Preparation of inert magnetic nano-particles for the directed immobilization of antibodies

Biosensors and Bioelectronics 20 (2005) 1380–1387

Preparation of inert magnetic nano-particles for the directed immobilization of antibodies Manuel Fuentes, Cesar Mateo, J.M. Guisán1 , Roberto Fernández-Lafuente∗ Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain Received 30 January 2004; received in revised form 9 June 2004; accepted 9 June 2004 Available online 13 July 2004

Abstract Various activated supports (cyanogen bromide, glutaraldehyde, epoxy-chelates, primary amino) were evaluated for the immobilization of IgG anti-horseradish peroxidase. Cyanogen bromide and glutaraldehyde supports greatly reduced the recognition capacity of the antigen, probably due to the incorrect orientation of the antibody on the support. Hetero-functional epoxy-chelate and immobilization by the sugar chain on primary amino groups had little effect on high recognition of the antigen (near to the theoretically expected value). However, the immobilization by the sugar chain resulted in a higher adsorption rate of horseradish peroxidase, possibly due to a favourable orientation on a flexible spacer arm). Antibodies immobilized on aminated surfaces showed two major drawbacks. Firstly, the biological activity of the immobilized antibody sharply decreased over several days when stored at low ionic strength, although this effect could be partially reversed by incubation at high ionic strength. Secondly, a high level of non-specific proteins adsorption on the support surface was observed. Both problems could be successfully resolved by controlling the coating of the support with aldehyde-aspartic-dextran. We propose that the loss of biological activity was related to the ionic adsorption of the immobilized antibody on the support surface, leading to a blocking of the recognition areas. This optimized protocol was applied to the immobilization of IgG anti-horseradish peroxidase from rabbit on magnetic nano-particles. A 10 ␮g preparation of nanoparticles was able to capture more than 75% of the 0.1 microgram of recombinant horseradish peroxidase present in 10 L of crude protein extract (1 g/L) from Escherichia coli. © 2004 Elsevier B.V. All rights reserved. Keywords: Magnetic nano-particles; Anti-horseradish peroxidase; Antibodies

1. Introduction For many years, the immunological antibody-antigen interaction has been used to determine concentrations of analytes (e.g., analytical chemistry, food technology, environment, medical diagnostics). There have been several recent developments aimed at improving the performance of immuno-sensors: antigen transport, reporter mechanisms, and readout systems (Nice and Catimel, 1999; Pancrazio et al., 1999; Nishi et al., 2000; Partington et al., 1999; König and Grätzel, 1994). However, a critical requirement ∗ Corresponding author. Tel.: +34 91 585 48 09; fax: +34 91 585 47 60. E-mail addresses: [email protected] (J.M. Guis´an), [email protected] (R. Fern´andez-Lafuente). 1 Co-corresponding auhtor.

0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.06.004

is the development of suitable immobilization protocols that yield active antibodies and inert surfaces that minimise non-specific adsorption of proteins. Several studies have compared methods of antibody attachment to biosensor surfaces for the development of immuno-surfaces for biosensors, including physicochemical adsorption and covalent attachment (Danczyk et al., 2003; Ahluwalia et al., 1991; Bhatia et al., 1993; Shriver-Lake et al., 1997; Shinkai, 2002; Koneracka et al., 1999; Aslam and Dent, 1998; Weetall and Lee, 1989). Covalent binding may increase antibody stability and control the available protein binding sites, but incorrect immobilization orientation may lead to complete loss of the biological activity of the antibody (Zull et al., 1994; Babacam et al., 2000). It has recently been proposed (Danczyk et al., 2003) that adsorption on immobilized protein A may be the favoured solution for antibody immobilization (Anderson et al., 1997).

