Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein.

A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.     

Genetic approaches to protein purification

A gene fusion system based on the protein A gene from Staphylococcus aureus has been developed to facilitate purification of recombinant proteins, both in large and small scale. Due to the strong interaction with IgG, it is possible to recover gene products fused to various protein A derivatives, in a one-step procedure with high yield and in purity. Site-directed mutagenesis was used to introduce enzymatic and chemical cleavage sites at the fusion point between the protein A derivative and the desired protein. The protein A “tail” can thereby be removed from the affinity purified fusion protein by the appropriate cleavage, releasing biologically active molecules. Recently, the system was improved by designing a synthetic DNA fragment encoding two IgG-binding domains derived from staphylococcal protein A which are resistant to various chemical cleavages. The gene fusion product is secreted to the culture medium of E.coli and can be recovered simply by passing the clarified culture medium through an IgG Fast Flow Sepharose. The system has been used to immobilize enzymes, to obtain monoclonal and polyclonal antibodies and to produce biologically active human peptide hormones in pilot plant scale.     

Immunochromatographic flow-injection method for detection of human serum immunoglobulin G antibodies to Helicobacter pylori

This study reports on the development of an immunochromatographic flow-injection (FI) method for the quantitation of human serum IgG antibodies to Helicobacter pylori. Patients’ sera were injected into the carrier stream of a FI manifold incorporating an H. pylori immuno-adsorbent reactor. The immuno-adsorbent was prepared by covalently linking H. pylori antigens to periodate oxidized Sepharose CL-4B. Any H. pylori antibody present in the serum is bound to the immuno-sorbent. The bound antibody was quantified by injecting into the carrier stream anti-human IgG peroxidase conjugate followed by TMB/H₂O₂ as the enzyme substrate. The FI system was optimized with respect to antigen loading (1.0 mg/ml gel), bioreactor length (3.0 cm), enzyme conjugate (0.2 mg/ml) and sample loop size (250 μl). One hundred clinical samples were analyzed for their H. pylori antibody concentrations and the results evaluated with respect to their receiver-operator characteristics (ROC). When a cut-off absorbance of between 0.7 and 0.75 was used for positive and negative samples a specificity (>95%), a sensitivity (>90%)and an overall accuracy (>94%) were obtained.     

Efficient high-performance liquid chromatographic system for protein purification

An efficient high-performance liquid chromatographic system, consisting of an affinity column and a high-performance size-exclusion column, was developed and applied to the purification of growth hormone receptors from rabbit livers. When a 6-ml sample of Triton X-100 extracts containing 16 mg of protein was applied to the system, 1200-fold purified receptor with a 10% recovery of binding activity from homogenates was obtained within 3-4 h. The purified receptor exhibited one main band on sodium dodecyl sulphate polyacrylamide gel electrophoresis, and the affinity constant (Ka = 6.0.10(9) M-1) was found to be comparable with that of 1% Triton X-100 extract (4.4.10(9) M-1). The injection of 1 ml of 3 M urea solution prior to receptor elution with 10 ml of 6 M urea solution was effective in removing non-specific binding proteins.     

Lysozyme refolding with immobilized GroEL column chromatography

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5 M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, there existed an optimum denaturant concentration of about 1 M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3 ml of the lysozyme (1 mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54 mg per 1 gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12 mg per 1 gel per h), indicating the advantage of the present system for producing a high throughput in protein refolding operations.     

