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Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand
Journal of Pharmaceutical and Biomedical Analysis 78–79 (2013) 154–160
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Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand Poonam Rajak, M.A. Vijayalakshmi, N.S. Jayaprakash ∗ Centre for Bioseparation Technology, VIT University, Vellore-632014, Tamil Nadu, India
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Article history: Received 16 October 2012 Received in revised form 9 February 2013 Accepted 13 February 2013 Available online 20 February 2013 Keywords: Anti-HSA monoclonal antibodies Immunoaffinity Oriented immobilization Albumin removal
a b s t r a c t Proteins present in human serum are of immense importance in the field of biomarker discovery. But, the presence of high-abundant proteins like albumin makes the analysis more challenging because of masking effect on low-abundant proteins. Therefore, removal of albumin using highly specific monoclonal antibodies (mAbs) can potentiate the discovery of low-abundant proteins. In the present study, mAbs against human serum albumin (HSA) were developed and integrated in to an immunoaffinity based system for specific removal of albumin from the serum. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the mouse immunized with HSA. Five clones (AHSA1-5) producing mAbs specific to HSA were established and characterized by enzyme linked immunosorbent assay (ELISA) and immunoblotting for specificity, sensitivity and affinity in terms of antigen binding. The mAbs were able to bind to both native albumin as well as its glycated isoform. Reactivity of mAbs with different mammalian sera was tested. The affinity constant of the mAbs ranged from 108 to 109 M−1 . An approach based on oriented immobilization was followed to immobilize purified anti-HSA mAbs on hydrazine activated agarose gel and the dynamic binding capacity of the column was determined. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Human serum albumin, the most abundant protein (30–50 mg/mL) in plasma, is a multifunctional non-glycosylated, negatively charged protein produced in the liver. It displays an extraordinary capacity of ligand binding, serving as a depot and carrier of several endogenous and exogenous compounds [1–3]. It also, by itself has been reported as a valuable biomarker in diseases such as cancer, HIV, diseases that need monitoring of glycemic control, etc. [1,4,5]. However, amidst all this HSA being the most abundant component of plasma/serum act as an impeding factor in analysis of serum, and other downstream process analysis for discovery/detection of less-abundant but possibly significant proteins. Hence, the removal of albumin from the serum proteins would enhance the sample loading capacity in an analytical method and improve the detection sensitivity of low-abundant proteins. Over the last three decades efforts are being made towards the efficient depletion of albumin from human serum. A variety of depletion methods have been reported for this purpose,
∗ Corresponding author. Tel.: +91 4162202377. E-mail addresses: [email protected], [email protected] (N.S. Jayaprakash). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.02.010
each with its own advantages and disadvantages. Albumin has been removed from serum by adsorption onto dye-affinity based columns. These methods can be effective in removal of albumin but the non-specificity and poor reproducibility make this technique undesirable [6–8]. Other methods reported include, a proprietary polypeptide affinity matrix that removes albumin and IgG but is apparently unavailable now [9] and a method based on size separation in a centrifugal filtration device that was, perhaps predictably unsuccessful [10]. The other reported methods were antibody based affinity systems which could utilize polyclonal or monoclonal antibodies [11,12]. The use of polyclonal antibodies based depletion systems can lead to unspecific binding and also lack of reproducibility [13]. Hence, monoclonal antibodies specific to albumin could avoid such problems and remove albumin specifically from the serum. Most often antibody-affinity based systems have used matrices such as Cynogen Bromide (CNBr) activated cellulose, Nhydroxysuccinimide activated sepharose, etc., where antibody is randomly immobilized via primary amino or carboxyl groups. The random coupling may take place involving domains which may be important for the specific recognition of the antigen. In addition, binding of antibodies at their antigen binding site or closer vicinity may also lead to a decrease or total loss of activity [14]. To overcome these effects, oriented immobilization approach is preferred where immobilization of antibodies occur via their carbohydrate
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moiety located in the CH2 domain of their Fc region. The immobilization process involves the oxidation of the carbohydrate moiety of the antibody and then covalently conjugating to the hydrazine activated matrices to form the stable hydrazone bonds [14–17]. In the present study, we generated anti-HSA mAbs and characterized them for selection of mAbs with high affinity and specificity. A mAb with high affinity to HSA was utilized to develop an immunoaffinity-depletion system exploring the site specific oriented immobilization of the antibody. The purified anti-HSA antibody [18] was immobilized on hydrazide activated agarose gel. The binding capacity of the system towards standard HSA and the removal of albumin from human serum were studied. 2. Materials and methods 2.1. Reagents and chemicals Disodium phosphate, monosodium phosphate, sodium chloride and potassium chloride, diamino benzidine was purchased from Sisco Research Laboratories (Mumbai, Maharashtra, India). Dulbecco’s Modified Eagle’s medium (DMEM), penicillin-streptomycin solution, hypoxanthine, azaserine, poly ethylene glycol (PEG), dimethyl sulfoxide, human serum albumin, Freund’s complete adjuvant, Freund’s incomplete adjuvant, bovine serum albumin (BSA), anti-mouse IgG-conjugated with horseradish peroxidase (HRP), anti-mouse IgG-conjugated with horse radish peroxidase were purchased form Sigma (St. Louis, MO, USA). Tetra methyl benzidine (TMB)/H2 O2 was purchased from Genei (Bangalore, Karnataka, India). Agarose hydrazide gel was obtained from BioRad, USA. ELISA plates were purchased from Nunc (Roskilde, Denmark) and other cell culture sterile plasticwares were purchased from Cellstar, Greiner Bio-one (Frickenhausen, Germany). 2.2. Immunization, fusion and screening Three female BALB/c mice were immunized subcutaneously with 100 g of human serum albumin (HSA) emulsified in Freund’s complete adjuvant at the ratio (1:1). Booster injections were given thrice at an interval of four weeks, with 100 g of HSA emulsified in Freund incomplete adjuvant. Three days prior to fusion, the mice were injected intraperitoneally with 300 g of HSA in saline. Spleen was excised from the mice; splenocytes were prepared as a single cell suspension and fused with Sp2/0 myeloma cells at the ratio of 5:1 using 50% polyethylene glycol (M.W. 4000) and 10% DMSO. Cells were then centrifuged, washed and resuspended in DMEM supplemented with 20% fetal bovine serum, 50 M -mercaptoethanol and HA (100 M hypoxanthine, 5.8 M azaserine). The fusion mixture was aliquoted in 96-well plates and placed in a 5% CO2 incubator at 37 ◦ C until the well contained visible growth of hybridoma cells. The screening of hybridomas secreting anti-HSA antibodies was done by indirect enzyme-linked immunosorbent assay (ELISA) method using microplates coated with HSA. The positive clones were expanded and subsequently subcloned to monoclonality by the method of limiting dilution. 2.2.1. Enzyme-linked immunosorbent assay ELISA plates were coated with HSA (1 g/well) suspended in 100 mM carbonate–bicarbonate buffer (pH 9.6) and incubated overnight at room temperature. The plates were then washed and blocked with 5% skim milk for 1 h at 37 ◦ C. After three washes, cell culture supernatants were added and incubated for 1 h at 37 ◦ C. The bound antibody was detected using goat anti-mouse IgG-HRP conjugate with tetramethylbenzidine (TMB)/H2 O2 as the enzyme substrate. The reaction was stopped with 2 M sulphuric acid and optical density (OD) was measured at 450 nm with a multiwell plate reader (FLUOstar Optima, BMG Labtech, Ortenberg, Germany). The
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clones showing strong reactivity against HSA which was tested by ELISA were further characterized and expanded for lab scale production and purification. 2.3. Characterization of anti-HSA monoclonal antibodies (mAbs) 2.3.1. Determination of monoclonal antibody isotype The isotypes of the monoclonal antibodies produced by the hybridoma clones was determined using a mouse mAb isotyping kit (Pierce, IL, USA) according to the manufacturer’s protocols. 2.3.2. Western blot analysis The protein preparations were electrophoresed in 10% polyacrylamide gel according to the standard method described by Laemli et al., 1970 [19]. The proteins were electrophoretically transferred at 90 V for 90 min onto a nitrocellulose membrane (Millipore, Bedford, MA, USA) using a Trans blot apparatus (Bio-Rad, Hercules, CA, USA). Western blot analysis was carried out using the cell culture supernatant containing anti-HSA monoclonal antibodies. Membrane was blocked with 5% skim milk in wash buffer (phosphate buffer saline (PBS) + 0.1% Tween-20) and incubated with mAb for 2 h at 37 ◦ C. The pre-immune serum from the mouse was used as a negative control. After extensive washing with PBS-Tween, horse radish peroxidase conjugated anti-mouse immunoglobulin was added and incubated at 37 ◦ C for 1 h. The bands were visualized by using diaminobenzidine as substrate. 