Functional monolithic platforms: Chromatographic tools for antibody purification

Polymer monoliths are an efficient platform for antibody purification. The use of monoclonal antibodies (mAbs) and engineered antibody structures as therapeutics has increased exponentially over the past few decades. Several approaches use polymer monoliths to purify large quantities of antibody with defined clinical and performance requirements. Functional monolithic supports have attracted a great deal of attention as they offer practical advantages for antibody purification, such as more rapid analysis, smaller sample volume requirements and the opportunity for a greater target molecule enrichment. This review focuses on the development of synthetic and natural polymer-based monoliths for antibody purification. The materials and methods employed in monolith production are discussed, highlighting the properties of each system. We also review the structural characterization techniques available using monolithic systems and their performance under different chromatographic approaches to antibody capture and release. Finally, a summary of monolithic platforms developed for antibody separation is presented, as well as expected trends in research to solve current and future challenges in this field. This review comprises a comprehensive analysis of proposed solutions highlighting the remarkable potential of monolithic platforms.     

Affinity chromatography of human anti-dextran antibodies isolation of two distinct populations

Affinity chromatography is a very efficient method for antibody purification. Two affinity chromatography supports were prepared to analyze the specificity of anti-dextran antibodies. Silica beads were grafted with native dextran or with functionalized dextran. The anti-dextran antibodies present in some human sera were analyzed by enzyme-linked immunosorbent assay method. These antibodies play an important role in severe dextran-induced anaphylactic reactions in humans by forming immune complexes with clinical dextran. The results indicated that two distinct populations of anti-dextran antibodies were purified from human serum, using dextran-coated silica beads. Elution from this support with an oligo-dextran of 4000 g/mol allowed the isolation of one population that only recognized native dextran as antigen. Functionalized dextran coated on dextran silica beads led to the purification, with a glycine-HCl buffer, of another subclass of antibodies that recognized substituted dextran derivatives. Furthermore, these antibodies could be useful tools for in vitro and in vivo investigations using dextran derivatives as bio-active polysaccharides.     

A monolithic lipase reactor for biodiesel production by transesterification of triacylglycerides into fatty acid methyl esters

An enzymatic reactor with lipase immobilized on a monolithic polymer support has been prepared and used to catalyze the transesterification of triacylglycerides into the fatty acid methyl esters commonly used for biodiesel. A design of experiments procedure was used to optimize the monolithic reactor with variables including control of the surface polarity of the monolith via variations in the length of the hydrocarbon chain in alkyl methacrylate monomer, time of grafting of 1-vinyl-4,4-dimethylazlactone used to activate the monolith, and time used for the immobilization of porcine lipase. Optimal conditions involved the use of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monolith, grafted first with vinylazlactone, then treated with lipase for 2 h to carry out the immobilization of the enzyme. Best conditions for the transesterification of glyceryl tributyrate included a temperature of 37°C and a 10 min residence time of the substrate in the bioreactor. The reactor did not lose its activity even after pumping through it a solution of substrate equaling 1,000 reactor volumes. This enzymatic reactor was also used for the transesterification of triacylglycerides from soybean oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.     

Ion-exchange macroporous hydrophilic gel monolith with grafted polymer brushes

The grafting of functional polymers to the pore surface of macroporous monolithic polyacrylamide cryogels was found to be an efficient and convenient method for the preparation of macroporous polyacrylamide gels, so-called cryogels (pAAm cryogels), with both controlled extent of functional group incorporated and with tailored surface chemistries. Anion-exchange polymer chains of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and poly([2-(methacryloyloxy)ethyl]-trimethylammonium chloride) (pMETA), and cation-exchange polymer chains of polyacrylate have been grafted onto pAAm cryogels using potassium diperiodatocuprate as initiator. It was possible to achieve the ion-exchange capacity up to 0.2-0.5 mmol/ml. The graft polymerization did not alter the macroporous structure of the pAAm cryogel, however the flow rate of solutes through the cryogel matrix decreased with increase in the density of polymer grafted. The sorption of low-molecular-weight (metal ion, dye) and high-molecular-weight (protein) substances on the grafted monolithic pAAm column has been studied. The results indicate that a 'tentacle'-type binding of protein to grafted polymer depended on the architecture of the grafted polymer layer and took place after a certain degree of grafting has been reached. The binding of proteins by tentacle-like polymer chains allowed for increasing the binding capacity for proteins on the grafted pAAm cryogels up to 6-12 mg/ml.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.     

Grafting of material-binding function into antibodies Functionalization by peptide grafting

Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials.     

Antibody exchange immunochemistry

Antibodies are found to transfer rapidly between antigen samples attached to separate solid supports. The half-times for this antibody exchange at 20 and 37 degrees C were 7 and 1 h, respectively. Antibody exchange was exhibited by all 17 polyclonal and monoclonal antibodies tested and was readily detected by enzyme-linked immunosorbent assay, immunoblotting, or immunocytochemical assays. Taking advantage of this phenomenon, antibodies can be affinity-purified using less than a picomole of antigen and small amounts of antibody.     

Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.     