Interaction of immunoglobulin G with N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia

Zirconia beads (25-38 microm in diameter) were modified with N,N,N'.N'-ethylenediaminetetramethylenephosphonic acid to generate a pseudo-biospecific support, r_PEZ. To better understand the force of interaction between the IgG and the r_PEZ, the equilibrium dissociation constant (Kd) was determined by static binding isotherms, as a function of temperature and by frontal analysis at different linear velocities. Temperature had no significant impact on the maximum static binding capacity (Q(max)) and the equilibrium-binding constant (Kd), whereas pH and the salt concentration had a noticeable impact on both Q(max) and Kd values. Q(max) was found to be in the range of 55-65 mg IgG per ml of beads and unaffected by temperature. The maximum dynamic binding capacity (Qx) was found to be in the range of 20-12 mg IgG per ml of beads. The adsorption rate constant (ka) was determined by a split-peak approach to be between 982 and 32421 mol(-1) s(-1) depending on the linear velocity. Adsorption rate of IgG on r_PEZ was studied as a function of both feed concentration and linear velocity. The standard enthalpy and entropy values were estimated for the interaction of IgG with this novel support. The binding constants were also determined by modeling the batch protein-uptake data.     

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.     

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′)₂ anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used.     

Microstructured layers of spherical biofunctional core-shell nanoparticles provide enlarged reactive surfaces for protein microarrays

Nanostructured core-shell particles with tailor-made affinity surfaces were used to generate microstructured affinity surfaces by microspotting the particles to form densely packed amorphous nanoparticle layers. These layers provided a large reactive surface for the specific binding of protein ligands from aqueous solution. Biofunctional core-shell particles were synthesized for this purpose that consisted of a silica core with a diameter of 100 nm and an organic shell a few nm thick. The nanoparticle core was prepared by sol-gel chemistry and the shell formed in suspension by organosilane chemistry. The shell provided amino groups or carbonyl groups at its outer surface for subsequent covalent immobilization of streptavidin, rabbit IgG antibodies or goat IgG antibodies. AlexaFluor 647-conjugated and biotinylated cytochrome C and CyDye-labeled anti-rabbit IgG and anti-goat IgG were probed as model analytes. The core-shell nanoparticles were spotted using a pin-ring micro-arrayer onto microscope glass slides that were coated with a polycation monolayer by dip-coating prior to nanoparticle deposition. Amorphous particle layers of well-defined thicknesses in the range of 100 nm to 2 microm were obtained by printing aqueous particle suspensions containing 5-500 mg/mL (0.5-50 wt%) of silica particles. The specific affinity of the plotted nanoparticulate capture surface was demonstrated by binding Cy3-labeled donkey anti-rabbit IgG and Cy5-labeled mouse anti-goat IgG to immobilized rabbit IgG and goat IgG particles. The signal intensity per spot increased for any given analyte concentration when the amount of particles per spot was augmented. This was attributed to the increasing integration of receptor molecules per surface footprint, which shifted the binding equilibrium towards the formation of the receptor-ligand complex. Additionally, the locally-increased supply of receptor molecules at the nanoparticulate microchip surface resulted in a wide dynamic range of 4 fM-20 nM (covering six orders of magnitude).     

重组蛋白A亲和填料的制备及其性能评价

为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。     

Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G

Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu²⁺, Ni²⁺ and Zn²⁺ were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu²⁺ was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14–16 mg/ml with Cu²⁺ and 7–9 mg/ml with Ni²⁺ for human IgG with MMA buffer. In the case of CIM-IDA-Zn²⁺ column, the binding capacity was only about 0.5 mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.     

Construction of a functional IgG-binding luciferase fusion protein for the rapid detection of specific bacterial strains

A fusion protein consisting of two IgG-binding domains of streptococcal protein G and firefly luciferase was constructed, and a simple and specific bioluminescent immunodetection system for bacterial strains was developed.     

Site-specific covalent attachment of an engineered Z-domain onto a solid matrix: An efficient platform for 3D IgG immobilization

Immobilized antibodies with oriented and homogeneous patterns are crucial to solid-phase molecular recognition assay. Antibody binding protein-based immobilization can effectively present the desired antibodies. However, steadily installing the stromatoid protein with site-specific attachment manner onto a matrix surface remains to be elucidated. In this study, we present an optimal protocol to tightly attach an immunoglobulin G (IgG)-binding protein (Z-domain) through covalent incorporation of Cys-tag and maleimide group onto polystyrene surface to guarantee site-specific, oriented, and irreversible attachment, resulting in a highly efficient platform for three-dimensional IgG immobilization. The actual IgG-binding characteristic of immobilized Z-Cys was investigated by employing affinity chromatography and size exclusion chromatography. And the efficacy and potential of this platform was demonstrated by applying it to the analysis of interaction between rabbit anti-HRP IgG and its binding partner HRP. The proposed approach may be an attractive strategy to construct high performance antibody arrays and biosensors given that the antibody is compatible with the Z-domain.     