2.3.3. Affinity constant determination An ELISA based procedure described by Friguet et al. [20] was used for the determination of the affinity constant of HSA-anti-HSA mAbs complexes. Different concentrations of HSA (1 g–1 ng) were pre-incubated with each mAb for 3 h at room temperature. Prior to that, an antibody double dilution curve was generated in order to select the dilution at which the antigen and the antibody are at equilibrium as indicated by a linear decline of the curve thereafter. The percentage inhibition of antibody binding to immobilized antibody on plate was determined using standard indirect-ELISA with wells coated with 1 g of HSA as discussed earlier. The affinity constants were determined using the reciprocal molar concentration of the antigen in solution required for 50% inhibition of the antibody binding to immobilized antigen. 2.3.4. Reactivity of mAbs with other mammalian sera Cross reactivity of the mAbs was checked by dot blot analysis with serum of mice, rat, rabbit, monkey and bovine. Specificity of the mAbs was also assayed against native (non-reduced, nonglycated) albumin and glycated albumin (Sigma, St. Louis, MO, USA) using ELISA. 2.4. T-flask growth curve and antibody production Cells were seeded at cell density of 1 × 105 /viable cells/mL into 25-cm2 T-flask (in triplicate) containing 5 mL of medium (Voigt et al., 1999) [21]. Cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 ◦ C in a humidified incubator with 5% CO2 . Cell culture samples were collected every 48 h over 12 days in order to determine the cell number and antibody production. Viable cell numbers were counted with (0.1% (w/v)) trypan blue on a hemocytometer. The antibody production was determined by indirect-ELISA. 2.5. Purification and stability characterization of purified anti-HSA mAbs The anti-HSA mAb was purified based on the procedure described by Rajak et al. [18]. Briefly, 0.34 mL monolith
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CIM-IDA-Co(II) disk (BIA Separations, Slovenia) was washed with equilibration buffer (25 mM MMA = MES + MOPS + Acetate + 0.5 M NaCl, pH 7.4). Cell culture supernatant, precipitated and dialyzed samples, containing anti-HSA mAbs were fed into the columns at a flow rate of 2 mL/min. The column was washed with equilibration buffer and the retained proteins were eluted with different molarities of imidazole (10, 50, 100 and 250 mM) in the equilibration buffer. The fractions were analyzed at 280 nm, and peak fractions were pooled and concentrated using 10 kDa membrane filters (Amicon, Millipore, USA). The chromatographic experiments were done on a fully automated AKTA FPLC system (Amersham Bioscience, Uppsala, Sweden). The purified antibody was taken further for stability characterization and immobilization studies. The antibodies were characterized for its stability at varying temperatures, pH and presence of common denaturants and commonly present metal ions in blood. The purified antibody solution was subjected to different temperatures in the range of 40–90 ◦ C and pH in the range of 3–9 (at 37 ◦ C) for 15 min. The antibody solution was made in 50 mM sodium acetate buffer for pH 3, 4 and 5. Sodium phosphate buffer (50 mM) was used for pH 6 and 7 and tris-HCl (50 mM) was used for pH 8 and 9. The percentage activity retained was determined by indirect ELISA considering the activity of antibody solution at 37 ◦ C without any treatment as 100%. The effect of denaturants on the antibody stability was studied by incubating the purified antibody with different concentrations of guanidinium chloride and urea upto 5 M and SDS, -mercaptoethanol and NaN3 upto 2 mM for 15 min. Similarly, the effect of metal ions on antibody stability was done by incubating the antibody with different metal ions (Al, Ca, Fe, Mg and Mn) upto 2 mM. The activity retained was checked by indirect ELISA. 2.6. Immunoprecipitation Protein-A (20 L) was incubated for 3 h at room temperature with ∼40 g of anti-HSA mAb. To the Protein-A bound antibody, 1 L of human serum (1:100 diluted) was added and incubated for 2 h. Then the complex was washed to remove any unbound HSA. SDS–PAGE loading dye containing beta-mercaptoethanol was added to the sample, boiled for ten minutes and subjected to SDSPAGE analysis. The protein bands were visualized using coomasie blue staining. 2.7. Generation of the anti-HSA immunoaffinity system Hydrazide activated agarose gel was equilibrated in sodium acetate buffer, pH 5.5 and mixed with a solution of oxidized antibody (3 mg/mL) and coupling was allowed to proceed overnight at room temperature on a rotary shaker. The antibody-immobilized gel suspension was packed into a cylindrical column and washed with the same buffer followed by 30 column volumes with phosphate buffer saline, pH 7.4.
as a difference of protein in the reaction mixture before and after immobilization. 2.8. Protein quantification The total protein concentration of the fractions collected in the chromatographic runs was quantified following Bradford, 1976 [22]. Human immunoglobulin G (IgG) (Sigma, St. Louis, MO, USA) was used as reference protein. 3. Results and discussion The study of human plasma proteome is complicated because of the presence of abundant proteins such as albumin and IgG which constitutes more than 70% of the total protein components. The removal of abundant proteins will help in a better way to detect the low-abundant proteins which could be of potential significant candidates in disease diagnosis and drug targets. Monoclonal antibodies because of high specificity can be used in the depletion of albumin from serum samples. Here we describe the production and characterization of monoclonal antibodies against human serum albumin for the development of a high affinity IgG based immunoaffinity system for albumin removal. More importantly, we explored the site specific oriented immobilization scheme for this purpose. The column capacity and selectivity were also tested using standard HSA and total human serum. 3.1. Production and characterization of anti-HSA monoclonal antibodies Several hybridomas producing monoclonal antibodies against HSA were obtained by the fusion of Sp2/0 myeloma cells with spleen cells from HSA immunized BALB/c mice. From the fusion experiments, wells which showed confluence growth of hybridomas were initially screened by means of indirect ELISA for the secretion of anti-HSA antibodies. The positive hybridomas were then subcloned to monoclonality by means of limiting dilution. The selected stable clones (AHSA1–5) were taken for further characterization studies. It was found that all the mAbs belonged to the same subclass IgG1 with kappa light chain. In Western blot analysis, all the anti-HSA mAbs displayed reactivity with membrane-blotted HSA (Fig. 1). Initially, we characterized the five mAbs in terms of antibody titer and antigen sensitivity. The titration curves of the five mAbs are shown in Fig. 2(a). The antibody titer was determined as its end point titer of a full length dilution curve and was taken as the highest dilution which gave 0.1 OD above the negative (Sp2/0 supernatant) control. The highest
2.7.1. Oxidation of antibody The antibody was oxidized by drop-wise addition of one tenth volume of freshly prepared 0.1 M sodium meta-periodate so as to attain a final concentration of 10 mM. Oxidation was performed for 1 h at room temperature with shaking and the reaction was terminated by adding ethylene glycol. The unreacted periodate was removed by dialysis against sodium acetate buffer, pH 5.5 over 12 h with one buffer exchange. 2.7.2. Determination of amount of immobilized antibody The concentration of the protein solution was determined spectrophotometrically at absorbance-280 nm taking an extinction coefficient of 1.4. The quantity of bound protein was determined
Fig. 1. Western blot analysis—Lane 1, 10% SDS-PAGE of native human serum albumin (Sigma); Lane 2–6, Western blot analysis of human serum albumin (Sigma) as probed with cell culture supernatant of different clones producing mAbs against HSA. 5 g of HSA was subjected to SDS-PAGE on 10% gels under non-reducing conditions.
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Fig. 3. Growth curve of anti-HSA hybridoma cells and the antibody production pattern. Cells were inoculated at an initial density of 1 × 105 /mL and cultured in 25 cm2 T-flask.
Fig. 2. (a): Antibody titre curve generated by double dilution of the cell culture supernatant of different clones. Antibody containing cell culture supernatant was titrated against constant amount of antigen. (b): Sensitivity of mAbs to different concentrations of HSA.
titer was shown by mAbs AHSA-2 and 5. The sensitivity of mAbs to HSA was determined by the antigen dilution curve of HSA antigen ELISA (Fig. 2b). The results showed that the antibodies were very sensitive and the lowest concentration of the antigen that could be detected with confidence was upto 0.5 ng for the mAb AHSA-2. Characterization of anti-HSA mAbs have been reported earlier by Omidfar et al. [23] with potential usage in designing immunoassay methods for screening of microalbuminuria. The affinity for the antibody–antigen complex in solution was determined following the method of Friguet et al. [20]. The antibody working dilutions for the experiment were chosen in the linear range such that the antigen and the antibody are at equilibria. The affinity constants were determined using the reciprocal molar concentration of the antigen in solution required for 50% inhibition of the antibody binding to immobilized antigen. The mAbs showed high affinity constants ranging from 108 to 109 M−1 . The initial hybridoma screening procedure utilized in our study detects antibodies by indirect ELISA thereby recognizing immobilized HSA on ELISA plates. However, our final aim is to develop an immunoaffinity system for removal of albumin from serum or other biological samples. Thus, this experiment also aided in determining whether these anti-HSA mAbs could also bind to the HSA protein in solution. The mAbs were also tested for its reactivity to the glycated isoform of HSA, using ELISA. It was observed that all the mAbs reacted with both normal and glycated HSA. The aim of removal of albumin necessitates the selection of suitable antibody which should not distinguish between the two isoforms. This property of antibody will be of use in the case of certain disease conditions, where the albumin undergoes increased glycation [24].
