Affinity of mouse immunoglobulin G subclasses for sialic acid derivatives immobilized on dextran-coated supports

High-performance liquid affinity chromatography is a powerful method for the purification of biological compounds owing to its specificity, rapidity and high resolution. In our laboratory, we develop chromatographic supports based on porous silica beads. However, in order to minimize non-specific interactions between the inorganic surface and proteins in aqueous solution, the silica beads are coated with modified dextran. As previously reported, many affinity ligands can be covalently grafted onto dextran-coated silica. In this study, N-acetylneuramic acid, which belongs to the sialic acid family and is present in immunoglobulin G (IgG) epitopes, is used as an active ligand. The interactions of this affinity support and IgG subclasses are analyzed. This immobilized ligand enables purification of IgG3 antibodies.     

Analyses of Idiotypes on Anti-α-1, 6-Dextran Antibodies in Mice

When mice of strains C57BL/6, C3H/He, and BALB/c were immunized with native dextran B512, only a small amount of IgM antibody was produced, but a substantial amount of anti-dextran antibody of IgG class was produced after immunization with a conjugate of dextran T10 and keyhole limpet hemocyanin regardless of the mouse strain used. Isoelectric focusing (IEF) spectra revealed limited heterogeneity of anti-dextran antibody of IgG class with strict consistency in all individual sera from C57BL/6 mice, even after secondary immunization, whereas antibodies from C3H/He and BALB/c mice showed more heterogeneous IEF spectra with some individual variations. Rabbit anti-idiotypic (Id) antibodies were raised by immunization with a subfraction of anti-dextran antibody of IgG class from C57BL/6 mice, which showed major bands focused at around pH 7.7 upon IEF. It was found by using the anti-Id antibodies that virtually all anti-dextran antibody molecules of both IgG and IgM classes from C57BL/6 mice possessed common Id determinants which can be classified into two specificities, one specific for antibody from C57BL/6 mice and the other cross-reactive with antibodies from BALB/c and C3H/He mice. About 80% of the antibody molecules from BALB/c and less than 20% of those from C3H/He mice were positive for the interstrain cross-reactive Id. Both Id determinants seemed to be closely related to the antigen binding sites, or at least to reside in the vicinity of the antigen binding sites of anti-dextran antibody.     

New antibody purification procedure using a thermally responsive poly(N-isopropylacrylamide)–dextran derivative conjugate

Through their specificity and affinity, antibodies are useful tools in research and medicine. In this study, we investigated a new type of chromatographic method using a thermosensitive polymer for the purification of antibodies against a dextran derivative (DD), as a model. The thermally reversible soluble–insoluble poly(N-isopropylacrylamide)–dextran derivative conjugate, named poly(NIPAAm)–DD, has been synthesized by conjugating amino-terminated poly(N-isopropylacrylamide) to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide. On one hand, this report describes the two steps of poly(NIPAAm)–DD conjugation and characterization. On the other hand, the poly(NIPAAm)–DD conjugate was used as a tool to purify polyclonal antibodies in serum samples from rabbits subcutaneously immunized with the derivatized dextran. Antibodies were purified and quantified by immunoenzymatic assays. Our results indicate that antibodies recognized both DD and poly(NIPAAm)–DD. In contrast, they did not bind to native poly(NIPAAm) or poly(NIPAAm) conjugated with another anionic dextran. We conclude that the conjugation of a polysaccharide to poly(NIPAAm) leads to an original and efficient chromatographic method to purify antibodies. Moreover, this novel method of purification is rapid, sensitive, inexpensive and could be used to purify various types of antibodies.     

Estimation of antibodies specific for dextran.

