A comparison of various commercially-available liquid chromatographic supports for immobilization of enzymes and immunoglobulins

A number of commercially-available, activated supports were evaluated and compared for the immobilization of enzymes (alkaline phosphatase, glucose oxidase and peroxidase) and human immunoglobulin G (IgG). The supports studied included pressure-stable, epoxy-activated acrylate-based supports (Separon HEMA 1000 and Eupergit C); agarosebased, epoxy-, cyanogen bromide-, glutaraldehyde- or N-hydroxysuccimide-activated supports (epoxy-activated or cyanogen bromide-activated Sepharose, ACT-Ultrogel AcA 22, Reacti-Gel 6X and Affi-Gel 10); and glass bead-based, activated supports (CDI- and NHS-Glycophase). As expected, the pH required for maximum protein immobilization and retention of activity varied with both protein and support. For example, the amount of alkaline phosphatase coupled was maximum at pH 3 or 5 for most supports, but retention of activity was greatest for immobilization at pH 7, 9 or 11. Glucose oxidase and peroxidase coupling and activity retention in general showed less variation in optimal coupling pH. Coupling of IgG and retention of anti-IgG binding activity were both optimal at a coupling pH of 9 or 11. The Separon HEMA-IgG support made in these studies was also utilized for rapid h.p.l.c, purification of anti-IgG from serum.     

Rapid determination of immobilized ConA lectin activity

ConA was immobilized on an epoxy-activated copolymer of 2-hydroxyethyl-methacrylate and ethylene-dimethacrylate and commercially available high-pressure liquid chromatography (HPLC) sorbents Separon HEMA 1000 EL, Separon HEMA 1000 E, and Separon HEMA 1000 EH (Tessek, Prague, CSFR Denmark). Specific, sensitive, and rapid method for determination of immobilized ConA lectin activity was developed. β-Galactosidase fromAspergilus oryzae oligomannosyl residues was used as specific affinant. After separation of bound and unbound β-galactosidase, enzyme activity was measured in supernatant and thus immobilized ConA lectin activity was calculated easily. The use of the method for evaluating the properties of immobilized ConA, efficiency of immobilization, specific activity, and thermostability is shown. The method developed could be generalized by using artificially glycosylated enzyme for any lectin.     

Preconcentration of pesticides from waters

Summary A procedure for the preconcentration of pesticides from water is described using the sorption on an organic sorbent with following desorption by means of an organic solvent. The usefulness of a few sorbents (Separon SE, Tenax GC, Porapak Q, Separon SI C 18) was compared on the basis of several factors influencing the sorption. The experiments showed the excellent suitability of Separon SI C 18 for this purpose.

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Anreicherung von Pesticiden aus Wässern

Zusammenfassung Die Anreicherung von Pesticiden aus verschiedenen Wässern und durch Sorption auf einem organischen Sorbent und anschließende Desorption mit einem organischen Lösungsmittel wird durchgeführt. Die entsprechenden Eigenschaften einiger Sorbentien (Separon SE, Tenax GC, Porapak Q, Separon SI C 18) wurden auf Grund von Parametern, die die Sorptionseffektivität beeinflussen, verglichen. Die Untersuchungen erwiesen die vorzüglichen Eigenschaften von Separon SI C 18 für den genannten Zweck.

     

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.     

Affinity chromatography of porcine pepsin on different types of immobilized 3,5-diiodo-L-tyrosine

The preparation of affinity sorbents containing immobilized iodinated derivatives of L-tyrosine for the affinity chromatography of porcine pepsin is described. The ligand was coupled either to Sepharose 4B or bead cellulose after the divinylsulfone activation or to Sepharose 4B after the activation with 2,4,6-trichloro-1,3,5-triazine. The highest capacity for porcine pepsin was found in the case of 3,5-diiodo-L-tyrosine coupled to divinylsulfone-activated Sepharose.     