The cross reactivity of the mAbs was checked by testing its reactivity to different available mammalian serum (mouse, rat, rabbit, monkey, bovine) using ELISA. Apart from human, all five mAbs also reacted with rat and mouse serum whereas there was no signal with rabbit and bovine serum. It was observed that the mAb AHSA2 varied from the others in showing no reactivity to monkey serum. This cross reactivity with other animal sera can be considered as an advantage as same mAbs can also be utilized in albumin related studies from other species since raising antibodies against albumin from every species can be costly. Based on high sensitivity, titre and the affinity constant, mAb AHSA-2 was considered for further expansion, characterization and immobilization studies. 3.2. T-flask growth curve The clone AHSA-2 was grown in 25 cm2 cell culture flask in DMEM supplemented with 10% fetal bovine serum. The cell density increased progressively and the growth curve was typical for murine hybridomas. There was no lag phase apparent for the cell line and the antibody production was found to be strongly growthassociated. The cell count reached its highest number on the fourth day and antibody production started increasing from the fourth day on, reaching its maximum on the ninth day of culture (Fig. 3). It was also observed that cells produced some additional antibody in the death phase. For further purification work, production of the mAb was carried out in a 175 cm2 T-flask and the cell culture supernatant was collected on the 9th day. 3.3. Purification and stability characterizations of the mAb The ant-HSA mAbs were purified using a high throughput metal chelate convective interaction media. Convective interaction media (CIM) is a methacrylate based monolith stationary phase possessing attractive features such as flow-independent binding capacity, convective mass transfer, mechanical stability and low pressure drops at higher flow rates enabling rapid separations required for increased productivity [25,26]. In a recent study, a systematic evaluation of convective interaction media immobilized with different metal ions (CIM-IDA-Me (II) (Me2+ : Cu2+ , Ni2+ , Zn2+ and Co2+ ) matrix was carried out for the purification of anti-HSA mAbs [18]. Here, we extended the application of the method for purification of anti-HSA mAbs for their use in development of an immunoaffinity system. Anti-HSA mAbs were thereby purified to homogenity using CIM-IDA-Co (II). The purified antibody was characterized for its thermal and pH stability. We observed that the anti-HSA antibody had optimal activity at 50 ◦ C and the activity reduced by almost 60%
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at 60 ◦ C. Most of the activity (>93%) was lost when the antibody was incubated at 70 ◦ C. The loss of antibody activity in terms of its antigen binding efficiency can be attributed to the changes in its conformational stability with the temperature variations. The pH stability assays showed that the antibody was stable over a broad range from pH 5 to 9. However, a significant loss was seen at pH 4.0. The antibody lost almost 50% of the total activity at pH 3.0. The changes observed in the antigen binding capacity of the anti-HSA antibodies with varying pH may be due to the changes occurring in the ionization state of the amino acids which would possibly effect its conformation [27]. Further stability characterizations of the mAbs were also done to study the effect of denaturants and metals on these antibodies. In case of denaturants, the treated antibody showed no loss of antigen recognition or any significant loss in activity on treatment with Guanidinium chloride and Urea up to 5 M concentrations and up to 2 mM of SDS, NaN3 , and -mercaptoethanol. Anti-HSA antibody treatment with up to 2 mM concentration of different metal ion solutions, which is a concentration much higher than in normal blood, showed no significant effect on antibody activity or any loss in the antigen recognition ability of the metaltreated antibodies. The action of urea involves a weakening of the association of adjacent polypetide linkages by competition of the urea for the hydrogen bonding affinity of the peptide linkages and also the breaking of hydrogen bonding between adjacent side chains [28]. This stability could be attributed to the disulfide network present in an antibody molecule. Since antibody molecule contains an intact disulphide network which appears to preserve the overall structure and increase its conformational stability.
3.4. Immunoprecipitation A preliminary validation of the ability of immobilized antibody to pick native albumin from human serum was demonstrated by immunoprecipitation using Protein A beads. The SDS-PAGE analysis of the immunoprecipitation revealed three bands (Fig. 4). In addition to the heavy (50 kDa) and light chain (25 kDa) of the antibody, an additional band was seen corresponding to the molecular weight of HSA at 66 kDa position. Therefore, this experiment indicated that the anti-HSA mAb was capable of binding to albumin in-solution when immobilized on a matrix.
Fig. 4. Immunoprecipitation/pull down assay using anti-HSA mAb coupled to agarose-Protein A beads for a qualitative determination of antigen–antibody complex with immobilized mAb and analysis of mAb specificity. Protein analyzed on 10% SDS-PAGE under reduced condition. Lane1, Std HSA; Lane 2, Std IgG; Lane 3, eluate from Protein A-mAb + serum.
3.5. Immobilization of anti-HSA mAb and generation of immunoaffinity system Anti-HSA monoclonal antibody was immobilized through its carbohydrate moiety on hydrazide activated matrix. The schematic representation of the immobilization procedure is shown in Fig. 5. Antibody was incubated with hydrazide activated agarose gel (0.5 mL) for a period of 24 h with constant shaking. The amount of bound protein was determined spectrophotometrically at Abs 280 nm and it was found to be 0.7 mg. This method of immobilization provides a site specific orientation with an excellent steric accessibility of the antigen binding site. The use of this approach for development of antibody-based immunoaffinity columns for various purposes has been reported by several authors [29–31]. Antibody affinity systems earlier reported for removal of albumin, were mostly based on polyclonal antibodies [11,13,32,33]. These polyclonal based systems may lead to non-specific binding and also lack of reproducibility due to batch to batch variation which may be overcome by the use of monoclonal antibodies. The use of mAbs for removal of albumin has utilized randomly coupled antibody using matrices activated with N-hydroxysuccinamide [12].
Fig. 5. Schematic representation of immobilization of antibody on hydrazide activated matrix. Step 1—Oxidation of antibody by sodium meta-periodate. Step 2—Immobilization of oxidized antibody on hydrazide activated matrix.
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Fig. 6. Dissociation of anti-HSA from HSA adsorbed on the microtiter wells. Absorbance at 450 nm indicates the amount of anti-HSA remaining after treatment with different eluents as detected by anti-IgG-peroxidase conjugate. Control, PBS; A, 0.5 M acetate buffer, pH2.5; B, 0.1 M Glycine, pH 2.5; C, 0.1 M Glycine + 1MnCl, pH2.5; D, 0.1 M glycine + 1%DMSO; E, 60% Methanol:H2 O; F, 80% Methanol:H2 O; G, PBS+ 2.5 M Guanidinium hydrochloride, pH 7.0; H, PBS + 2.5 M Guanidinium hydrochloride, pH 5.0; I, PBS+ 5 M Guanidinium hydrochloride, pH 7.0.
Such random coupling of antibody may lead to a decrease or complete loss of activity. In comparison to these methods, the present method of immobilization provides a site specific orientation with an excellent steric accessibity of the antigen binding site. The agarose-hydrazide gel with immobilized anti-HSA monoclonal antibody was then characterized in terms of its dynamic binding capacity using pure HSA and also removal of albumin from human serum. Prior to the experiment of albumin binding to the column immobilized with anti-HSA monoclonal antibody, different elution buffers were screened for their capacity to dissociate the antigen–antibody interaction. We assessed the efficiency of different elution buffers by means of modified ELISA method similar to Kummar et al. [34] by incorporating an elution step. Although there is no general rule to the selection of an eluent for the dissociation of antigen–antibody interaction the commonly used eluents are pH, chaotropic salts, ionic strengths, organic solvents and denaturants. The degree of dissociation achieved by different eluents varies with difference in the antibody [35].We analyzed nine different elution buffers involving conditions like varying pH, ionic strength, organic solvent, denaturant or a combination of these (Fig. 6). The difference in absorbance at 450 nm between the control and different eluent treated wells was utilized for determining the efficiency of the respective eluents used. Acetate buffer (0.5 M, pH 2.5) was found to be the best for dissociation of anti-HSA-HSA complex followed by PBS + 2.5 M Guanidinium hydrochloride, pH 5.0. For the determination of the dynamic binding capacity of the system, a 0.2 mg/mL solution of HSA in PBS, pH 7.4 was fed into the column until the column was saturated and the concentration of protein in effluent from the column was same as the feed concentration. The amount of protein bound was measured taking a difference of protein in the feed and the outlet until 50% breakthrough (Fig. 7) and it was found to be 0.71 mg. As the column volume was 0.5 mL the dynamic binding capacity of agarose-hydrazide-anti-HSA was found to be 1.42 mg/mL. A similar study has been reported by Brne et al. [30] where the authors have Table 1 Protein mass balance of human serum fractionation on Agarose-hydrazide-anti-HSA IgG chromatography. Protein injected (mg/mL)
Flow through (mg)
Elution (mg)
Recovery (%)
∼0.8
0.38
0.345
90.6%
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Fig. 7. Breakthrough curve generated using 0.5 mL of Agarose-hydrazide gel immobilized with anti-HSA monoclonal IgG. HSA feed concentration, 0.2 mg/mL in PBS; Flow rate, 1 mL/min.