QDs-labeled microspheres for the adsorption of rabbit immunoglobulin G and fluoroimmunoassay

This paper presents a study on the adsorption of rabbit immunoglobulin G onto CdTe quantum dots (QDs)/polystyrene microspheres. The adsorption appears to be sensitive to pH conditions and ionic strength. Maximum adsorption for protein was obtained near the isoelectric point. Adsorption isotherm analysis demonstrated that the electrostatic interaction plays an important role in the adsorption of protein. The thickness of adsorbed layer calculated from the maximal adsorption amounts (q(m)) is 6.5 nm, which indicates that the rabbit IgG molecules exist between the side-on and end-on mode in the monolayer. The bio-functional rabbit IgG/fluorescent microspheres were further used for the detection of antibody in fluoroimmunoassays. This approach allowed detection of goat anti-rabbit IgG in the range of 1-100 ng/mL.     

Serum protein immunogenicity: implications for liver xenografting

The objectives of this study were threefold: (i) assess immunogenicity of donor plasma proteins following hepatic xenotransplantation, (ii) identify potential immunogens, and (iii) consider the implications of antibody formation against these plasma proteins in xenograft survival. We studied liver and heart xenografts in a concordant combination, hamster to rat. All grafts were examined at necropsy for evidence of rat immunoglobulin G (IgG) deposition. Cardiac xenografts were placed in recipients who had, or had not, been sensitized with hamster serum. Hepatic xenografts were placed in naive recipients to see if antibodies to hamster serum proteins could be eluted from the rejecting organ. Sera of immunized rats were examined for the presence of anti-hamster antibodies by immunoelectrophoresis and by Western blotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of hamster serum. Antibodies in sera of immunized rats were compared with those eluted from rejecting livers. Candidate antigens were identified by tandem mass spectrometry, sequence analysis, and reference to protein databases. Results showed that sera of immunized rats recognized a minimum of four different antigens in hamster serum by immunoelectrophoresis, and a minimum of seven by the more sensitive SDS-PAGE Western blot. IgG eluted from rejecting livers bound three of seven candidate antigens recognized by sera of the immunized animals. Sequence analysis searches revealed proteinase inhibitors in each of the three SDS-PAGE bands common to the above samples. All of these candidate proteinase inhibitor immunogens share a common catabolic fate, uptake via the lipoprotein-related protein (LRP/alpha 2-macroglobulin receptor (CD91). Sensitization to hamster serum proteins hastened cardiac xenograft rejection in 30-50% of recipients (depending on sensitization protocol). Vascular deposition of rat IgG occurred in all rejecting xenografts. Antibody binding to proteinase inhibitors could disturb their functional activity and contribute to the pathogenesis of delayed xenograft rejection.     

Characterization of IgG Langmuir-Blodgett films immobilized on functionalized polymers

The bioactivity of anti-human IgG Langmuir-Blodgett (LB) films, the non-specific adsorption of protein and the topography of anti-IgG LB films have been studied for application in immunosensors. The antibody (AB) LB films were horizontally deposited on glass and functionalized polymers, such as carboxy-poly(vinyl chloride) (PVC-COOH), chloropropyl and aminopropyl sol-gel. The LB films were characterized by means of ellipsometry, atomic force microscopy (AFM) and bicinchoninic acid (BCA) protein test. The interpretation of ellipsometric data was performed using a one-layer model. Non-specifically adsorbed protein was desorbed by washing the IgG film in 0.5 M NaCl, 2 M NaCl and 1% N-cetyl-N,N,N-trimethylammoniumbromide detergent solution resulting in a 50% reduction of the film thickness. The mean thickness of an anti-IgG film on glass measured by ellipsometry, PVC-COOH and aminopropyl sol-gel was 9+/-2, 11+/-1 and 23+/-8 nm, respectively. According to the BCA test 6-8 mug antibody (AB) per slide was bound to the functionalized polymers, but only 3 mug AB per slide was adsorbed on glass. The average distance of anti-IgG granules as indicated by AFM measurements on PVC-COOH, chloropropyl and aminopropyl sol-gel was 42+/-20, 34+/-3 and 23+/-4 nm. The average distance of granular AB structures on glass, however, was 150+/-50 nm.     