An evaluation of a ceramic monolith as an enzyme support material

Catalase was immobilized on commercially available monolithic catalyst supports and also on participate support obtained by crushing the monolith. The kinetics of the monolith- and particulate-supported enzymes were analyzed in a continuous tubular reactor system and pressure drop was also monitored. Analysis of the results indicates that the monolith-supported system presents very little resistance to flow which results in a considerably smaller pressure drop than is obtained in flow through particulate-supported systems under comparable conversion conditions. Ceramic monoliths thus appear to be very suitable for use as enzyme supports in continuous tubular reactor applications, particularly where high pressure drops might be expected.     

Use of Radioactive Antibodies for Characterizing Antigens and Application to the Study of Flagella Synthesis

A simple rapid immunochemical procedure has been developed which provides information about the qualitative and quantitative nature of antigens. It involves the use of purified radioactive ((125)I-labeled) antibodies. The amount of antibody bound to the antigen is determined by filtering the mixture through diethylaminoethyl (DEAE)-cellulose paper. All of the antigen, as well as the antibody complexed with it, is trapped on the paper, whereas free antibody is removed by repeated washing. This technique has been applied to the study of three immune systems, bovine serum albumin, Escherichia coli tryptophan synthetase B protein, and Bacillus subtilis flagella. The results obtained by the DEAE-antibody binding technique were comparable, in terms of sensitivity, specificity, and accuracy, to data obtained by microcomplement fixation and precipitin methods. The assay was used to measure the kinetics of flagella regeneration in B. subtilis.     

Affinity puricication of polyclonal antibodies using immobilized multimeric peptides

The possibility of using multiple antigenic peptides (MAP) not only for the production and characterisation of antibodies but also for their purification by affinity chromatography, has been explored with two different tetrametic MAPs synthesised starting from a tetradentate lysine core. Recognition selectivity and specificity of the mulltimeric antigents were retained after immobilization on preactivated affinity supports, allowing convenient antibody purification directly from crude sera in a single chromatographic step. Since antibodies raised against MAPs recognise very frequently the N-terminal portion of the peptide antigen, results suggest that only a limited number of peptide chains remains covalently linked to the solid phase, leaving the others uncoupled and free to interact fully with the antibody. Recovery of antibody immunoreactivity from affinity purifications on MAP-columns was much higher than that obtained from columns prepared by immobilizing at the same density the corresponding linear peptide antigen. The purity of thus obtained antibodies is also far superior, as detected by SDS-PAGE analysis. Retention of the multimeric peptide recognition properties for the corresponding antibodies after immobilization on solid supports suggests that production, characteriszation, and even the affinity purification of anti-peptide antibodies, could be carried out simply and conveniently via the synthesis of a single multimeric antigen, without additional steps.     

Membrane chromatographic immunoassay method for rapid quantitative analysis of specific serum antibodies

This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.     

Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering

By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient folding. This is particularly important when antibodies are to be used under conditions where the disulfide bonds cannot form, i.e., in intracellular applications (as "intrabodies"). We describe in detail two methods for the knowledge-based improvement of antibody stability and folding efficiency. While CDR grafting from a non-human to the most closely related human antibody framework is an established technique to reduce the immunogenicity of a therapeutic antibody, CDR grafting for stabilization implies the use of a more distantly related acceptor framework with superior biophysical characteristics. The use of such dissimilar frameworks requires particular attention to antigen contact residues outside the classical CDR definition and to residues capable of indirectly affecting the conformation of the antigen binding site. As a second alternative, the stability of a suboptimal framework can be improved by the introduction of point mutations designed to optimize key residue interactions. We describe the analysis methods used to identify such point mutations, which can be introduced all at once, while maintaining the framework features necessary for antigen binding. These rational approaches render the continued "rediscovery" of certain mutations by directed evolution unnecessary, but they can also be used in conjunction with such methods to discover even better molecules.     

Production and characterization of a monoclonal antibody to dog hepatic lipase

Partially purified dog hepatic lipase was used as antigen to produce monoclonal antibodies in mice. In addition to enzyme-linked immunosorbent assay (ELISA), a reliable and efficient procedure for screening antibodies reacting to hepatic lipase has been developed. A method to distinguish antibodies directing to active site or non-active site epitopes has also been described. We obtained three positive clones that survived after subcloning and expansion. All three monoclonal antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. Specificity of monoclonal antibody LDHL No. 537 to dog hepatic lipase was demonstrated by passing post-heparin plasma through its immunoaffinity column. Only dog hepatic lipase was removed by LDHL No. 537 from post-heparin plasma. The immunoaffinity chromatography also demonstrated the co-existence of three enzyme activities (mono- and triacylglycerol lipase and phospholipase A1) on the dog hepatic lipase molecule. The subunit weight of dog hepatic lipase has been estimated at 57500 +/- 600 (n=3) by using immunoaffinity chromatography and the combination of immunoprecipitation and autoradiography methods.     

Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content.