Comparison of chromatographic ion-exchange resins. II. More strong anion-exchange resins

A comparative study was performed on strong anion exchangers to investigate the pH dependence, titration curves, efficiency, binding strength, particle size distribution, and static and dynamic capacity of the chromatographic resins. The resins tested included Q Sepharose XL, UNO Q-1, Poros 50 HQ, Toyopearl QAE 550c, Separon HemaBio 1000Q, Q-Cellthru Bigbeads Plus, Q Sepharose HP and Toyopearl SuperQ 650s. Testing was performed with five different proteins: anti-Factor VII monoclonal antibody (immunoglobulin G), aprotinin, bovine serum albumin, lipolase and myoglobin. The dependence of pH on retention varies from generally low to very high for proteins with a low isoelectric point (pl). An unexpected binding at pH 7-8 of aprotinin with pI >11 was observed on Separon HemaBio 1000Q. No link between pH dependence on retention and titration curves of the different resins was observed. Efficiency results show the expected trend of higher dependence of the plate height with increasing flow-rate of soft resins compared to resins for medium- and high-pressure operation. No or a very small difference in particle size distribution was obtained between new and used resins. Binding to anion-exchange resins as a function of ionic strength varies to some extent depending on the specific protein. Generally, binding and elution at high salt concentration may be performed with Q Sepharose XL, Toyopearl QAE 550c, Q Sepharose HP and Poros 50 HQ, while binding and elution at low salt concentration may be performed with Q-Cellthru Bigbeads Plus. A very high binding capacity was obtained with Q Sepharose XL. Comparison of static capacity and dynamic capacity at 10% breakthrough shows approx. 50-80% utilization of the total available capacity during chromatographic operation. A general good agreement was obtained between this study and data obtained by the suppliers. The results of this study may be used for selection of resins for testing in process development.     

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.     

Oriented Covalent Immobilization of Engineered ZZ-Cys onto Maleimide-Sepharose: An Affinity Platform for IgG Purification

Protein A affinity chromatography is an important technique that is widely used in purifying polyclonal and monoclonal antibodies. However, improving the IgG loading capacity of protein A affinity materials remains crucial. In this study, a smaller divalent IgG binding molecule derived from the B domain of protein A, i.e., ZZ-domain, was used to develop an affinity adsorbent with high IgG loading capacity by improving the unit area yield of the site-specific immobilization affinity ligand. The engineered ZZ-Cys was tightly immobilized onto Sepharose support via the covalent incorporation of a cysteine handle and a maleimide group, with oriented manner and divalent IgG binding capacity, thereby resulting in homogenous conjugates, namely, Sepharose–ZZ^SA. Approximately 1.19 mg of ZZ-Cys was coupled onto wet Sepharose g⁻¹ and the maximum saturation binding capacity of Sepharose–ZZ^SA g⁻¹ was approximately 23.80 mg of IgG. The smaller engineered ZZ-Cys can be produced at a lower cost than protein A and covalently conjugated onto matrix surface with high density and full IgG binding capacity. Thus, the proposed platform may be of general use for IgG purification in an efficient and economical manner.

     

Anion chromatography on hydroxyethyl methacrylate-based sorbents

The selectivity of sorbents plays an important role in the design of ion chromatographic separations. The selectivity of the hydroxyethylmethacrylate-based sorbent Tessek Separon HEMA-S 1000 Q-L was compared with those of other commonly used sorbents and the role of the eluent charge was investigated. The selectivity of this sorbent was found to be satisfactory for most commonly used ion chromatographic detection modes, including indirect photometry and both suppressed and non-suppressed conductivity detection.     

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.     

Evaluation of the mode of binding of immunoglobulin to activated agarose.

Determining the orientation of the immobilization of proteins to solid-phase matrices is of critical importance in the development of systems that employ immobilized proteins. Among these are enzyme-linked immunoassays, immobilized enzymes and affinity chromatography matrices. To determine the orientation of immunoglobulin G (IgG) on activated agaroses, we coupled the immunoglobulin covalently to various activated matrices. The IgG was then cleaved with papain and the liberated fragments collected and analyzed using high-performance liquid chromatography. Only Fab fragments could be detected regardless of the activation method used. This implies that IgG binds to these matrices predominantly via the Fc domain. In order to develop a quantitative method of measuring the Fab and Fc fragments, we compared the binding of IgG and its papain cleavage fragments to S-Zephyr columns and Mono-S columns. Comparison between these columns showed that IgG is bound more tightly to the S-Zephyr column and, in contrast, its retention on Q-Zephyr is less than on a comparable Mono-Q column. The resolution of IgG and its fragments was better in all cases on S-Zephyr than on Mono-S under the conditions employed.     