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Magnetic nanoparticles are now commercially available and have special relevance in analytical chemistry because they permit a rapid purification in one step-concentration of analytes by the possibility of capturing the magnetic nanoparticles using magnetic fields (Bangs, 1996; Meza, 1997; Shinkai, 2002; Haukanes and Kvam, 1993; Koneracka et al., 2002; Martins, 1998) that could be “the beginning” or “the end” in any analytical protocol. The immobilization of antibodies on this kind of materials will be the final aim of this work. 1.1. Antibody immobilization The immobilization of biologically active molecules requires good steric accessibility to active binding sites (Turkova, 1999). Typically, when the antibody is immobilized, the recognition sites (Fab region) should be oriented away from the support surface to preserve the full function of the antibody. This paper focuses on comparing immuno-surface functionality for three covalent protein immobilization techniques. Each of these techniques utilizes a different method of attaching proteins to the surface of support. The first is the immobilization of the antibody on conventional immobilization supports via the protein amino groups using, for example, glutaraldehyde (Bickerstaff, 1997) or cyanogen bromide activated supports (March et al., 1974). The second is the use of a new generation of hetero-functional supports, iminodiacetic-metal chelate-epoxy (IDA-metal chelate-epoxy) that bind the protein via high density His residues (Mateo et al., 2000, 2001a,b; Pessela et al., 2003). It has been reported that such an area exists in the Fc region of immunoglobulins (Chaga, 2001; Gaber-Porekar and Menart, 2001; Lu et al., 1996). The third is via the sugar chain (Shriver-Lake et al., 1997; Tijseen and Kurstak, 1984). The use of amino supports with very low pKa values (Fernández-Lafuente et al., 1993) and the mild oxidation of the sugar chains to generate aldehyde groups (Abraham et al., 1991; Sanderson and Wilson, 1971) is proposed. 1.2. Problems related to the non-specific adsorption of proteins Another important parameter to consider when preparing a suitable biosensor is the presence of a fully inert surface in order to prevent non-specific protein adsorption that may yield a false positive signal. This problem is greater where the target is a very low concentration protein and where the support may become fully blocked with non-specific proteins, thus preventing the specific adsorption. We propose the use of dextran-coated support surface after antibody immobilization. This technique has been shown to reduce protein adsorption (Mateo et al., 2001a,b; Patent, 2004).

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2. Materials and methods 2.1. Materials Cross-linked 4% agarose and monoamino-N-aminoethylagarose (MANAE-agarose) were supplied by Hispanagar SA (Burgos, Spain). Eupergit 250 was supplied from Röhm Pharma (Darmstadt, Germany). Bromocyanogen Sepharose was from Pharmacia (Uppsala, Sweden). Glutaraldehyde support was prepared from MANAE-groups as described (Bickerstaff, 1997). IDA-Cu support was prepared from Eupergit 250 as previously described (Armisen et al., 1999; Mateo et al., 2002). Polyclonal anti-horseradish peroxidase (developed in Rabbit), and horseradish peroxidase (HRP) were purchased from Sigma (Illinois, USA). Polyclonal IgG anti-horseradish peroxidase (anti-HRP) was purified by passage through an affinity column (Lane, 1988) of HRP immobilized on glyoxyl-agarose; HRP was purified by passing the commercial preparation through an affinity column (Lane, 1988) of anti-HRP immobilized on MANAE-agarose. Magnetic particles EM/100-30 were supplied by Estapor (Merck Co., France). Dextrans (various molecular weights) from Leuconostoc mesenteroides, ethylendiamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and 2,2 -azino-bis(3-ethylbenz-thiazoline-6sulfonic acid) (ABTS) were from Sigma (Illinois, USA). Dextran-aldehyde and aspartic-aldehyde-dextran (various molecular weights and degrees of activation) (Penzol et al., 1998) were prepared as reported (Fuentes et al., 2004; Patent, 2004). Proteins extracts from Escherichia coli and Acetobacter turbidans (nucleic acid free) were a kind gift from Prof. Berenguer (CBM, UAM, Madrid). Other reagents were all of analytical grade. 2.2. Methods 2.2.1. Determination of horseradish peroxidase activity Horseradish peroxidase (HRP) activity was determined using H2 O2 as the oxidizing substrate and ABTS as the reducing substrate. Activity was followed spectrophotometrically by recording the increase in the absorbance at 430 nm. The reaction mixture contained 1 mM ABTS and 1 mM H2 O2 in 50 mM sodium phosphate buffer at pH 6 and 25 ◦ C. The activities of enzymes are given in ␮mol of oxidized substrate/min/mg protein under the specified conditions. The experiments were carried out at least in triplicate, yielding an experimental error of less than 5%. 2.2.2. Oxidation of immunoglobulin G anti-horseradish peroxidase (anti-HRP) from rabbit IgG anti-HRP was incubated with 10 mM sodium periodate at 4 ◦ C. After 2 h, the oxidized immunoglobulin was dialyzed against distilled water at 4 ◦ C. It has been reported that this treatment retains the full functionality of the antibodies (Abraham et al., 1991).