Fig. 8. Human serum fractionation on Agarose-hydrazide-anti-HSA IgG column. Equilibration and washing buffer, phosphate buffer saline, pH 7.4; Elution buffer, 0.1 M sodium acetate, pH 2.5. Column was restored by washing with the equlibration buffer Insert: SDS-PAGE analysis. Lane L, human serum; lane 1, flow through; lane 2, elution; lane M, standard HSA.
used acid dihydrazide activated convective interaction media for immobilization of anti-HSA and antibody directed towards inter-␣inhibitor protein (IaIP) individually. However, the dynamic binding capacity of anti-HSA CIM-HZ column was found to be 0.3 mg/mL which is approximately 5 times lesser and a need for improvement in the binding capacity has been mentioned by the authors. The binding of native albumin from human serum with the agarose-hydrazide-anti-HSA column was tested by injecting 10 L with a corresponding protein concentration of ∼0.8 mg of total human serum in PBS, pH 7.4. The column elution showed 0.345 mg of bound HSA (Table 1). The SDS-PAGE analysis (gel stained with silver nitrate [36]) of the concentrated protein peak fraction showed a clear band at 66 kDa corresponding to albumin. Additionally, very faint bands of higher molecular weight were also seen which could be the possible oligomers of HSA. The flow through lane (Fig. 8, lane 1) indicated a slight overloading of the column as some quantity of albumin was seen in it. Recirculation of the sample could be one possible way of total removal of the albumin. Study of other adsorption buffers and conditions may also enhance the binding. Further studies would be directed in this direction. 4. Conclusion In the present study we discuss the production and characterization of anti-HSA monoclonal antibodies. Further, high affinity monoclonal antibodies were used for the development of an immunoaffinity matrix. The site specific oriented immobilization of the antibody was explored thereby ensuring that the
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immunobinding efficacy of the ligand antibody remains unaffected. The binding capacity and the selectivity of the column was tested using pure HSA and native HSA from human serum, respectively, and it showed high selectivity for the protein of interest. In addition to the immunoaffinity depletion system, the developed high affinity mAbs could also be used for the detection of albumin in human serum, urine and other biological fluids. Acknowledgments The authors thank the Department of Biotechnology, Government of India (DBT), for funding the project (BT/01/COE/06/18). The first author thanks DBT for a fellowship. Authors also thank Dr. Anjali A. Karande, Professor, Indian Institute of Science, Bangalore, India for her help rendered during mAb development. References [1] M.E. Baker, Beyond carrier proteins, J. Endocrinol. 175 (2002) 121–127. [2] G. Fanali, A. Masi, V. Trezza, M. Marino, M. Fasano, P. Ascenzi, HSA-bench to bedside, Mol. Aspects Med. 33 (3) (2012) 209–290. [3] G.J. Quinlan, G.S. Martin, T.W. Evans, Albumin-biochemical properties and therapeutic potential, Hepatology 41 (6) (2005) 1211–1219. [4] J.G. Feldman, S.J. Gange, P. Bachheti, M. Cohen, M. Young, K.E. Squires, C. Williams, P. Goldwasser, K. Anastos, Serum albumin is a powerful predictor ofsurvival aong HIV-1-infected women, J. Acquir. Immune Defic. Syndr. 33 (1) (2003) 66–73. [5] E. Glattre, A. Engeland, E. Jellum, A.T. Hostmark, Serum albumin and risk of thyroid cancer: a population based-matched case control study, Nor Epidemiol. 11 (2) (2001) 197–200. [6] E.B. Altintas, A. Denizli, Efficient removal of albumin from human serum by monosize dye-affinity beads, J. Chromatogr. B 832 (2006) 216–223. [7] E. Gianazza, P. Arnaud, Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA mechanism of molecular interaction, Biochem. J. 203 (1982) 637–641. [8] J. Travis, J. Bowen, D. Tewksbury, D. Johnson, R. Pannell, Isolation of albumin from whole human plasma and fractionation of albumin depleted plasma, Biochem. J. 157 (1976) 302–306. [9] B.A. Lollo, S. Harvey, J. Liao, A.C. Stevens, R. Wagenknecht, R. Sayen, J. Whaley, F.G. Sajjadi, Improved two-dimensional gel electrophoresis representation of serum proteins by using ProtoClearTM, Electrophoresis 20 (1999) 854–859. [10] H.M. Georgiou, G.E. Rice, M.S. Baker, Proteomic analysis of human plasma: failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins, Proteomics 1 (2001) 1503–1506. [11] R. Pieper, Q. Su, C.L. Gatlin, N.L. Anderson, S. Huang, S. Steiner, Proteomics 3 (2003) 422–432. [12] L.F. Steel, M.G. Trotter, P.B. Nakajima, T.S. Mattu, G. Gonye, T. Block, Efficient and specific removal of albumin from human serum, Mol. Cell Proteomics 2.4 (2003) 262–270. [13] K. Bjorhall, T. Miliotis, P. Davidsson, Comparision of different depletion strades for improved resolution in proteomic analysis of human serum samples, Proteomics 5 (2005) 307–317. [14] M. Nesnevitch, M.A. Firer, The solid phase in affinity chromatography: strategies for antibody attachment, J. Biochem., Biophys. Methods 49 (2001) 467–480. [15] J.H. Kang, H.J. Choi, S.Y. Hwang, S.H. Han, J.Y. Jeon, E.K. Lee, Improving immunobinding using oriented immobilization of an oxidised antibody, J. Chromatogr. A 1161 (2007) 9–14.
[16] J. Turkova, Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function, J. Chromatogr. B 722 (1999) 11–31. [17] R. Vankova, A. Guadinova, H. Sussenbekova, P. Dobrev, M. Strnad, J. Holik, J. Lenfeld, Comparision of oriented and random antibody immobilization in immunoaffinit chromatography of cytokinins, J. Chromatogr. A 811 (1998) 77–84. [18] P. Rajak, M.A. Vijayalakshmi, N.S. Jayaprakash, Purification of monoclonal antibodies, IgG1, from cell culture supernatant by use of metal chelate convective interaction media monolithic columns, Biomed. Chromatogr. (2012), http://dx.doi.org/10.1002/bmc.2721. [19] U.K. Laemli, Cleavage of structural proteins during the assembly of head of the head of bacteriophage T4, Nature 227 (1970) 680–685. [20] B. Friguet, A.F. Chaffote, L.D. Ohaniance, M.E. Goldberg, Measurement of true affinity constant in solution of antigen-antibody complexes by Enzyme linked immunosorbent assay, J. Immunol. Methods 77 (1984) 305–309. [21] A. Voigt, F. Zintl, Hybridoma cell growth and antineuroblastoma monoclonal antibody production spinner flasks using a protein-free medium with microcarriers, J. Biotech. 68 (1999) 213–226. [22] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72 (1976) 248–254. [23] K. Omidfar, S. Kashnian, M. Paknejad, S. Kashnian, B. Larijani, H. Roshanfekr, Production and characterization of monoclonal antibodies against Human serum albumin, Hybridoma 26 (2007), http://dx.doi.org/10.1089/hyb.2007.01. [24] C.G. Schwalkwijk, N. Ligtvoet, H. Twaalfhoven, A. Jager, H.G.T. Blaauwgeers, R.O. Schlingemann, L. Tarnow, H.H. Parving, C.D.A. Stehouwer, V.W.M. Hinsberg, Amadori albumin in Type 1 Diabetic patients correlation of markers with endothelial function, association with diabetic nephropathy and localization in retinal capillaries, Diabetes 48 (1999) 2446–2453. ´ A. Buchacher, Application of monoliths as supports for affinity chro[25] D. Josic, matography and fast enzymatic conversion, J. Biochem. Biophys. Methods 49 (2001) 153–174. ˇ cek, M. Barut, [26] A. Podgornik, J. Janˇcar, M. Merhar, S. Kozamernik, D. Glover, K. Cuˇ ˇ Large-scale methacrylate monolithic columns: design and properA. Strancar, ties, J. Biochem. Biophys. Methods 60 (2004) 179–189. [27] A.L. Lehninger, D.L. Nelson, M.M. Cox, Principles of Biochemistry, fourth ed., W.H. Freeman and Co, 2005. [28] R.K. Scopes, Protein Purification: Principles and Practice, third ed., SpringerVerlag, pl. 1994. [29] Z. Bilkova, J. Mazurova, J. Churacek, D. Horak, J. Turkova, Oriented immobilisation of chymotrypsin by use of suitable antibodies coupled to a non porous solid support, J. Chromatogr. A 852 (1999) 141–149. [30] P. Brne, Y.P. Lim, A. Podgornik, M. Barut, B. Pihlar, A. Strancer, Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilisation of antibodies, J. Chromatogr. A 1216 (2009) 2658–2663. [31] Q. Weiping, X. Bin, W. Lei, W. Chunxiao, Zengdong, W. Yu, Orientation of antibodies on a 3-aminopropyltriethoxylsilane-modified silicon water surface, J. Incl. Phenom. Macrocycl. Chem. 35 (1999) 419–429. [32] N. Seam, D.A. Gonzales, S.J. Kern, G.L. Hortin, G.T. Hoehn, A.F. Suffredini, Quality control of serum albumin depletion for proteomic analysis, Clin. Chem. 53 (2007) 1915–1920. [33] Y.Y. Wang, P.C.P. Cheng, D.W. Chan, A simple affinity spin tube filter method for removing high-abundant common proteins or enriching low-abundant biomarkers for serum proteomic analysis, Proteomics 3 (2003) 243–248. [34] A. Kummer, E.C.Y. Li-Chan, Application of an ELISA elution as a screening tool for dissociation of yolk antibody-antigen complex, J. Immunol. Methods 211 (1998) 125–137. [35] M. Bonde, H. Frokier, D.S. Pepper, Selection of monoclonal antibodies for immunoaffinity chromatography: model studies with antibodies against soy bean trypsin inhibitor, J. Biochem. Biophys. Methods 23 (1991) 23–73. [36] M.V. Nesterenko, T. Michael, S.J. Upton, A simple modification of Blum’s silver stain method allows for 30 minute detection for proteins in polyacrylamide gels, J. Biochem. Biophys. Methods 28 (1994) 239–242.
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand Poonam Rajak, M.A. Vijayalakshmi, N.S. Jayaprakash ∗ Centre for Bioseparation Technology, VIT University, Vellore-632014, Tamil Nadu, India
a r t i c l e
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Article history: Received 16 October 2012 Received in revised form 9 February 2013 Accepted 13 February 2013 Available online 20 February 2013 Keywords: Anti-HSA monoclonal antibodies Immunoaffinity Oriented immobilization Albumin removal
a b s t r a c t Proteins present in human serum are of immense importance in the field of biomarker discovery. But, the presence of high-abundant proteins like albumin makes the analysis more challenging because of masking effect on low-abundant proteins. Therefore, removal of albumin using highly specific monoclonal antibodies (mAbs) can potentiate the discovery of low-abundant proteins. In the present study, mAbs against human serum albumin (HSA) were developed and integrated in to an immunoaffinity based system for specific removal of albumin from the serum. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the mouse immunized with HSA. Five clones (AHSA1-5) producing mAbs specific to HSA were established and characterized by enzyme linked immunosorbent assay (ELISA) and immunoblotting for specificity, sensitivity and affinity in terms of antigen binding. The mAbs were able to bind to both native albumin as well as its glycated isoform. Reactivity of mAbs with different mammalian sera was tested. The affinity constant of the mAbs ranged from 108 to 109 M−1 . An approach based on oriented immobilization was followed to immobilize purified anti-HSA mAbs on hydrazine activated agarose gel and the dynamic binding capacity of the column was determined. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Human serum albumin, the most abundant protein (30–50 mg/mL) in plasma, is a multifunctional non-glycosylated, negatively charged protein produced in the liver. It displays an extraordinary capacity of ligand binding, serving as a depot and carrier of several endogenous and exogenous compounds [1–3]. It also, by itself has been reported as a valuable biomarker in diseases such as cancer, HIV, diseases that need monitoring of glycemic control, etc. [1,4,5]. However, amidst all this HSA being the most abundant component of plasma/serum act as an impeding factor in analysis of serum, and other downstream process analysis for discovery/detection of less-abundant but possibly significant proteins. Hence, the removal of albumin from the serum proteins would enhance the sample loading capacity in an analytical method and improve the detection sensitivity of low-abundant proteins. Over the last three decades efforts are being made towards the efficient depletion of albumin from human serum. A variety of depletion methods have been reported for this purpose,
∗ Corresponding author. Tel.: +91 4162202377. E-mail addresses: [email protected], [email protected] (N.S. Jayaprakash). 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.02.010
each with its own advantages and disadvantages. Albumin has been removed from serum by adsorption onto dye-affinity based columns. These methods can be effective in removal of albumin but the non-specificity and poor reproducibility make this technique undesirable [6–8]. Other methods reported include, a proprietary polypeptide affinity matrix that removes albumin and IgG but is apparently unavailable now [9] and a method based on size separation in a centrifugal filtration device that was, perhaps predictably unsuccessful [10]. The other reported methods were antibody based affinity systems which could utilize polyclonal or monoclonal antibodies [11,12]. The use of polyclonal antibodies based depletion systems can lead to unspecific binding and also lack of reproducibility [13]. Hence, monoclonal antibodies specific to albumin could avoid such problems and remove albumin specifically from the serum. Most often antibody-affinity based systems have used matrices such as Cynogen Bromide (CNBr) activated cellulose, Nhydroxysuccinimide activated sepharose, etc., where antibody is randomly immobilized via primary amino or carboxyl groups. The random coupling may take place involving domains which may be important for the specific recognition of the antigen. In addition, binding of antibodies at their antigen binding site or closer vicinity may also lead to a decrease or total loss of activity [14]. To overcome these effects, oriented immobilization approach is preferred where immobilization of antibodies occur via their carbohydrate
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moiety located in the CH2 domain of their Fc region. The immobilization process involves the oxidation of the carbohydrate moiety of the antibody and then covalently conjugating to the hydrazine activated matrices to form the stable hydrazone bonds [14–17]. In the present study, we generated anti-HSA mAbs and characterized them for selection of mAbs with high affinity and specificity. A mAb with high affinity to HSA was utilized to develop an immunoaffinity-depletion system exploring the site specific oriented immobilization of the antibody. The purified anti-HSA antibody [18] was immobilized on hydrazide activated agarose gel. The binding capacity of the system towards standard HSA and the removal of albumin from human serum were studied. 2. Materials and methods 2.1. Reagents and chemicals Disodium phosphate, monosodium phosphate, sodium chloride and potassium chloride, diamino benzidine was purchased from Sisco Research Laboratories (Mumbai, Maharashtra, India). Dulbecco’s Modified Eagle’s medium (DMEM), penicillin-streptomycin solution, hypoxanthine, azaserine, poly ethylene glycol (PEG), dimethyl sulfoxide, human serum albumin, Freund’s complete adjuvant, Freund’s incomplete adjuvant, bovine serum albumin (BSA), anti-mouse IgG-conjugated with horseradish peroxidase (HRP), anti-mouse IgG-conjugated with horse radish peroxidase were purchased form Sigma (St. Louis, MO, USA). Tetra methyl benzidine (TMB)/H2 O2 was purchased from Genei (Bangalore, Karnataka, India). Agarose hydrazide gel was obtained from BioRad, USA. ELISA plates were purchased from Nunc (Roskilde, Denmark) and other cell culture sterile plasticwares were purchased from Cellstar, Greiner Bio-one (Frickenhausen, Germany). 2.2. Immunization, fusion and screening Three female BALB/c mice were immunized subcutaneously with 100 g of human serum albumin (HSA) emulsified in Freund’s complete adjuvant at the ratio (1:1). Booster injections were given thrice at an interval of four weeks, with 100 g of HSA emulsified in Freund incomplete adjuvant. Three days prior to fusion, the mice were injected intraperitoneally with 300 g of HSA in saline. Spleen was excised from the mice; splenocytes were prepared as a single cell suspension and fused with Sp2/0 myeloma cells at the ratio of 5:1 using 50% polyethylene glycol (M.W. 4000) and 10% DMSO. Cells were then centrifuged, washed and resuspended in DMEM supplemented with 20% fetal bovine serum, 50 M -mercaptoethanol and HA (100 M hypoxanthine, 5.8 M azaserine). The fusion mixture was aliquoted in 96-well plates and placed in a 5% CO2 incubator at 37 ◦ C until the well contained visible growth of hybridoma cells. The screening of hybridomas secreting anti-HSA antibodies was done by indirect enzyme-linked immunosorbent assay (ELISA) method using microplates coated with HSA. The positive clones were expanded and subsequently subcloned to monoclonality by the method of limiting dilution. 2.2.1. Enzyme-linked immunosorbent assay ELISA plates were coated with HSA (1 g/well) suspended in 100 mM carbonate–bicarbonate buffer (pH 9.6) and incubated overnight at room temperature. The plates were then washed and blocked with 5% skim milk for 1 h at 37 ◦ C. After three washes, cell culture supernatants were added and incubated for 1 h at 37 ◦ C. The bound antibody was detected using goat anti-mouse IgG-HRP conjugate with tetramethylbenzidine (TMB)/H2 O2 as the enzyme substrate. The reaction was stopped with 2 M sulphuric acid and optical density (OD) was measured at 450 nm with a multiwell plate reader (FLUOstar Optima, BMG Labtech, Ortenberg, Germany). The
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clones showing strong reactivity against HSA which was tested by ELISA were further characterized and expanded for lab scale production and purification. 2.3. Characterization of anti-HSA monoclonal antibodies (mAbs) 2.3.1. Determination of monoclonal antibody isotype The isotypes of the monoclonal antibodies produced by the hybridoma clones was determined using a mouse mAb isotyping kit (Pierce, IL, USA) according to the manufacturer’s protocols. 2.3.2. Western blot analysis The protein preparations were electrophoresed in 10% polyacrylamide gel according to the standard method described by Laemli et al., 1970 [19]. The proteins were electrophoretically transferred at 90 V for 90 min onto a nitrocellulose membrane (Millipore, Bedford, MA, USA) using a Trans blot apparatus (Bio-Rad, Hercules, CA, USA). Western blot analysis was carried out using the cell culture supernatant containing anti-HSA monoclonal antibodies. Membrane was blocked with 5% skim milk in wash buffer (phosphate buffer saline (PBS) + 0.1% Tween-20) and incubated with mAb for 2 h at 37 ◦ C. The pre-immune serum from the mouse was used as a negative control. After extensive washing with PBS-Tween, horse radish peroxidase conjugated anti-mouse immunoglobulin was added and incubated at 37 ◦ C for 1 h. The bands were visualized by using diaminobenzidine as substrate. 2.3.3. Affinity constant determination An ELISA based procedure described by Friguet et al. [20] was used for the determination of the affinity constant of HSA-anti-HSA mAbs complexes. Different concentrations of HSA (1 g–1 ng) were pre-incubated with each mAb for 3 h at room temperature. Prior to that, an antibody double dilution curve was generated in order to select the dilution at which the antigen and the antibody are at equilibrium as indicated by a linear decline of the curve thereafter. The percentage inhibition of antibody binding to immobilized antibody on plate was determined using standard indirect-ELISA with wells coated with 1 g of HSA as discussed earlier. The affinity constants were determined using the reciprocal molar concentration of the antigen in solution required for 50% inhibition of the antibody binding to immobilized antigen. 2.3.4. Reactivity of mAbs with other mammalian sera Cross reactivity of the mAbs was checked by dot blot analysis with serum of mice, rat, rabbit, monkey and bovine. Specificity of the mAbs was also assayed against native (non-reduced, nonglycated) albumin and glycated albumin (Sigma, St. Louis, MO, USA) using ELISA. 2.4. T-flask growth curve and antibody production Cells were seeded at cell density of 1 × 105 /viable cells/mL into 25-cm2 T-flask (in triplicate) containing 5 mL of medium (Voigt et al., 1999) [21]. Cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 ◦ C in a humidified incubator with 5% CO2 . Cell culture samples were collected every 48 h over 12 days in order to determine the cell number and antibody production. Viable cell numbers were counted with (0.1% (w/v)) trypan blue on a hemocytometer. The antibody production was determined by indirect-ELISA. 2.5. Purification and stability characterization of purified anti-HSA mAbs The anti-HSA mAb was purified based on the procedure described by Rajak et al. [18]. Briefly, 0.34 mL monolith
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CIM-IDA-Co(II) disk (BIA Separations, Slovenia) was washed with equilibration buffer (25 mM MMA = MES + MOPS + Acetate + 0.5 M NaCl, pH 7.4). Cell culture supernatant, precipitated and dialyzed samples, containing anti-HSA mAbs were fed into the columns at a flow rate of 2 mL/min. The column was washed with equilibration buffer and the retained proteins were eluted with different molarities of imidazole (10, 50, 100 and 250 mM) in the equilibration buffer. The fractions were analyzed at 280 nm, and peak fractions were pooled and concentrated using 10 kDa membrane filters (Amicon, Millipore, USA). The chromatographic experiments were done on a fully automated AKTA FPLC system (Amersham Bioscience, Uppsala, Sweden). The purified antibody was taken further for stability characterization and immobilization studies. The antibodies were characterized for its stability at varying temperatures, pH and presence of common denaturants and commonly present metal ions in blood. The purified antibody solution was subjected to different temperatures in the range of 40–90 ◦ C and pH in the range of 3–9 (at 37 ◦ C) for 15 min. The antibody solution was made in 50 mM sodium acetate buffer for pH 3, 4 and 5. Sodium phosphate buffer (50 mM) was used for pH 6 and 7 and tris-HCl (50 mM) was used for pH 8 and 9. The percentage activity retained was determined by indirect ELISA considering the activity of antibody solution at 37 ◦ C without any treatment as 100%. The effect of denaturants on the antibody stability was studied by incubating the purified antibody with different concentrations of guanidinium chloride and urea upto 5 M and SDS, -mercaptoethanol and NaN3 upto 2 mM for 15 min. Similarly, the effect of metal ions on antibody stability was done by incubating the antibody with different metal ions (Al, Ca, Fe, Mg and Mn) upto 2 mM. The activity retained was checked by indirect ELISA. 2.6. Immunoprecipitation Protein-A (20 L) was incubated for 3 h at room temperature with ∼40 g of anti-HSA mAb. To the Protein-A bound antibody, 1 L of human serum (1:100 diluted) was added and incubated for 2 h. Then the complex was washed to remove any unbound HSA. SDS–PAGE loading dye containing beta-mercaptoethanol was added to the sample, boiled for ten minutes and subjected to SDSPAGE analysis. The protein bands were visualized using coomasie blue staining. 2.7. Generation of the anti-HSA immunoaffinity system Hydrazide activated agarose gel was equilibrated in sodium acetate buffer, pH 5.5 and mixed with a solution of oxidized antibody (3 mg/mL) and coupling was allowed to proceed overnight at room temperature on a rotary shaker. The antibody-immobilized gel suspension was packed into a cylindrical column and washed with the same buffer followed by 30 column volumes with phosphate buffer saline, pH 7.4.
as a difference of protein in the reaction mixture before and after immobilization. 2.8. Protein quantification The total protein concentration of the fractions collected in the chromatographic runs was quantified following Bradford, 1976 [22]. Human immunoglobulin G (IgG) (Sigma, St. Louis, MO, USA) was used as reference protein. 3. Results and discussion The study of human plasma proteome is complicated because of the presence of abundant proteins such as albumin and IgG which constitutes more than 70% of the total protein components. The removal of abundant proteins will help in a better way to detect the low-abundant proteins which could be of potential significant candidates in disease diagnosis and drug targets. Monoclonal antibodies because of high specificity can be used in the depletion of albumin from serum samples. Here we describe the production and characterization of monoclonal antibodies against human serum albumin for the development of a high affinity IgG based immunoaffinity system for albumin removal. More importantly, we explored the site specific oriented immobilization scheme for this purpose. The column capacity and selectivity were also tested using standard HSA and total human serum. 3.1. Production and characterization of anti-HSA monoclonal antibodies Several hybridomas producing monoclonal antibodies against HSA were obtained by the fusion of Sp2/0 myeloma cells with spleen cells from HSA immunized BALB/c mice. From the fusion experiments, wells which showed confluence growth of hybridomas were initially screened by means of indirect ELISA for the secretion of anti-HSA antibodies. The positive hybridomas were then subcloned to monoclonality by means of limiting dilution. The selected stable clones (AHSA1–5) were taken for further characterization studies. It was found that all the mAbs belonged to the same subclass IgG1 with kappa light chain. In Western blot analysis, all the anti-HSA mAbs displayed reactivity with membrane-blotted HSA (Fig. 1). Initially, we characterized the five mAbs in terms of antibody titer and antigen sensitivity. The titration curves of the five mAbs are shown in Fig. 2(a). The antibody titer was determined as its end point titer of a full length dilution curve and was taken as the highest dilution which gave 0.1 OD above the negative (Sp2/0 supernatant) control. The highest
2.7.1. Oxidation of antibody The antibody was oxidized by drop-wise addition of one tenth volume of freshly prepared 0.1 M sodium meta-periodate so as to attain a final concentration of 10 mM. Oxidation was performed for 1 h at room temperature with shaking and the reaction was terminated by adding ethylene glycol. The unreacted periodate was removed by dialysis against sodium acetate buffer, pH 5.5 over 12 h with one buffer exchange. 2.7.2. Determination of amount of immobilized antibody The concentration of the protein solution was determined spectrophotometrically at absorbance-280 nm taking an extinction coefficient of 1.4. The quantity of bound protein was determined
Fig. 1. Western blot analysis—Lane 1, 10% SDS-PAGE of native human serum albumin (Sigma); Lane 2–6, Western blot analysis of human serum albumin (Sigma) as probed with cell culture supernatant of different clones producing mAbs against HSA. 5 g of HSA was subjected to SDS-PAGE on 10% gels under non-reducing conditions.
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Fig. 3. Growth curve of anti-HSA hybridoma cells and the antibody production pattern. Cells were inoculated at an initial density of 1 × 105 /mL and cultured in 25 cm2 T-flask.
Fig. 2. (a): Antibody titre curve generated by double dilution of the cell culture supernatant of different clones. Antibody containing cell culture supernatant was titrated against constant amount of antigen. (b): Sensitivity of mAbs to different concentrations of HSA.
titer was shown by mAbs AHSA-2 and 5. The sensitivity of mAbs to HSA was determined by the antigen dilution curve of HSA antigen ELISA (Fig. 2b). The results showed that the antibodies were very sensitive and the lowest concentration of the antigen that could be detected with confidence was upto 0.5 ng for the mAb AHSA-2. Characterization of anti-HSA mAbs have been reported earlier by Omidfar et al. [23] with potential usage in designing immunoassay methods for screening of microalbuminuria. The affinity for the antibody–antigen complex in solution was determined following the method of Friguet et al. [20]. The antibody working dilutions for the experiment were chosen in the linear range such that the antigen and the antibody are at equilibria. The affinity constants were determined using the reciprocal molar concentration of the antigen in solution required for 50% inhibition of the antibody binding to immobilized antigen. The mAbs showed high affinity constants ranging from 108 to 109 M−1 . The initial hybridoma screening procedure utilized in our study detects antibodies by indirect ELISA thereby recognizing immobilized HSA on ELISA plates. However, our final aim is to develop an immunoaffinity system for removal of albumin from serum or other biological samples. Thus, this experiment also aided in determining whether these anti-HSA mAbs could also bind to the HSA protein in solution. The mAbs were also tested for its reactivity to the glycated isoform of HSA, using ELISA. It was observed that all the mAbs reacted with both normal and glycated HSA. The aim of removal of albumin necessitates the selection of suitable antibody which should not distinguish between the two isoforms. This property of antibody will be of use in the case of certain disease conditions, where the albumin undergoes increased glycation [24].