Influence of Calixarenes on Chromatographic Separation of Benzene or Uracil Derivatives

The addition of calix[4]arenes to MeCN/H₂O or MeOH/MeCN/THF/H₂O mobile phases improves LC separation of benzene or uracil derivatives on Separon SGX C18 or Separon SGX NH₂ supports. Structure of the calixarenes and their host–guest supramolecular complexes with the analytes are discussed in context of the LC separation.     

Secretory immunoglobulin a from human milk catalyzes milk protein phosphorylation

This article presents evidence that protein kinase activity is an intrin sic property of secretory immunoglobulin A (sIgA) from milk of healthy human mothers. Polyclonal sIgA was purified by sequential chromatog raphy on protein A-Sepharose, DEAE-cellulose, and gel filtration on Toyopearl HW-55 and Sepharose 4B columns. Its purity was established by one-and two-dimensional SDS-PAGE. The protein kinase activity was inhibited by specific antibodies (Abs) against sIgA, and was stable to acidic and alkaline conditions. Catalytic sIgA showed optimal reaction conditions (pH and MgCl₂ concentration) and substrate specificity dif ferent from those of known protein kinases; i.e., sIgA phosphorylated the serine residues of various milk proteins in the presence of different γ-[³²P]nucleoside-and deoxynucleoside-5’-triphosphates. The homoge neous Fab fragment of sIgA also showed kinase activity. An ATP-binding activity of fractions of sIgA was demonstrated by affinity chromatog raphy on ATP-Sepharose and by covalent binding of an affinity analog of ATP; this activity was mediated by the L chain of sIgA. The authors believe these observations are the first example of the catalytic activity of IgA Abs and of natural catalytic Abs with synthetic activity. In addition, the findings suggest the likelihood that catalytic Abs are generated by the immune system of healthy mothers.     

Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography

Summary A high-performance liquid gel-permeation chromatographic method is described for the determination of human serum immunoglobulin G (IgG) by separating the fluorescent immuno complex from the free fluorescence-labeled antibody. Fluorescence-labeled antibody used in this study was fluorescein isothiocyanate (FITC)-labeled Fab fragment goat anti-human IgG (anti-IgG Fab). Immuno complexes and antibody of different molecular sizes can be separated. FITC-labeled anti-IgG Fab was added to the serum and the mixture is passed through the column. An immuno complex separates as well-delineated peak in the column void volume, and was measured by the fluorescence of the column eluate (Ex=490nm, Em=520nm). The total analysis time for a serum sample was approximately 15min. The minimum detection limit was 25 mg/dl. The relative standard deviation was below 2% (peak area). The results of the HPL-GPC analysis correlate well with those obtained by laser nephelometric assay (r=0.992).     

Synthesis and properties of dendrimers possessing the same fluorophore(s) located either peripherally or off-center

Two series of phosphorus dendrimers functionalized by maleimide derivatives are synthesized, as well as three new monomeric maleimide derivatives, of which two are characterized by X-ray diffraction. The first series of phosphorus dendrimers possesses maleimide derivatives as end groups (6-48, from generation 0 to generation 3). The second series of dendrimers possesses a single copy of the same maleimide derivative linked "off-center" to a cyclotriphosphazene core, leading to dissymmetrical dendrimers; this series is synthesized from generation 0 to generation 2. The fluorescence properties of both series of dendrimers and of monomers are studied, affording new information. First, the presence of labile hydrogen extinguishes the fluorescence. Second, the grafting of the fluorophore(s) directly to the core affords highly fluorescent compounds. Finally, an original influence of the branches possessing phosphorhydrazone linkages toward the fluorescence properties is shown.     

Production of a humanized Fab fragment of a neutralizing antibody against rabies virus

This paper describes the production of a functional humanized Fab fragment of a neutralizing antibody against the rabies virus. It is a prototype of a therapeutic agent that could be an alternative to equine anti-rabies immunoglobulins and anti-rabies serum immunoglobulins obtained from the blood of vaccinated human donors. Variable fragments of the high-affinity light and heavy chains neutralizing an antibody against the rabies virus were cloned and sequenced. Mouse constant regions were replaced by human constant regions followed by expression of the humanized Fab fragments in a yeast expression system. The immunochemical properties of the Fab fragments were evaluated using ELISA, polyacrylamide gel electrophoresis, and the Western blot assay. The binding capability of the produced humanized Fab fragments surpasses that of the parental antibody. A high degree of humanization was confirmed using sera against human immunoglobulins. The yield of the humanized Fab fragments was 21 mg per liter of culture medium. The purified Fab fragment preparation did not contain traces of the isolated light and heavy chains of the humanized Fab fragments.     