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2.2.3. Coating the supports with aldehyde-dextran Hundred milligrams of dextran-aldehyde was added to 10 mL of activated amino support (agarose or magnetic nano-particles; Bickerstaff, 1997) suspended in 80 mL of 100 mM phosphate buffer, pH 7.5 and the suspension gently stirred at room temperature. After 16 h the composites were reduced by addition of solid sodium borohydride until a concentration of 10 mg/mL at pH 10 was reached. The reaction was left to proceed under moderate stirring for a further 2 h. The composites were then washed with 100 mM sodium acetate, pH 4, 100 mM sodium borate, pH 9, 1 M sodium chloride, and finally with an excess of distilled water to eliminate any dextran adsorbed on the support. 2.2.4. Coating the supports with aldehyde-aspartic-dextran Different amounts of aldehyde-aspartic-dextran (from 10 to 100 mg) were added to 10 mL of activated primary amino supports (agarose or magnetic nano-particles) suspended in 80 mL of 100 mM phosphate pH 7.5, and the suspension gently stirred at room temperature. After 16 h, the composites were reduced by adding solid sodium borohydride to a concentration of 10 mg/mL at pH 10. The reaction was left to proceed under mild stirring for 2 h. The composites were then washed with 100 mM sodium acetate, pH 4, 100 mM sodium borate, pH 9, 1 M sodium chloride, and finally with an excess of distilled water to eliminate any aspartic-dextran adsorbed on the support (Fuentes et al., 2004). 2.2.5. Antibody immobilization onto different supports 2.2.5.1. MANAE-agarose. Oxidized anti-HRP (10 mg) in 150 mM sodium phosphate buffer pH 7.5 was incubated with 2 mL of MANAE-agarose 4BCL at 4 ◦ C for 14 h. The Schiff’s bases formed and the remaining aldehyde groups were reduced by addition of sodium borohydride until final concentration of 1 mg/mL at pH 8.5 and 4 ◦ C. The preparation was then washed extensively with distilled water.

2.2.5.4. Amino magnetic nano-particles. Modification of magnetic particles with ethylendiamine. Ten milligrams per milliliter of magnetic particles (with carboxylic groups) was incubated (during 90 min) with 1 M ethylendiamine pH 4.75. Solid EDCI was then added to a final concentration of 10 mM and the reaction allowed to proceed for 90 min before extensive washing with distilled water. Immobilization of IgG anti-HRP. Oxidized anti-HRP (10 mg) in 150 mM sodium phosphate buffer pH 7.5 was added to 2 mL of amino magnetic particles (10 mg/mL) at 4 ◦ C and incubated overnight. The Schiff’s bases formed and the remaining aldehyde groups were reduced by addition of solid sodium borohydride until final concentration 1 mg/mL at pH 8.5 and 4 ◦ C. The preparation was then washed extensively with distilled water. In all cases, the immobilized anti-HRP was determined by quantitating the difference in protein concentration in the supernatant before and after immobilization, using the Bradford method (Bradford, 1976). The experiments were carried out at least in triplicate, typically yielding an experimental error of less than 5%. 2.2.6. Determination of the biological activity of immuno-preparations Different immobilized antibody preparations were incubated in the presence of a solution of 0.5 mg/mL HRP in 25 mM sodium phosphate buffer pH 7 for different times at room temperature (Mishell and Shiige, 1980). The extent of antigen-antibody binding was determined by measurement of the HRP activity adsorbed on the different immobilized antibody-derivatives as described above. The experiments were carried out at least in triplicate, typically yielding an experimental error of less than 5%.