Avid AL, a synthetic ligand affinity gel mimicking immobilized bacterial antibody receptor for purification of immunoglobulin G.

Avid AL is an affinity gel designed for the purification of immunoglobulin G (IgG). The gel was prepared by first reacting Sepharose with 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine and then with 2-mercaptoethanol. The IgG purified by Avid AL is about 95% pure. The binding parameters of Avid AL for the whole IgG, Fab and Fc fragment and the stability of gel were investigated. The IgG bound to Avid AL can be eluted with an acidic buffer or with a novel neutral buffer containing electron donors. The development of such a mild neutral elution buffer is described. Application of Avid AL in a rapid gram-scale IgG purification was demonstrated. The possible mechanism of IgG binding is discussed.     

Determination of immunoglobulin G in bovine colostrum and milk by direct biosensor SPR-immunoassay

An automated biosensor surface-plasmon resonance-based assay was developed for the determination of immunoglobulin G (IgG) in bovine milk and colostrum with either goat or rabbit antibovine IgG or protein G used as detecting molecule. The method is configured as a direct and nonlabeled immunoassay, with quantitation against an authentic IgG calibrant. Whole colostrum or milk is prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flowrate, contact time, and regeneration, were optimized, and nonspecific binding was evaluated. Performance parameters included working range of 15-10 000 ng/mL, method detection limit of 0.08 mg/mL, overall instrument response reproducibility relative standard deviation (RSD(R)) of 0.47%, mean between-run RSD(R) of 10.5% for colostrum, and surface stability over 200 analyses. The proposed method was compared with independent alternative methods. The technique was applied to the measurement of IgG content during early lactation transition from colostrum to milk, as well as in consumer milk, colostrum, and hyperimmune milk powders.     

Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography. II. Immobilization of synthetic metal-binding peptides from metal ion transport proteins as model bioactive protein surface domains.

This preliminary investigation tests the premise that biologically relevant (1) peptide-metal ion interactions, and (2) metal ion-dependent macromolecular recognition events (e.g., peptide-peptide interactions) may be modeled by biomimetic affinity chromatography. Divinylsulfone-activated agarose (6%) was used to immobilize three different synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein histidine-rich glycoprotein (HRG). The synthetic peptides represented 1-3 multiple repeat units of the 5-residue sequence (Gly-His-His-Pro-His) found in the C-terminal of HRG. By frontal analyses, immobilized HRG peptides of the type (GHHPH)nG, where n = 1-3, were each found to have a similar binding capacity for both Cu(II) ions and Zn(II) ions (31-38 mumol/ml gel). The metal ion-dependent interaction of a variety of model peptides with each of the immobilized HRG peptide affinity columns demonstrated differences in selectivity despite the similar internal sequence homology and metal ion binding capacity. The immobilized 11-residue HRG peptide was loaded with Cu(II) ions and used to demonstrate selective adsorption and isolation of proteins from human plasma. These results suggest that immobilized metal-binding peptides selected from known solvent-exposed protein surface metal-binding domains may be useful model systems to evaluate the specificity of biologically relevant metal ion-dependent interaction and transfer events in vitro.     

A preliminary study for isolation of catalytic antibodies by histidine ligand affinity chromatography as an alternative to conventional protein A/G methods

Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies.