Solid-phase biotinylation of antibodies

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels.     

One-step purification of murine IgM and human alpha 2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 M methyl alpha-D-mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl alpha-D-mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. alpha 2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man(alpha 1,3)Man units are bound to the immobilized lectin.     

Design and performances of immunoassay based on SPR biosensor with magnetic microbeads

A surface plasmon resonance (SPR) biosensor system was developed for immunoassay, based on the conjugates of magnetic microbeads coupling with antibody which could be trapped on the Au film firmly due to the magnetic force. The magnetic microbeads were used as the solid support for the heat shock protein 70 (Hsp 70) antibody and antibody immobilized magnetic microbeads were utilized instead of the single antibody for the determination of Hsp 70. Since the magnetic bead is coated with dextran, the antibodies and some specific biomolecular receptors can be immobilized using a variety of chemical reactions. Compared to traditional antibody immobilization on the sensing film, there is not a covalent link between the Au film and the antibody. There is a great advantage in that sensor can be stripped and reused, and the same chemistry used to derivative dextran-coated SPR sensors can be used for the magnetic bead-coated sensors. The sensing layer was formed well. Different dilution ratios (v/v) of the conjugates result in different detectable ranges. When the dilution ratios of the conjugate are 1:10 and 1:5, the lowest concentrations of Hsp 70 that can be detected are 1.50 and 0.30 microg ml(-1), respectively.     

Improved immunomatrix methods to detect protein:protein interactions

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.     

Specific interchain cross-linking of antibodies using bismaleimides. Repression of ligand leakage in immunoaffinity chromatography.

The extensive use of antibody-containing affinity columns in the purification of biologically active compounds (e.g., genetically engineered proteins) is severely hampered by the leaching of antibody (or portions thereof) from the immunoaffinity resin during elution of the target antigen. One of the major problems in this context is the combined use of reducing (i.e., thiols) and chaotropic (e.g., detergents and denaturants) agents in the elution step, which causes the disassociation of heavy and/or light chains from the immobilized antibody, thereby contaminating the resultant product. In order to overcome this problem, we have cross-linked the four antibody chains at their sites of disulfide interlinkage, thus producing a single antibody chain. To accomplish this, interchain disulfide bonds were reduced, and the resultant thiol groups were cross-linked by using bifunctional SH-specific reagents (particularly bismaleimides). Cross-linking of up to 95% of the available SH groups produced was achieved with concomitant retention of antigen-binding activity. The cross-linked antibody was immobilized onto CNBr-activated Sepharose, and the resultant column was found to be substantially more stable to harsh elution conditions than similar columns which contain the un-cross-linked antibody.     

Development of a capacitive immunosensor: a comparison of monoclonal and polyclonal capture antibodies as the primary layer

There is widespread interest in capacitance immunosensor systems which directly detect antigen binding to immobilized antibody. Our system comprises an active biolayer of antibodies bound to a silicon--silicon dioxide--silicon nitride (Si-SiO2-Si3N4) surface. As with other groups, our system initially gave poorly reproducible responses on addition of antigen. We mechanically degraded the Si-SiO2-Si3N4 surface, and the responses on addition of transferrin were monitored. The mechanical degradation allowed the affinity reaction to be 'seen' capacitively. Once the system was established, a comparison of capture antibodies was performed to establish the most effective biolayer. Three affinity reactions were examined: (a) 1D2A4, monoclonal antibody (mAb) to human transferrin, as the capture layer; (b) polyclonal goat anti-human transferrin antibody (PcAb) as the capture layer; and (c) 1D2A4 with transferrin (Tf) prebound as the capture layer. There was no response to addition of transferrin where 1D2A4 was the capture layer. Addition of transferrin when the polyclonal antibody was used as the primary layer resulted in a drop in measured capacitance. Addition of goat anti-human transferrin antibody to a device with 1D2A4 plus transferrin as the capture layer also resulted in a measured capacitance decrease. There is a difference in dielectric/blocking effectiveness between the monoclonal and polyclonal antibodies.     

Antibody‐immobilized column for quick cell separation based on cell rolling

Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label‐free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody‐immobilized cell‐rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell‐rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34‐positive and ‐negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34‐positive and CD34‐negative cells on antibody‐immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell‐sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. 125I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained an Mr 134,000 125I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of the fibroblast form. O-Phosphotyrosine prevented 125I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with [32P]Pi, GH was shown to stimulate formation of a 32P-labeled protein which bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114,000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. The Mr 114,000 phosphorylated protein could be immunoprecipitated with anti-GH antibody, indicating that GH remained noncovalently bound to this protein during absorption to and elution from the immobilized phosphotyrosyl binding antibody. Phosphoamino acid analysis after both limited acid hydrolysis and extensive base hydrolysis of the Mr 114,000 phosphoprotein confirmed the presence of phosphotyrosyl residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

疏水吸附固定化天冬氨酸酶及其性质的研究

本文论述了一种N-烷基琼脂珠衍生物的合成方法,研究了pH,离子强度、载体上疏水基团含量等因素对载体吸附天冬氨酸酶的影响以及固定化天冬酸酶的性质。结果表明邻甲苯胺基琼脂珠在pH5.5,0.1mol/L磷酸缓冲液(含有0.25mol/LKCL)中,每克湿载体可吸附15—25mg酶蛋白,酶活力回收达90％以上。固定化酶的性质有所改变,其热稳定性和操作稳定性明显增强。 固定化天冬氨酸酸柱可以用于连续化生产L-天冬氮酸,在pH8.0,1.0mol/L及丁烯二酸铵(含0.02mol/L MgCl_2),30℃条件下,以空间流速SV=3.5操作2个月,固定化酶活力仍保持79.5％。     

Study of antibody/antigen binding kinetics by total internal reflection ellipsometry

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.     