The cross reactivity of the mAbs was checked by testing its reactivity to different available mammalian serum (mouse, rat, rabbit, monkey, bovine) using ELISA. Apart from human, all five mAbs also reacted with rat and mouse serum whereas there was no signal with rabbit and bovine serum. It was observed that the mAb AHSA2 varied from the others in showing no reactivity to monkey serum. This cross reactivity with other animal sera can be considered as an advantage as same mAbs can also be utilized in albumin related studies from other species since raising antibodies against albumin from every species can be costly. Based on high sensitivity, titre and the affinity constant, mAb AHSA-2 was considered for further expansion, characterization and immobilization studies. 3.2. T-flask growth curve The clone AHSA-2 was grown in 25 cm2 cell culture flask in DMEM supplemented with 10% fetal bovine serum. The cell density increased progressively and the growth curve was typical for murine hybridomas. There was no lag phase apparent for the cell line and the antibody production was found to be strongly growthassociated. The cell count reached its highest number on the fourth day and antibody production started increasing from the fourth day on, reaching its maximum on the ninth day of culture (Fig. 3). It was also observed that cells produced some additional antibody in the death phase. For further purification work, production of the mAb was carried out in a 175 cm2 T-flask and the cell culture supernatant was collected on the 9th day. 3.3. Purification and stability characterizations of the mAb The ant-HSA mAbs were purified using a high throughput metal chelate convective interaction media. Convective interaction media (CIM) is a methacrylate based monolith stationary phase possessing attractive features such as flow-independent binding capacity, convective mass transfer, mechanical stability and low pressure drops at higher flow rates enabling rapid separations required for increased productivity [25,26]. In a recent study, a systematic evaluation of convective interaction media immobilized with different metal ions (CIM-IDA-Me (II) (Me2+ : Cu2+ , Ni2+ , Zn2+ and Co2+ ) matrix was carried out for the purification of anti-HSA mAbs [18]. Here, we extended the application of the method for purification of anti-HSA mAbs for their use in development of an immunoaffinity system. Anti-HSA mAbs were thereby purified to homogenity using CIM-IDA-Co (II). The purified antibody was characterized for its thermal and pH stability. We observed that the anti-HSA antibody had optimal activity at 50 ◦ C and the activity reduced by almost 60%
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at 60 ◦ C. Most of the activity (>93%) was lost when the antibody was incubated at 70 ◦ C. The loss of antibody activity in terms of its antigen binding efficiency can be attributed to the changes in its conformational stability with the temperature variations. The pH stability assays showed that the antibody was stable over a broad range from pH 5 to 9. However, a significant loss was seen at pH 4.0. The antibody lost almost 50% of the total activity at pH 3.0. The changes observed in the antigen binding capacity of the anti-HSA antibodies with varying pH may be due to the changes occurring in the ionization state of the amino acids which would possibly effect its conformation [27]. Further stability characterizations of the mAbs were also done to study the effect of denaturants and metals on these antibodies. In case of denaturants, the treated antibody showed no loss of antigen recognition or any significant loss in activity on treatment with Guanidinium chloride and Urea up to 5 M concentrations and up to 2 mM of SDS, NaN3 , and -mercaptoethanol. Anti-HSA antibody treatment with up to 2 mM concentration of different metal ion solutions, which is a concentration much higher than in normal blood, showed no significant effect on antibody activity or any loss in the antigen recognition ability of the metaltreated antibodies. The action of urea involves a weakening of the association of adjacent polypetide linkages by competition of the urea for the hydrogen bonding affinity of the peptide linkages and also the breaking of hydrogen bonding between adjacent side chains [28]. This stability could be attributed to the disulfide network present in an antibody molecule. Since antibody molecule contains an intact disulphide network which appears to preserve the overall structure and increase its conformational stability.
3.4. Immunoprecipitation A preliminary validation of the ability of immobilized antibody to pick native albumin from human serum was demonstrated by immunoprecipitation using Protein A beads. The SDS-PAGE analysis of the immunoprecipitation revealed three bands (Fig. 4). In addition to the heavy (50 kDa) and light chain (25 kDa) of the antibody, an additional band was seen corresponding to the molecular weight of HSA at 66 kDa position. Therefore, this experiment indicated that the anti-HSA mAb was capable of binding to albumin in-solution when immobilized on a matrix.
Fig. 4. Immunoprecipitation/pull down assay using anti-HSA mAb coupled to agarose-Protein A beads for a qualitative determination of antigen–antibody complex with immobilized mAb and analysis of mAb specificity. Protein analyzed on 10% SDS-PAGE under reduced condition. Lane1, Std HSA; Lane 2, Std IgG; Lane 3, eluate from Protein A-mAb + serum.
3.5. Immobilization of anti-HSA mAb and generation of immunoaffinity system Anti-HSA monoclonal antibody was immobilized through its carbohydrate moiety on hydrazide activated matrix. The schematic representation of the immobilization procedure is shown in Fig. 5. Antibody was incubated with hydrazide activated agarose gel (0.5 mL) for a period of 24 h with constant shaking. The amount of bound protein was determined spectrophotometrically at Abs 280 nm and it was found to be 0.7 mg. This method of immobilization provides a site specific orientation with an excellent steric accessibility of the antigen binding site. The use of this approach for development of antibody-based immunoaffinity columns for various purposes has been reported by several authors [29–31]. Antibody affinity systems earlier reported for removal of albumin, were mostly based on polyclonal antibodies [11,13,32,33]. These polyclonal based systems may lead to non-specific binding and also lack of reproducibility due to batch to batch variation which may be overcome by the use of monoclonal antibodies. The use of mAbs for removal of albumin has utilized randomly coupled antibody using matrices activated with N-hydroxysuccinamide [12].
Fig. 5. Schematic representation of immobilization of antibody on hydrazide activated matrix. Step 1—Oxidation of antibody by sodium meta-periodate. Step 2—Immobilization of oxidized antibody on hydrazide activated matrix.
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Fig. 6. Dissociation of anti-HSA from HSA adsorbed on the microtiter wells. Absorbance at 450 nm indicates the amount of anti-HSA remaining after treatment with different eluents as detected by anti-IgG-peroxidase conjugate. Control, PBS; A, 0.5 M acetate buffer, pH2.5; B, 0.1 M Glycine, pH 2.5; C, 0.1 M Glycine + 1MnCl, pH2.5; D, 0.1 M glycine + 1%DMSO; E, 60% Methanol:H2 O; F, 80% Methanol:H2 O; G, PBS+ 2.5 M Guanidinium hydrochloride, pH 7.0; H, PBS + 2.5 M Guanidinium hydrochloride, pH 5.0; I, PBS+ 5 M Guanidinium hydrochloride, pH 7.0.
Such random coupling of antibody may lead to a decrease or complete loss of activity. In comparison to these methods, the present method of immobilization provides a site specific orientation with an excellent steric accessibity of the antigen binding site. The agarose-hydrazide gel with immobilized anti-HSA monoclonal antibody was then characterized in terms of its dynamic binding capacity using pure HSA and also removal of albumin from human serum. Prior to the experiment of albumin binding to the column immobilized with anti-HSA monoclonal antibody, different elution buffers were screened for their capacity to dissociate the antigen–antibody interaction. We assessed the efficiency of different elution buffers by means of modified ELISA method similar to Kummar et al. [34] by incorporating an elution step. Although there is no general rule to the selection of an eluent for the dissociation of antigen–antibody interaction the commonly used eluents are pH, chaotropic salts, ionic strengths, organic solvents and denaturants. The degree of dissociation achieved by different eluents varies with difference in the antibody [35].We analyzed nine different elution buffers involving conditions like varying pH, ionic strength, organic solvent, denaturant or a combination of these (Fig. 6). The difference in absorbance at 450 nm between the control and different eluent treated wells was utilized for determining the efficiency of the respective eluents used. Acetate buffer (0.5 M, pH 2.5) was found to be the best for dissociation of anti-HSA-HSA complex followed by PBS + 2.5 M Guanidinium hydrochloride, pH 5.0. For the determination of the dynamic binding capacity of the system, a 0.2 mg/mL solution of HSA in PBS, pH 7.4 was fed into the column until the column was saturated and the concentration of protein in effluent from the column was same as the feed concentration. The amount of protein bound was measured taking a difference of protein in the feed and the outlet until 50% breakthrough (Fig. 7) and it was found to be 0.71 mg. As the column volume was 0.5 mL the dynamic binding capacity of agarose-hydrazide-anti-HSA was found to be 1.42 mg/mL. A similar study has been reported by Brne et al. [30] where the authors have Table 1 Protein mass balance of human serum fractionation on Agarose-hydrazide-anti-HSA IgG chromatography. Protein injected (mg/mL)
Flow through (mg)
Elution (mg)
Recovery (%)
∼0.8
0.38
0.345
90.6%
159
Fig. 7. Breakthrough curve generated using 0.5 mL of Agarose-hydrazide gel immobilized with anti-HSA monoclonal IgG. HSA feed concentration, 0.2 mg/mL in PBS; Flow rate, 1 mL/min.