Substituent effects on the reversibility of furan-maleimide cycloadditions

The effects of furan and maleimide substitution on the dynamic reversibility of their Diels-Alder reactivity have been investigated computationally and by (1)H NMR spectroscopy. Furan and furan derivatives bearing methoxy, methyl, or formyl groups at their 2- or 3-positions were investigated with maleimide and maleimide derivatives bearing N-methyl, N-allyl, and N-phenyl substituents. Computational predictions indicate that electronic and regiochemical effects of furan substitution significantly influence their Diels-Alder reactivity with maleimide, with reaction free energies of exo adduct formation ranging from ΔG = -9.4 to 0.9 kcal/mol and transition state barriers to exo adduct formation ranging from ΔG(?) = 18.9 to 25.6 kcal/mol. Much less variation was observed for the reactivity of N-substituted maleimide derivatives and furan, with reaction and transition state free energies each falling within a range of 1.1 kcal/mol. Dynamic exchange experiments monitored by (1)H NMR spectroscopy support computational predictions. The results indicate the reactivity and reversibility of furan-maleimide cycloadditions can be tuned significantly through the addition of appropriate substituents and have implications in the use of furan and maleimide derivatives in the construction of thermally responsive organic materials.     

Application of polymaleimidostyrene as a convenient immobilization reagent of enzyme in biosensor

Sulfhydryl groups of glucose oxidase (GOD) were reacted with maleimide groups of polymaleimidostyrene (PMS) which was coated onto the porous carbon sheet, and the carbon sheet immobilized by GOD was combined with an oxygen electrode to fabricate a glucose sensor. The activity of thiolated GOD immobilized to PMS is much larger than that of native GOD immobilized to PMS. The good linear relationship of glucose and oxygen current response was obtained in a concentration range from 0.1 to 2 mM and upper limit of linear range was found to be 3.0 mM. The immobilized GOD activity is highly dependent on pH at immobilization and the maximum activity was obtained at pH 5.5, probably because the SH groups of GOD that are indispensable for generation of enzyme activity is not exposed at this pH. It was found that PMS is very effective reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups.     

Reactivity of double bonds of substituted <Emphasis Type="Italic">p</Emphasis>-(methacryloyloxy)-<Emphasis Type="Italic">N</Emphasis>-phenylmaleimides in free-radical polymerization

The thermoinduced free-radical polymerization of model systems, namely, methyl methacrylate and N-phenylmaleimide derivatives, has been studied. It has been shown that, in the polymerization of p-(methacryloyloxy)-N-phenylmaleimides, the yield of the polymer and its molecular-mass distribution (mono-or bimodal) and polydispersity depend on the degree of substitution and the nature of a substituent in a double bond of a maleimide fragment. The structure of the maleimide fragment, in turn, determines its capability to copolymerize with the methacryloyl group of a bifunctional monomer as well as the occurrence of chain-transfer and termination reactions.     

Sorption and immobilization of cellulase on silicate clay minerals

The interaction of organic molecules with mineral surfaces is a subject of interest in a variety of disciplines. Enzymes are able to be sorbed and immobilized by clay minerals and humic colloids in soil environment. The present study was done to elucidate some aspects of sorption and immobilization of cellulase on soil components by analysis of the sorption, and immobilization of cellulase on Avicel, a soil sample, illite, kaolinite, montmorillonite, and palygorskite. Palygorskite displayed the highest sorption capacity. Sorbents coated with hydroxyaluminum displayed significantly higher capacity than uncoated sorbents. The positive effects of Al(OH)(x) coating on sorption capacities of the different sorbents were not equal. The effect decreased in the order soil > palygorskite > kaolinite > Avicel > montmorillonite > illite. The amount of sorbed cellulase desorbed from external surfaces of soil was quite low (about 16%), especially in coated samples (about 6%). X-ray diffraction analysis of K-montmorillonite and Ca-montmorillonite showed that Al(OH)(x) was intercalated between the montmorillonite layers. Immobilization of cellulase on the sorbents did not result in expansion of their crystal structures. Therefore, it may be concluded that the amount of cellulase immobilized on internal surfaces of the sorbents was negligible.