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab')2-IgG antibody (70 microg/mL) was immobilized on the QCM. In case of anti-CRP F(ab')2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab')2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 microg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab')2-IgG antibody and MPC polymer.     

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.     

Covalent immobilization of antibodies on finally inert support surfaces through their surface regions having the highest densities in carboxyl groups

The correct immobilization of antibodies is one of the most critical steps in the preparation of immunosensors and immunochromatography matrices. In addition, the final support has to be chemical and physically inert to avoid the unspecific adsorption of proteins that can reduce the sensitivity of the biosensor or the purification achieved by the chromatography. The solution to both problems is one of the major challenges in the field. Here, we have presented two different novel and simple alternatives to have the unmodified antibody anionically exchanged to a support, further covalently immobilized with more than 90% of the antibodies bonded to the support by the four subunits, retaining a high functionality and giving a final "inert" surface. The first solution was the use of supports having a low superficial density of amino groups activated with glutaraldehyde. Here, the inertness was achieved by the use of a very low density of amino groups, unable to adsorb proteins at 100 mM sodium phosphate, while immobilization proceeds mainly via a first adsorption of the antibody and a further reaction with the glutaraldehyde groups. The second solution implies the design of a novel support (amino-epoxy). This support again produces a first ionic exchange of the antibody on the support and a further reaction with the epoxy groups, but because the epoxy groups can be finally blocked with aspartic groups (annulling the charge), the initial density of amino-epoxy groups can be as high as possible. Both systems permitted the correct and oriented immobilization of IgG. The immobilized antibody showed high-functionality (65-75%) and a final inert support surface. This immobilized antibody (antiperoxidase) was able to capture fully specifically HRP contaminating a protein crude extract from E. coli.     

New antibody immobilization method via functional liposome layer for specific protein assays

A specific protein assay system based on functional liposome-modified gold electrodes has been demonstrated. To fabricate such assay system, a liposome layer was initially grown on top of a gold layer. The liposome layer contained two kinds of functional molecules: biotin molecules for the binding sites of streptavidin and N-(10,12-pentacosadiynoic)-acetylferrocene molecules for the facile redox probe in electrochemical detections. Then, streptavidin was attached on the functional liposme-modified layer using the interaction of streptavidin-sbiotin complex. On the streptavidin-attached surface, antibody molecules, anti-human serum albumin antibodies could be immobilized without any secondary antibodies. AFM imaging of the streptavidin-attached liposome surface revealed a uniform distribution of closely packed streptavidin molecules. In situ quartz-crystal microbalance and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity. Our specific antibody assay system, based on a functional liposome modified electrode, can be developed further to yield sophisticated structures for numerous protein chips and immunoassay sensors.     

Development of a piezoelectric immunosensor for the detection of Salmonella typhimurium.

A piezoelectric biosensor has been developed for the detection of Salmonella typhimurium. The antibody to Salmonella was immobilized on the crystal by various immobilization procedures. The best result was obtained when antibody was immobilized on the crystal precoated with a thin layer of polyethyleneimine. The response of the coated crystal for S. typhimurium in a microbial suspension was in the range of 10(5) to 10(9) cells ml-1. The time required for a complete interaction between the crystal and the cells appeared to depend upon the cell concentration of the analyzed sample. The antibody-bound crystal lost no activity over 4 days at 4 degrees C and it could be reused for 6-8 consecutive assays.     

Automated kinetic exclusion assays to quantify protein binding interactions in homogeneous solution.

A method was developed for the quantification of protein-ligand interactions in which the free protein present in homogeneous reaction mixtures was separated and quantified using a KinExA immunoassay instrument. Separation was achieved by rapid percolation of the reaction mixture over a column of microbeads whose surfaces were coated with an immobilized form of the ligand. The protein thus captured was quantified using a fluorescently labeled anti-protein antibody. The features of this new method were illustrated using a model system in which each of the principal reagents was covalently labeled with a different fluorescent molecule: mouse monoclonal anti-biotin primary antibody (fluorescein), biotin (B-phycoerythrin), and goat anti-mouse polyclonal secondary antibody (indodicarbocyanin). Values for the equilibrium and kinetic rate constants for the binding between the anti-biotin antibody and biotin conjugated with B-phycoerythrin were determined and shown to be independent of whether the fluorescent label was located on the primary or secondary antibody. Equilibrium binding experiments conducted with (F(AB))(2) and corresponding F(AB) fragments showed that the valency of the binding protein had no influence on the value of the dissociation constant. The values of the equilibrium and rate constants obtained by this new method are those for the binding reaction in homogeneous solution; the immobilized ligand is only a tool exploited for the separation and quantification of the free protein.     

Simple immunoaffinity method to purify recombinant hepatitis B surface antigen secreted by transfected mammalian cells

Purification of recombinant hepatitis B surface antigen (recHBsAg) produced in a stable Chinese hamster ovary (CHO) cell line was evaluated using Linx Affinity Purification System (Invitrogen, USA). To purify HBsAg secreted by this cell line, a murine monoclonal antibody (MAbAH1) raised against native HBsAg was used. The purified AH1MAb was conjugated with phenyldiboronic acid (PDBA) and immobilized on the immunoaffinity chromatographic support. Using an optimized protocol the affinity column was able to purify recHBsAg from supernatant of mammalian cells cultures with more than 80% purity. This method showed to be simple and quicker than the current ultracentrifugation methods. The method is also efficient and economical in obtaining purified recHBsAg.     

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K(a): approximately 10(15) M(-1)) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.     

Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.