Methods are described for the isolation and characterization of picogram quantities of anti-dextran antibodies. 14C-dextrans produced by using the dextransucrases of Leuconostoc mesenteroides strains B1355 and B512 were used in a radioimmunoassay. The specificity of this assay was verified by using cell cytoplasmic lysates from mouse plasmacytomas, J558 (anti-alpha 1 leads to 3 dextran) and W3129 (anti-alpha 1 leads to 6 dextran). Dextran produced by strain B1355 and insolubilized with epichlorohydrin was used as an immunoabsorbent.     

Analysis of the diversity of murine antibodies to dextran B1355. I. Generation of a larger, pauci-clonal response by a bacterial vaccine.

Mice immunized with a combination of dextran B1355 in adjuvant followed by three injections of 2 x 10(9) Escherichia coli B organisms produced an average of 14.5 mg/ml of anti-dextran antibodies. It was demonstrated that the stimulating effect of E. coli B was due to antigenic determinants cross-reactive with B1355 and not solely because of adjuvant properties of the organism. The anti-dextran antibodies were distributed among both 7S and 19S components. Isoelectric focusing of the 7S antibodies showed several spectrotypes of antibody, most of which were shared by the majority of the individual sera. The limited spectrotypic heterogeneity of the 7S antibodies was supported by idiotypic studies. Thus, a heterologous, anti-idiotypic serum, rabbit anti-M104, was prepared which distinguished between two closely related myeloma proteins, M104 and J558,with specificity for alpha-(1 leads to 3) dextran. This antiserum demonstrated that some, but not all, of the 7S and 19S anti-dextran antibodies possessed variable region determinants cross-reactive with M104.     

Expression of a cross-reactive idiotope on naturally occurring murine antibodies against 3-fucosyllactosamine, levan, and dextran

We recently identified a cross-reactive Id (6C4) that is expressed on the H chain of many BALB/c mAb against the 3-fucosyllactosamine (3-FL) determinant, Gal(beta 1-4) (Fuc(alpha 1-3] GlcNAc-R. The VH segments of seven mAb that we recently sequenced are encoded by VH441, which also encodes VH segments of antibodies against galactan, levan, and dextran. To analyze the expression of the 6C4 Id on naturally occurring anti-carbohydrate antibodies, we isolated 6C4+ antibodies by affinity chromatography from pools of normal BALB/c serum. Approximately 20 to 30% of antibodies against 3-FL and levan, and all antibodies against dextran, were removed from the sera by passage over a column containing mAb 6C4. Absorption of the eluate with 3-FL beads removed anti-3-FL antibodies but not anti-dextran or anti-levan. The expression of a cross-reactive Id on naturally occurring antibodies against several carbohydrate Ag suggests that these antibodies may participate in an Id network. We also reported previously that BALB/c mice have naturally occurring anti-3-FL antibodies and respond well to immunization against this determinant, whereas C57BL/6 mice do neither. To examine the role of the Igh-C allotype in the regulation of the anti-3-FL response, we studied congenic strains of BALB/c and C57BL/6 mice. Both congenic strains produced anti-3-FL antibodies in response to immunization, but only C.B-20 mice exhibited naturally occurring antibodies. These data suggest that the naturally occurring and elicited antibody responses against 3-FL are differentially regulated.     

Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.     

Immunochemical similarity of synthetic alpha-(1----3)-branched D-glucans to dextran B1375

Anti-dextran B1375 antibodies were raised in rabbits by injecting formalin-killed Leuconostoc mesenteroides strain NRRL B1375, and the anti-dextran serum was used to examine native dextran B1375, and synthetic linear and four alpha-(1----3)-branched alpha-(1----6)-D-glucopyranans for similarities. The antiserum reacted with the homologous dextran B1375 and also with all the synthetic linear and branched glucans. Precipitation and precipitation-inhibition studies indicated that the antiserum contained at least three groups of antibodies with different specificities, the first specific for linear alpha-(1----6)-D-glucopyranan structure, the second specific for alpha-D-glycopyranosyl-(1----3)-branching and the last specific for another, unknown structure present in the dextran B1375 molecule. Two samples of the synthetic branched glucans were shown to be immunochemically the most similar to natural dextran B1375 by inhibition experiments.     

用免疫亲和层析法纯化萝卜 PHGPx 天然蛋白

萝卜磷脂氢谷胱甘肽过氧化物酶 (RsPHGPx) 是一个定位于线粒体的蛋白质 . 为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的 RsPHGPx. 用重组 RsPHGPx 蛋白免疫兔子获得了抗 RsPHGPx 的多克隆抗血清,以重组 RsPHGPx 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗 RsPHGPx 的抗体 . 将纯化好的抗体偶联到一个 N- 羟基琥珀酰亚胺 (NHS) 预先激活的琼脂糖柱子上,装配成一个以单特异性的抗 RsPHGPx 抗体为配体的免疫亲和层析柱 . 经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案 . 按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质 . 免疫印迹分析表明,该蛋白质被抗 RsPHGPx 的抗血清特异识别 . 酶活性分析表明,该蛋白质具有显著的 PHGPx 活性 . 这些结果表明,纯化到的特异蛋白质是萝卜的 RsPHGPx 天然蛋白 . 这是首个关于定位于植物细胞器的 PHGPx 蛋白纯化的报道 . 这一结果为准确测定 RsPHGPx 信号肽的切割位点奠定了基础,并将有助于对植物 PHGPx 的亚细胞定位机制及其生理功能的深入研究 .     

Human alfa-fetoprotein: isolation and production of monoclonal antibodies.

Alfa-fetoprotein from human cord serum was purified in a single step by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B with a final recovery of alfa-fetoprotein of about 90% and a purification factor of 900. The purified preparation was homogeneous on SDS-PAGE and native PAGE running with a relative molecular weight of 72,000. Monoclonal antibodies against this purified preparation were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. 50% of culture wells exhibited hybrid growth and 7% of these wells contained anti-AFP secreting hybrids. Positive hybrid cells were cloned twice by the limiting dilution method and 8 clones were obtained that secreted monoclonal antibodies. Five of these cell lines (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were selected at random for purification and characterization purposes. All 5 cell lines secreted monoclonal antibodies of IgG1 subclass which were purified by affinity chromatography on Protein A- Sepharose CL-4B column with a final recovery of 80% and a purification factor of about 13. The purified preparations were homogeneous on SDS-PAGE, native PAGE and IEF. The monoclonal antibodies were highly specific for human alfa-fetoprotein as determined by Western blotting. The affinity constants (K) of these Mab ranged from 10(6) to 10(9) l/mol.     

Influence of the nature of coupling agents on insulin adsorption on supports grafted with sialic acid for high-performance affinity chromatography

Porous silica exhibits excellent mechanical properties for use as a stationary phase for high-performance liquid chromatography. However, negative surface charges make it unusable in its native state. For this reason, silica beads are coated with dextran polymers carrying a calculated amount of diethylaminoethyl groups. Both the minimization of non-specific interactions and the hydrophilic character of such supports allow their functionalization with biospecific ligands and finally their use in high-performance affinity chromatography of biological products. The use of these modified supports in high-performance affinity chromatography requires a better understanding of various characteristics of stationary phases. For this purpose, several techniques were utilized, in particular, size-exclusion chromatography and adsorption of radiolabelled albumin. These methods provided complementary information on the structure of these supports. Coated silica-based supports were functionalized with sialic acid by means of different coupling agents. The affinity of these supports for insulin was determined by the establishment of adsorption isotherms and by high-performance affinity chromatography, to evidence the relationships between structural characteristics of the supports and their separation properties. The study of interactions between these supports and insulin allowed us to show the importance of the coupling method on the performances of supports in affinity chromatography.     

Antibody response of BALB/c mice to dextran B1355S: alterations in the expression of an idiotype associated with the depletion of idiotype-binding cells

In this study, we established that BALB/c mice recognize and respond to the idiotype (M104E IdI) of a major dextran-specific clonotype within the BALB/c mouse repertoire. This idiotype recognition is established by demonstrating the presence of idiotype-binding cells and by the production of antibodies specific for the private M104E idiotype. To determine whether or not the idiotype-recognizing cells play a regulatory role during an immune response to dextran, the idiotype-binding cells were selectively removed either by panning or by radiation-induced killing. Two significant effects are observed when the depleted spleen cells are immunized with dextran. First, there is a substantial increase in the proportion of anti-dextran antibodies that are M104E IdI+. The second effect of the idiotype-specific cell depletion is the production of significant amounts of M104E IdI+ immunoglobulin molecules which do not bind dextran. The depletion experiments produced no alteration in the concentration of anti-dextran antibodies found in the serum or in the number of dextran-specific PFC in the spleen. The data indicate that idiotype-reactive cells can play a role in regulating the level of individual clonotype expression (i.e., the M104E clonotype), but that an alternative mechanism must exist for regulating the absolute amount of anti-dextran antibody produced after immunization.     

Serum antibody and cellular immune response in mice to dextran B512.

Serum antibodies to dextran started to appear 3 days after immunization of C57BL/6 mice. Synthesis of IgM antibodies was followed by IgG3 and IgGA. Other immunoglobulin classes (IgG1, IgG2b, and IgG2a) were very low or absent. The immune response to dextran was also thymus independent with regard to IgG3 and IgA synthesis as demonstrated by the use of nu/nu mice. CBA and C57BL/6 mice were high responders to dextran with regard to IgM synthesis. C57BL/6 mice produced high levels of IgG3 and IgA antibodies, whereas CBA, A/J, and A.TL only synthesized IgM antibodies. A/J and A.TL strains were most frequently low responders with regard to IgM synthesis and CBA/N mice were completely nonresponders with regard to all immunoglobulin classes. The ability to produce anti-dextran antibodies increased with age in high responder strains. This was most pronounced for IgG3 and IgA antibodies, which reached adult levels 3 months after birth. The affinity of anti-dextran antibodies was high and homogeneous in antisera from C57BL/6 mice. Preimmune matural antibodies and antibodies from immunized low responder strains had a low and variable affinity for dextran.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

CRF immunoreactive peptides in the human hypophysis: a cautionary note

By monitoring with a non competitive enzyme linked immunosorbent assay (ELISA), corticotropin releasing factor (CRF)-like immunoreactive material was isolated from the human hypophysis. After acid extraction of peptides from frozen human hypophyses, the purification was achieved by affinity chromatography using purified anti-ovine-CRF IgG bound to a solid phase and then by two HPLC steps using an alkylsilane-bonded large pore size silica. Two CRF-like peptides were purified: discrete immunoreactive peaks coinciding with an optical density peak at 215 nm. Although these peptides were recognized by ELISA, they were not recognized in an RIA using the same anti-ovine-CRF serum and ovine CRF-41 as tracer. Neither of these CRF-immunoreactive peptides had any effect on either the spontaneous or stimulated ACTH release in the perfused isolated anterior pituitary cell bioassay.     

Monoclonal anti-alpha (1----3) dextran antibodies of Igha BALB/c and Ighb C.B20 mice display striking similarities

Allotype Ighb congenic C.B20 mice when immunized with dextran B1355S are unable to produce anti-alpha (1----3) dextran antibodies that express the VH-associated cross-reactive IdX idiotype. This intrastrain-specific idiotype is normally associated only with the anti-dextran response of Igha mice of which BALB/c is a prototype strain. In this study we have obtained monoclonal hybridoma antibodies specific for the alpha (1----3) glucosidic linkage of dextran from C.B20 mice that were presensitized with rabbit anti-IdX antibodies. These antibodies display the light chain isotype distribution, the H chain amino terminal sequence, share VH-associated IdX idiotypic determinants, and finally the similar fine specificity for dextrans observed for anti-alpha (1----3) dextran antibodies of BALB/c mice.     