Fig. 8. Human serum fractionation on Agarose-hydrazide-anti-HSA IgG column. Equilibration and washing buffer, phosphate buffer saline, pH 7.4; Elution buffer, 0.1 M sodium acetate, pH 2.5. Column was restored by washing with the equlibration buffer Insert: SDS-PAGE analysis. Lane L, human serum; lane 1, flow through; lane 2, elution; lane M, standard HSA.
used acid dihydrazide activated convective interaction media for immobilization of anti-HSA and antibody directed towards inter-␣inhibitor protein (IaIP) individually. However, the dynamic binding capacity of anti-HSA CIM-HZ column was found to be 0.3 mg/mL which is approximately 5 times lesser and a need for improvement in the binding capacity has been mentioned by the authors. The binding of native albumin from human serum with the agarose-hydrazide-anti-HSA column was tested by injecting 10 L with a corresponding protein concentration of ∼0.8 mg of total human serum in PBS, pH 7.4. The column elution showed 0.345 mg of bound HSA (Table 1). The SDS-PAGE analysis (gel stained with silver nitrate [36]) of the concentrated protein peak fraction showed a clear band at 66 kDa corresponding to albumin. Additionally, very faint bands of higher molecular weight were also seen which could be the possible oligomers of HSA. The flow through lane (Fig. 8, lane 1) indicated a slight overloading of the column as some quantity of albumin was seen in it. Recirculation of the sample could be one possible way of total removal of the albumin. Study of other adsorption buffers and conditions may also enhance the binding. Further studies would be directed in this direction. 4. Conclusion In the present study we discuss the production and characterization of anti-HSA monoclonal antibodies. Further, high affinity monoclonal antibodies were used for the development of an immunoaffinity matrix. The site specific oriented immobilization of the antibody was explored thereby ensuring that the
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immunobinding efficacy of the ligand antibody remains unaffected. The binding capacity and the selectivity of the column was tested using pure HSA and native HSA from human serum, respectively, and it showed high selectivity for the protein of interest. In addition to the immunoaffinity depletion system, the developed high affinity mAbs could also be used for the detection of albumin in human serum, urine and other biological fluids. Acknowledgments The authors thank the Department of Biotechnology, Government of India (DBT), for funding the project (BT/01/COE/06/18). The first author thanks DBT for a fellowship. Authors also thank Dr. Anjali A. Karande, Professor, Indian Institute of Science, Bangalore, India for her help rendered during mAb development. References [1] M.E. Baker, Beyond carrier proteins, J. Endocrinol. 175 (2002) 121–127. [2] G. Fanali, A. Masi, V. Trezza, M. Marino, M. Fasano, P. Ascenzi, HSA-bench to bedside, Mol. Aspects Med. 33 (3) (2012) 209–290. [3] G.J. Quinlan, G.S. Martin, T.W. Evans, Albumin-biochemical properties and therapeutic potential, Hepatology 41 (6) (2005) 1211–1219. [4] J.G. Feldman, S.J. Gange, P. Bachheti, M. Cohen, M. Young, K.E. Squires, C. Williams, P. Goldwasser, K. Anastos, Serum albumin is a powerful predictor ofsurvival aong HIV-1-infected women, J. Acquir. Immune Defic. Syndr. 33 (1) (2003) 66–73. [5] E. Glattre, A. Engeland, E. Jellum, A.T. Hostmark, Serum albumin and risk of thyroid cancer: a population based-matched case control study, Nor Epidemiol. 11 (2) (2001) 197–200. [6] E.B. Altintas, A. Denizli, Efficient removal of albumin from human serum by monosize dye-affinity beads, J. Chromatogr. B 832 (2006) 216–223. [7] E. Gianazza, P. Arnaud, Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA mechanism of molecular interaction, Biochem. J. 203 (1982) 637–641. [8] J. Travis, J. Bowen, D. Tewksbury, D. Johnson, R. Pannell, Isolation of albumin from whole human plasma and fractionation of albumin depleted plasma, Biochem. J. 157 (1976) 302–306. [9] B.A. Lollo, S. Harvey, J. Liao, A.C. Stevens, R. Wagenknecht, R. Sayen, J. Whaley, F.G. Sajjadi, Improved two-dimensional gel electrophoresis representation of serum proteins by using ProtoClearTM, Electrophoresis 20 (1999) 854–859. [10] H.M. Georgiou, G.E. Rice, M.S. Baker, Proteomic analysis of human plasma: failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins, Proteomics 1 (2001) 1503–1506. [11] R. Pieper, Q. Su, C.L. Gatlin, N.L. Anderson, S. Huang, S. Steiner, Proteomics 3 (2003) 422–432. [12] L.F. Steel, M.G. Trotter, P.B. Nakajima, T.S. Mattu, G. Gonye, T. Block, Efficient and specific removal of albumin from human serum, Mol. Cell Proteomics 2.4 (2003) 262–270. [13] K. Bjorhall, T. Miliotis, P. Davidsson, Comparision of different depletion strades for improved resolution in proteomic analysis of human serum samples, Proteomics 5 (2005) 307–317. [14] M. Nesnevitch, M.A. Firer, The solid phase in affinity chromatography: strategies for antibody attachment, J. Biochem., Biophys. Methods 49 (2001) 467–480. [15] J.H. Kang, H.J. Choi, S.Y. Hwang, S.H. Han, J.Y. Jeon, E.K. Lee, Improving immunobinding using oriented immobilization of an oxidised antibody, J. Chromatogr. A 1161 (2007) 9–14.
[16] J. Turkova, Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function, J. Chromatogr. B 722 (1999) 11–31. [17] R. Vankova, A. Guadinova, H. Sussenbekova, P. Dobrev, M. Strnad, J. Holik, J. Lenfeld, Comparision of oriented and random antibody immobilization in immunoaffinit chromatography of cytokinins, J. Chromatogr. A 811 (1998) 77–84. [18] P. Rajak, M.A. Vijayalakshmi, N.S. Jayaprakash, Purification of monoclonal antibodies, IgG1, from cell culture supernatant by use of metal chelate convective interaction media monolithic columns, Biomed. Chromatogr. (2012), http://dx.doi.org/10.1002/bmc.2721. [19] U.K. Laemli, Cleavage of structural proteins during the assembly of head of the head of bacteriophage T4, Nature 227 (1970) 680–685. [20] B. Friguet, A.F. Chaffote, L.D. Ohaniance, M.E. Goldberg, Measurement of true affinity constant in solution of antigen-antibody complexes by Enzyme linked immunosorbent assay, J. Immunol. Methods 77 (1984) 305–309. [21] A. Voigt, F. Zintl, Hybridoma cell growth and antineuroblastoma monoclonal antibody production spinner flasks using a protein-free medium with microcarriers, J. Biotech. 68 (1999) 213–226. [22] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72 (1976) 248–254. [23] K. Omidfar, S. Kashnian, M. Paknejad, S. Kashnian, B. Larijani, H. Roshanfekr, Production and characterization of monoclonal antibodies against Human serum albumin, Hybridoma 26 (2007), http://dx.doi.org/10.1089/hyb.2007.01. [24] C.G. Schwalkwijk, N. Ligtvoet, H. Twaalfhoven, A. Jager, H.G.T. Blaauwgeers, R.O. Schlingemann, L. Tarnow, H.H. Parving, C.D.A. Stehouwer, V.W.M. Hinsberg, Amadori albumin in Type 1 Diabetic patients correlation of markers with endothelial function, association with diabetic nephropathy and localization in retinal capillaries, Diabetes 48 (1999) 2446–2453. ´ A. Buchacher, Application of monoliths as supports for affinity chro[25] D. Josic, matography and fast enzymatic conversion, J. Biochem. Biophys. Methods 49 (2001) 153–174. ˇ cek, M. Barut, [26] A. Podgornik, J. Janˇcar, M. Merhar, S. Kozamernik, D. Glover, K. Cuˇ ˇ Large-scale methacrylate monolithic columns: design and properA. Strancar, ties, J. Biochem. Biophys. Methods 60 (2004) 179–189. [27] A.L. Lehninger, D.L. Nelson, M.M. Cox, Principles of Biochemistry, fourth ed., W.H. Freeman and Co, 2005. [28] R.K. Scopes, Protein Purification: Principles and Practice, third ed., SpringerVerlag, pl. 1994. [29] Z. Bilkova, J. Mazurova, J. Churacek, D. Horak, J. Turkova, Oriented immobilisation of chymotrypsin by use of suitable antibodies coupled to a non porous solid support, J. Chromatogr. A 852 (1999) 141–149. [30] P. Brne, Y.P. Lim, A. Podgornik, M. Barut, B. Pihlar, A. Strancer, Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilisation of antibodies, J. Chromatogr. A 1216 (2009) 2658–2663. [31] Q. Weiping, X. Bin, W. Lei, W. Chunxiao, Zengdong, W. Yu, Orientation of antibodies on a 3-aminopropyltriethoxylsilane-modified silicon water surface, J. Incl. Phenom. Macrocycl. Chem. 35 (1999) 419–429. [32] N. Seam, D.A. Gonzales, S.J. Kern, G.L. Hortin, G.T. Hoehn, A.F. Suffredini, Quality control of serum albumin depletion for proteomic analysis, Clin. Chem. 53 (2007) 1915–1920. [33] Y.Y. Wang, P.C.P. Cheng, D.W. Chan, A simple affinity spin tube filter method for removing high-abundant common proteins or enriching low-abundant biomarkers for serum proteomic analysis, Proteomics 3 (2003) 243–248. [34] A. Kummer, E.C.Y. Li-Chan, Application of an ELISA elution as a screening tool for dissociation of yolk antibody-antigen complex, J. Immunol. Methods 211 (1998) 125–137. [35] M. Bonde, H. Frokier, D.S. Pepper, Selection of monoclonal antibodies for immunoaffinity chromatography: model studies with antibodies against soy bean trypsin inhibitor, J. Biochem. Biophys. Methods 23 (1991) 23–73. [36] M.V. Nesterenko, T. Michael, S.J. Upton, A simple modification of Blum’s silver stain method allows for 30 minute detection for proteins in polyacrylamide gels, J. Biochem. Biophys. Methods 28 (1994) 239–242.