect on the antibody stability, or a stronger resistance to antibody inactivation, provided by the hydroxyl groups of the PEG [34, 35]. Fig. 9 3.4 Response of the constructed immunosensor to CL An FIA system was constructed, and its response to CL was determined using the immobilized CL mAb as an immuno-reactor with a CL concentrations range of 0.01 to 1000 ng/ml. As shown in Fig. 10, the response signal decreased linearly with increasing CL concentrations from 0.05 to 25 ng/ml because a competitive immunoassay mode was used. A linear regression equation of y= - 0.1258Ln(x) +0.6219 was obtained with a correlation coefficient (R2) of 0.9874, and the detection limit (calculated from multiplying the background signal by three) was 0.0859 ng/ml. Compared with the detection limit of 20 ng/ml for other immunosensor, the detection limit of 0.26 ng/ml for capillary electrophoresis and the detection limit of 0.05 ng/ml for LC-MS, the immunosensor method using in-situ immobilized CL mAb as sensing elements was found to be highly sensitive for detecting CL [36-38]. To further investigate the reproducibility of this method, we repeatedly tested three CL standards at different concentrations (0.10 ng/ml, 0.20 ng/ml and 1.0 ng/ml) five times in parallel. The RSD was all less than 7.41%, suggesting that the proposed method 14

exhibited acceptable reproducibility (data not shown). Fig. 10 3.5 Measurement of CL in pork samples To further evaluate the feasibility of the proposed method, 50 pork samples collected from different markets were analyzed by immunosensor. No CL residues were detected in all collected samples. So five different concentrations of CL were spiked in the pork samples by using the standard addition method and their recoveries were determined. As shown in Table 1, the recoveries of CL in the samples and the RSD were in the range of 90.5-102.6% and 2.6-4.8%, respectively, suggesting that the proposed immunosensor could be feasible for the analysis of CL in real samples.

Table 1 Conclusion

In this report, we described the purification of CL mAb by an ATPS and its in-situ immobilization directly in the PEG phase. This novel process could allow antibody recovery and immobilization in the PEG phase simultaneously, avoid the use of other complicated steps, and allow recycling of the top PEG phase. In addition, a rougher surface and network micelles formed by PEG helped the antibody to maintain its activity during storage and operation by modifying the properties of the matrices. To further improve on this process, it can be considered in future study to use the process coupled counter-current ATPS and in-situ immobilization for separation and immobilization of CL mAb with the advances in ATPS technology. This method might help to improve the partition efficiency and phase components recycling, and provide better scale-up and continuous operation capabilities as counter-current ATPS can achieve a multistage separation of biomolecules [39, 40]. The immunosensor, which was constructed based on the immobilized CL mAb coupled with FIA, exhibited a sensitive response to CL over the concentration range of 0.05 to 25 ng/ml, with a lower detection limit of 0.0859 ng/ml. Furthermore, the proposed immunosensor was applied to the quantitative determination of CL in pork samples with satisfactory recoveries ranging from 90.5% to 102.6%. This technique, 15

with good reproducibility, could be a very attractive alternative to the existing methods for the detection of CL. Acknowledgments

This work was financially supported by the National Natural Science Funds (No. 31101283), National High Technology Research and Development Program of China (863 Program) (No. 2013AA102207) and National Natural Science Funds (No. 31201421). Reference

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Figure Captions

Fig. 1 Schematic diagram of the experimental procedure Fig. 2 Effect of pH on the CL mAb partition parameters using a PEG/phosphate ATPS Fig. 3 Effect of NaCl concentration on the CL mAb partition parameters using a PEG/phosphate ATPS Fig. 4 Partition of CL mAb in different molecular weights PEG/ phosphate ATPS Fig. 5 Effect of PEG concentrations on the CL mAb partition parameters using a PEG/phosphate ATPS Fig. 6 SDS-PAGE patterns of the top and bottom phases from ATPS partitioning. Lane M- molecular weight markers, Lane A- top phase from the extraction step, Lane Bbottom phase from the extraction step Fig. 7 Operational stability of immobilized CL mAb-PSMS and PEG-CL mAb-PSMS Fig. 8 Storage stability of immobilized CL mAb-PSMS and PEG-CL mAb-PSMS Fig. 9 AFM images of PSMS (a), CL mAb-PSMS (b) and PEG-CL mAb-PSMS (c), and resulting surface roughness (d) of PSMS (1), CL mAb-PSMS (2), and PEG-CL mAb-PSMS (3) Fig. 10 (a) Calibration curve for the competitive immunoassay of the CL standard solution with the concentrations of 0.01-1000 ng/ml, (b) calibration curve of the CL standard solution with concentrations of 0.05-25 ng/ml. B/B0 represents the activity intensity ratio of different concentrations of CL to 0 ng/ml standard solutions

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Table 1 Application of the proposed immunosensor for CL detection in pork samples Added (µg/kg) Found (µg/kg) Recovery (%) RSD (%) (n=4) 0.5 0.51 102.0 4.3 1 0.92 92.0 3.1 2 1.81 90.5 4.8 4 3.73 93.3 2.6 8 8.21 102.6 3.7

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