High-performance liquid chromatography of proteins on deformed nonporous agarose beads: immuno-affinity chromatography, exemplified with human growth hormone as ligand and a combination of ethylene glycol and salt for desorption of the antibodies

An affinity chromatography column packed with nonporous agarose beads derivatized with human growth hormone via carbonyldiimidazol was used for the purification of antibodies against human growth hormone from antiserum. Desorption with 1 M sodium chloride in 60% ethylene glycol at pH 9.8 gave 100% total recovery of the antibodies, as measured by radioimmunoassay. The adsorption/desorption process is discussed in terms of hydrophobic and electrostatic interaction (these interactions may be involved in the bond between antibody and antigen in a cooperative fashion). The binding capacity of the column was estimated at about 50 micrograms of antibodies per gram sedimented agarose beads.     

High-performance affinity chromatography for the purification of heparin-binding proteins from detergent-solubilized smooth muscle cell membranes

Heparin and heparan sulfates are regulators of cellular events including adhesion, proliferation and migration. In particular, the antiproliferative effect of heparin on smooth muscle cell (SMC) growth is well described. However, its mechanism of action remains unclear. Numerous results suggest an endocytosis mediated by a still unknown heparin receptor on vascular SMCs. In order to identify a putative heparin receptor on SMCs that could be involved in heparin signalling, affinity chromatography supports were developed. In this paper, we describe high-performance liquid affinity chromatography (HPLAC) supports obtained from silica beads coated with dextran polymer substituted by a calculated amount of diethylaminoethyl functions. With a polysaccharide dextran layer, this type of support can be grafted with specific ligands, such as heparin, using conventional coupling methods. In a previous work, we demonstrated, using butanedioldiglycidyl ether, that silica stationary phases coupled to heparin could be used for the fast elution and good peak resolution of heparin-binding proteins. In the present work, an affinity chromatographic fraction of SMC membrane extracts was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and six heparin-binding proteins from dodecyloctaethyleneglycol monoether-solubilized SMCs were observed. Their M_r values were between 40 and 70 kDa, with three major protein bands at 66, 45 and 41 kDa. These results indicate the usefulness of the chromatographic method for purifying heparin binding proteins from SMC membrane.     

Role of dextran-specific suppressor T cells in the regulation of the immune response by a reactive form of dextran

Reactive forms of antigens or haptens have been shown to induce a state of hyporesponsiveness mediated in part by suppressor T cells. Injection of Balb/c x C57B16 F1 (CB6F1) mice with a reactive form of dextran B1355S (periodate oxidized dextran, dex-P) specifically reduced responses to dextran immunization within 1 day after dex-P treatment. This unresponsiveness lasted at least 23 days and required a reactive form of dextran for its induction since native dextran and oxidized/reduced dextran failed to induce tolerance. Furthermore, hyporesponsiveness could be induced by iv injection of dextran-coupled cells, especially peripheral blood lymphocytes, a result which suggests that in vivo coupling to cellular antigens is involved in dex-P-induced hyporesponsiveness. Suppression of the anti-dextran response could be transferred to normal mice with T-cell-enriched spleen cell populations from dex-P-injected mice. Interestingly, the presence of B cells in the transferred cell preparations interfered with detection of suppression. Both Lyt 1+2- and Lyt 1-2+ cells were involved in the dex-P-induced suppression; indeed, mixtures of these types of T cells led to the most profound degree of suppression. The suppressive activity of spleen cells from dex-P-injected mice could be removed by passage over dextran-coated plates. Moreover, cells eluted from the plates specifically suppressed anti-dextran responses of normal mice, indicating that dex-P injection induces a population of antigen-binding suppressor cells. This system will allow the study of the suppressor-T-cell receptors in a well-defined idiotypic system.