The bicinchoninic acid (BCA) assay method for the determination of protein has been investigated for its utility in measuring the protein content of plasma lipoproteins. Although other methods, principally those based on the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275) have been extensively used for this purpose, the tolerance of the BCA method to many commonly encountered detergents and buffers offers a definite advantage over the Lowry-based methods. In this study, lipoprotein protein values obtained by the BCA method were compared to a standard modification of the Lowry et al. procedure since this assay forms the basis of much of the relevant literature. The standard BCA assay was found to overestimate the protein content of very low density lipoprotein by approximately 70% and low density lipoprotein by approximately 30%; high density lipoprotein values compared favorably. Overestimations by the BCA assay paralleled the relative phospholipid content of the lipoprotein fractions. This apparent lipid effect was eliminated by the addition of 2% sodium dodecyl sulfate to samples prior to the analysis. In the presence of this detergent, BCA assay measurements for these three lipoprotein fractions were 97, 90, and 98%, respectively, of the reference assay values.     

An enzyme-linked immunoassay for the detection of antibodies to endoplasmic reticulum

A simple, sensitive solid-phase assay for the detection of antibodies to endoplasmic reticulum is described. The assay is dependent upon the amount of antigen bound to the solid support and upon the amount of antibody bound to the support via the relevant antigen. The assay can be used to measure both polyclonal and monoclonal antibody to endoplasmic reticulum. It has been used to isolate several monoclonal antibodies which can recognize and precipitate specific proteins of the endoplasmic reticulum. In addition, it has been used to probe the membrane orientation of endoplasmic reticulum antigens.     

Terminally functionalized thermoresponsive polymer brushes for simultaneously promoting cell adhesion and cell sheet harvest

For preparing cell sheets effectively for cell sheet-based regenerative medicine, cell-adhesion strength to thermoresponsive cell culture surfaces need to be controlled precisely. To design new thermoresponsive surfaces via a terminal modification method, thermoresponsive polymer brush surfaces were fabricated through the surface-initiated reversible addition-fragmentation chain transfer (RAFT) radical polymerization of N-isopropylacrylamide (IPAAm) on glass substrates. The RAFT-mediated grafting method gave dithiobenzoate (DTB) groups to grafted PIPAAm termini, which can be converted to various functional groups. In this study, the terminal carboxylation of PIPAAm chains provided high cell adhesive property to thermoresponsive surfaces. Although cell adhesion is generally promoted by a decrease in the grafted PIPAAm amount, the decrease also decelerated thermally-induced cell detachment, whereas the influence of terminal modification was negligible on the cell detachment. Consequently, the terminally modified PIPAAm brush surfaces allowed smooth muscle cells (SMCs) to simultaneously adhere strongly and detach themselves rapidly. In this study, SMCs were unable to reach a confluent monolayer on as-prepared PIPAAm brush surfaces (grafted amount: 0.41 μg/cm(2)) without terminal carboxylation due to their insufficient cell-adhesion strength. On the other hand, though a decrease in the PIPAAm amount allowed SMCs to form a confluent cell monolayer on the PIPAAm brush surface, the SMCs were unable to be harvested as a monolithic cell sheet by low-temperature culture at 20 °C. Because of their unique property, only terminal-carboxylated PIPAAm brush surfaces achieved rapid harvesting of complete cell sheets by low-temperature culturing.     

沙丁胺醇人工抗原的合成及抗体制备

沙丁胺醇是一种β-兴奋剂,常被很多畜禽水产养殖户非法用于动物养殖。为建立沙丁胺醇在食品中残留的快速检测方法,研究了沙丁胺醇免疫原的合成和抗体的制备方法。采用对氨基苯甲酸法合成了沙丁胺醇（SAL）免疫原SAL-cBSA,采用重氮化法合成的克伦特罗（CL）偶合物CL-cOVA作为包被抗原,用紫外光谱法分析了所合成免疫原和包被抗原。用免疫原SAL-cBSA免疫新西兰大白兔获得多克隆抗体,抗体效价达到32000。采用间接ELISA法检测抗体IC50值为8.79ng/ml,SAL的浓度在1ng/ml~100ng/ml区间时,SAL与对抗体的竞争结合力呈直线关系。表明所制备的沙丁胺醇免疫原具有良好的免疫原性,所制备的抗体拥有很高的灵敏度。     

Comparison of IgG4 assays using whole parasite extract and BmR1 recombinant antigen in determining antibody prevalence in brugian filariasis

BACKGROUND: Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. METHODS: Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid trade mark ]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. RESULTS: There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. CONCLUSIONS: Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.     

狂犬病毒抗体ELISA检测试剂盒的改进研究

为了提高狂犬病毒抗体检测的灵敏度和特异性,采用狂犬病毒单克隆抗体包被酶标板,再分别加入重组的狂犬病毒糖蛋白或细胞培养抗原做固相层的方法(抗体捕捉法),用传统的间接ELISA法做对照,按常规方法检测抗狂犬病毒抗体。结果显示,抗体捕捉法的非特异性反应低于间接法,而灵敏度达到0．51U水平,高于间接法。在800份临床标本检测中,检出率明显高于间接法。用15份阳性血清作小鼠中和试验,并和抗体捕捉ELISA法比较具有高度的一致性。试验结果充分表明,该方法优于传统的ELISA间接法。因此可作为临床注射狂犬疫苗后检测血清中狂犬病毒抗体的常规方法。