2.2.5.2. Eupergit-epoxy-IDA-Cu. Ten milligrams of anti-HRP in 50 mM sodium phosphate buffer pH 7 was added to 2 mL Eupergit-IDA-Cu. After 72 h the remaining epoxy groups were blocked by incubation with 3 M Glycine, pH 8.5 for 24 h, and the matrix washed with distilled water.

2.2.7. Study of the loading capacity of amino magnetic nano-particles Different amounts of anti-HRP IgG in 150 mM sodium phosphate buffer pH 7.5 were added to 2 mL volumes of amino magnetic particles (10 mg/mL) at 4 ◦ C, and incubated overnight. The amount of antibody immobilized was determined by the Bradford method (Bradford, 1976). The Schiff’s bases formed were reduced by addition of 1 mg of sodium borohydride per milliliter of solution at pH 8.5 and 4 ◦ C. The preparation was then washed extensively with distilled water.

2.2.5.3. BrCN—agarose or glutaraldehyde agarose. Ten milligrams of anti-HRP was incubated at pH 7.5 in 150 mM sodium phosphate buffer with 2 mL cyanogen bromide or glutaraldehyde supports as described by the manufacturer (Technical documentation Pharmacia (Uppsala, Sweden)).

2.2.8. SDS-PAGE analysis Samples of antibody-immobilized magnetic nano-particles were analysed by SDS-PAGE as described by Laemmli (1970) using an SE 250-Mighty small II electrophoretic unit (Hoefer Co.) with of 12% polyacrylamide gels. Gels were silver stained. Low molecular weight markers (14–94 kDa) from Pharmacia were used.

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Table 1 Biological recognition capacity of different IgG anti-horseradish peroxidase derivatives IgG anti-HRP immobilized on

Immunoactivity expressed (%)a

Cyanogen bromide support Glutaraldehyde support Amino support epoxy-IDA-Cu support

20 25 >90 >90

Immunoactivity of IgG anti-HRP immobilized onto different activated surfaces was determined as described in Section 2.2. a Ten milligrams of IgG equivalence 0.25 mg of purified horseradish peroxidase.

3. Results 3.1. Immobilization of antibodies onto different activated surfaces Purified IgG anti-HRP was immobilized on supports activated with different reactive groups (MANAE, glutaraldehyde, chelate-epoxides and cyanogen bromide). In all cases, protein could not be detected in the supernatant, suggesting that the immobilization of the antibody was quantitative. The recognition capacity of the different immobilized preparations was then studied. When the antibodies were immobilized on supports activated with glutaraldehyde and cyanogen bromide groups, the antibodies almost had no capacity to recognize HRP (Table 1), suggesting that the orientation of the antibodies on these supports was not adequate. This result is not surprising as these supports generally interact with amino-terminal groups, which in antibodies are mainly found in the Fab region (Amzel and Poljak, 1979; Amit et al., 1986).

Fig. 1. Adsorption course of peroxidase by different immobilized IgG anti-HRP preparations. Experiments were performed as described in Section 2.2. Circles: using IgG anti-HRP immobilized via sugar chain. Squares: using IgG anti-horseradish peroxidase immobilized via His residues.

Conversely, when antibodies were immobilized via highest density His regions (found in the Fc region) (Amzel and Poljak, 1979; Amit et al., 1986) using epoxy-chelate supports or the oxidized sugar chains of MANAE-supports, they retained almost unaltered binding affinity (Table 1) (Scheme 1). A comparison of the capture rates of HRP by the different immobilized antibodies showed that HRP adsorption was much more rapid using oxidized anti-HRP immobilized on MANAE-agarose. This may suggest that even although the HRP could access the Fab region of the antibody immobilized via His residues, steric hindrance might reduce the kinetics of the binding process (Fig. 1). Immobilization via the sugar chain, which acts as a natural spacer arm (Shriver-Lake et al., 1997; Abraham et al., 1991),

Scheme 1. Immobilization of IgG anti-HRP on different activated supports.

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Table 2 Effect of coating in the ionic adsorption of proteins on MANAE-agarose Support

Agarose Agarose Agarose Agarose

Percentage of adsorbed protein at pH 7 amino amino + dextran monolayer amino + dextran bilayer amino + dextran trilayer

10 min

1h

24 h

40 14 0 0

50 15 0 0

75 18 0 0

The ionic adsorbed protein was determined by the Bradford method as described in Section 2.2.

did not apparently inhibit either the extent or rate of the antibody-antigen recognition process. 3.2. Non-specific interactions 3.2.1. Coating supports with aldehyde-dextran The non-specific ionic adsorption of proteins to aminated supports is a significant problem, since amino surfaces exhibit a very high level of non-specific proteins adsorption. MANAE-agarose is an excellent ionic exchanger; approximately 70% of the proteins present in a crude cytoplasmic extract of E. coli could be adsorbed at pH 7 and low ionic strength onto the support surface (Table 2). The coating of this surface with aldehyde-dextran (MW = 282 kDa) reduced this percentage to 15%. A further coating of the previously modified support using a smaller dextran (MW = 72 kDa) effectively eliminated the ionic adsorption of the proteins onto the amino support (Table 2). However, when trying to perform this strategy on the support with immobilized anti-HRP, even a single coating with aldehyde-dextran (20 kDa) reduced the recognition capacity dramatically. Changes in the size of the dextran, degree of support activation or modification conditions did not significantly alter the results. One possible explanation was that the dextran aldehyde groups were reacting with terminal amino groups in the region Fab of the antibody, destroying its recognition capacity. 3.2.2. Coating of the aminated support surface with aldehyde-aspartic-dextran To overcome this problem, we decided to use aldehydeaspartic-dextran (20 kD) as a hetero-functional polymer. This strategy is based on the large difference between an electrostatic adsorption (very rapid) and a covalent reaction between aldehydes and amino groups (significantly slower). Similar strategies have been used to direct the immobilization of proteins on other supports (Mateo et al., 2000, 2003; Pessela et al., 2003). Thus, the poly-aspartic polymer is rapidly adsorbed on the positively charged surface and then reacts covalently with nearby amino groups of the support, limiting antibody modification (Scheme 2). However, it is essential that only sufficient aspactic-aldehyde-dextran to cover the support surface is used. Excess aspartic-aldehyde-dextran could react with the antibody and promote its inactivation. This may be

Fig. 2. Effect of the coating on the stability of IgG anti-HRP immobilized on aminated supports. Different IgG anti-HRP preparations immobilized on amino supports (see Section 2.2) were incubated at 4 ◦ C and their functionality evaluated at different times by measuring the HRP activity adsorbed after 10 h. Squares: anti-HRP immobilized on amino supports coated by aldehyde-aspartic-dextran. Circles: anti-HRP immobilized on amino supports stored in 25 mM phosphate buffer pH 7. Triangles: anti-HRP immobilized on amino support stored in 1 M sodium chloride. Rhombus: anti-HRP immobilized onto amino supports coated by aldehyde-dextran.

achieved either by calculating the amount of aldehyde necessary to cover the support surface or by controlling the reaction time. Due to the relative simplicity of the latter, this approach was employed. The immobilized antibody was incubated for 30 min with 10 mg/mL aldehyde-aspartic-dextran, thereby prevented the non-specific adsorption of proteins while inducing only a small reduction of HRP recognition. Longer incubation times resulted in a substantial reduction of the biological activity (data not shown). 3.3. Maintenance of the biological recognition capacity of IgG anti-HRP immobilized on MANAE-agarose Different preparations of IgG anti-HRP immobilized on MANAE-agarose were incubated at 4 ◦ C and their immunoactivities evaluated at different times. Fig. 2 demonstrates that immunoactivity decreased rapidly such that the

Fig. 3. Loading capacity of amino magnetic nano-particles. Immobilization was performed in 150 mM sodium phosphate buffer pH 7.5 as described in Section 2.2. The amount of IgG anti-HRP immobilized was determined by the Bradford method.

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Scheme 2. Polymer coating of the surface of aminated supports containing immobilized IgG.

immobilized antibody was incapable of binding HRP after only 36 h. However, when the immobilized preparations were stored in 1 M sodium chloride at pH 7, the decrease in recognition capacity was negligible. Similarly, incubation in 1 M NaCl of antibody pre-inactivated by storing at low ionic strength resulted in a partial recovery of the activity within minutes (results not shown). This suggests that the loss of biological recognition may be due to the progressive adsorption of the antibody onto the cationic support surface of the support (Balcao et al., 2001; Mieglo et al., 2003).

No loss of immunological activity was detected even after 1 month when the coating was performed using aldehydeaspartic-dextran, suggesting that the support-antibody interactions were prevented. 3.4. Immobilization of the antibody on magnetic nano-particles Based on these results, we decided to use aminated magnetic nano-particles coated with aspartic-aldehyde-dextran to prepare an immuno-biosensor.

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Fig. 4. SDS-PAGE gels of the proteins adsorbed on IgG anti-HRP immobilized on magnetic particles. Lane 1: molecular weight marker (94, 67, 43, 30, 20.1 kDa.). Lane 2: 10 ␮g of IgG anti-HRP immobilized via sugar chain on amino magnetic particles and further coated. Lane 3: 10 ␮g of IgG anti-HRP immobilized via sugar chain on amino magnetic particles and further coated, incubated 24 h in 10 L containing 10 g of proteins and 0.1 ␮g of HRP. Other details are described in Section 2.2.

Fig. 3 shows the loading capacity of amino magnetic nano-particles. More than 100 mg of purified IgG anti-HRP was immobilized per gram of amino magnetic nano-particles. However, to ensure a proper blocking with the aspartic-aldehyde-dextran, we used half the maximum loading for further experiments. Immobilization of the antibody on aminated nano-particles at 50% of the maximum loading and further blocking with aldehyde-aspartic-dextran retained more than 80% of initial recognition capacity. This preparation could be stored for several weeks without any significant loss of recognition capacity. 3.5. Performance of the IgG anti-HRP magnetic nano-particles: concentration and purification of the antigen 0.1 micrograms of pure HRP plus 10 g of a crude E. coli cell extract were dissolved in 10 L of 10 mM sodium phosphate buffer at pH 7 and 4 ◦ C. After addition of 10 ␮g of optimized antibody-magnetic nano-particles and incubation for 24 h, the nano-particles were recovered. Analysis of HRP activity indicated that over 75% of HRP was effectively recovered with the immobilized antibody. SDS-PAGE of the supernatant obtained after boiling the washed antibody-magnetic particle–antigen composite suggested that the main proteins present were the HRP and the IgG anti-HRP (Fig. 4). The ability of only 10 ␮g of immobilized antibody nano-particles to bind much of the HRP present in 10 L of heterogeneous protein solution attests to the high binding affinity of these systems.

4. Conclusion In conclusion, we have developed a methodology that permit the immobilization of IgG on a proper orientation, and that yield a fully inert surface. This strategy has been applied

at different supports, including magnetic nano-particles, with very promising results.

Acknowledgements This work has been partially funded by Genomica (SAU., Spain) MIC SL and the project PACTI-CICYT (COO1999AX011). Interesting suggestions from Dr. Sultan (Estapor, Merck, France) and Dr. Tercero (GENOMICA, SAU. Spain) Mr. Rodriguez and Dr. Caballero (Biosensores SL, Spain) are gratefully acknowledged. The help of A. Berenguer during the writing of this manuscript is gratefully recognized. Specially, we would like to thank the highly valued suggestions from D.A. Cowan (UWC, Cape Town).

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