口蹄疫病毒免疫活性串联片段FB的表达及免疫原性测定(英)

将口蹄疫病毒(FMDV)免疫串联片段FB克隆至原核表达载体pBAD/TOPO中,经鉴定后得到重组质粒pBAD-FB,将此重组质粒转化到受体菌TOP10中,用诱导剂阿拉伯醛糖分别以不同的浓度进行诱导,并在不同诱导时间进行采样,经处理后做SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹分析.结果发现以终浓度为0.002%的阿拉伯醛糖进行诱导,4 h后表达可达到高峰,其大小约为26 ku,软件扫描结果显示,FB融合蛋白的表达量占细菌总蛋白的28.9%,能与抗FMDV抗体发生特异性反应,融合蛋白以包涵体和可溶形式存在.将融合蛋白的可溶性组分用50% Ni-NTA树脂过柱纯化并抽提融合蛋白的包涵体,经过洗涤后分别制成油乳剂疫苗,经皮下注射免疫豚鼠,用乳鼠中和试验测定豚鼠血清中和指数,并用口蹄疫病毒对豚鼠进行攻毒.结果表明,用此融合蛋白的纯化产物和包涵体免疫豚鼠能诱导产生高滴度的中和抗体,对病毒的攻击提供100%的免疫保护.     

Purification, specificity, and hypervariable region sequence of anti-pneumococcal polysaccharide antibodies elicited in a single rabbit.

Four homogeneous antibodies to type VIII pneumococcal polysaccharide (S8) were isolated from the serum of a single rabbit (3322) by affinity chromatography on an S8 immunoadsoebent by utilizing gradient elution with cellobiose and NaCl. The binding properties of these antibodies were determined by a radioimmunoassay with 125I-bovine gamma-globulin-S8. Cellobiose (a disaccharide unit of S8) was the immunodominant group of each of the four antibodies, but each antibody bound to this disaccharide with different relative affinities. The amino acid sequences (positions 0-40) of three of the four antibody light chains were each different both in framework and first hypervariable region sequences. The fourth antibody light chain has a blocked amino terminus. These findings indicate that antibodies elicited by a relatively simple antigen and examined at one time during the course of immunization in a single rabbit may exhibit common specificities for an oligosaccharide determinant, yet have different binding affinities for that determinant as well as different primary structures in the complementarity (hypervariable) regions and framework regions.     

Characterization of an arabinogalactan-protein from suspension culture of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Suspension cultures of Echinacea purpurea have been established in MS medium supplemented with 2,4-D and an arabinogalactan-protein (AGP) was purified from the secreted soluble polymers by precipitation with ethanol, followed by precipitation with β-glucosyl Yariv reagent. It revealed typical features of AGPs: a high amount of polysaccharide (90% w/w) with the dominating monosaccharides galactose and arabinose and some glucuronic acid, and a small protein moiety (10% w/w) with the main amino acids Ala, Hyp, Glx, Ser, Asx and Thr. Linkage- and NMR-analyses showed the polysaccharide part to be composed of a branched core-polysaccharide of 3-, 6- and 3,6-linked Galp residues with terminal Araf, Arap, Galp and GlcAp residues. Compared to an AGP from pressed juice of the aerial parts of Echinacea purpurea, differences particularly in terminal arabinose mono- and oligosaccharides in arabinogalactan (AG) side branches could be detected. Testing of different AGP-antibodies with both AGPs confirmed the results of the analytical investigations. Binding of AGPs from plant and cell cultures to LM2, a monoclonal AGP-antibody reacting with a GlcA containing epitope, was comparable. The reactivity of a monoclonal antibody raised against the AGP from the plant recognizing a galactan epitope was also nearly similar with both AGPs. In contrast, polyclonal antibodies raised against the AGP from the plant and directed against an Araf-containing epitope of the AG side branches showed nearly no cross reactivity with the AGP from cell culture.     

Isolation of an antigenic oligosaccharide fraction from Leptospira interrogans serovar canicola with a monoclonal antibody

An oligosaccharide fraction containing the antigenic determinant of lipopolysaccharide antigen (TM antigen) from Leptospira interrogans serovar canicola, recognized by a monoclonal antibody (CT3) which agglutinates serovars canicola and broomi, was isolated by formic acid and successive sulphuric acid hydrolyses. Separation of the antigenic compounds was done by Bio-Gel P-2 and Sephadex G-25 gel filtration, and high-performance liquid chromatography with two different columns. The fraction finally obtained was a mixture of two oligosaccharides, both of which migrated as a single spot having a slightly higher mobility than an authentic tetrasaccharide (stachyose) on thin layer chromatography. The fraction contained rhamnose, arabinose and two major and two minor unknown sugars which were shown to be N- or O-acetylated and/or O-methylated sugars by nuclear magnetic resonance. The fraction inhibited the binding of CT3 antibody with TM antigen in enzyme-linked immunosorbent assay and microscopic agglutination of serovar canicola with the antibody. The inhibitory activity was destroyed by periodate oxidation or mild alkaline treatment, but was resistant to sodium borohydride reduction.     

Heavy and light chain variable region sequences and antibody properties of anti-phosphotyrosine antibodies reveal both common and distinct features.

Phosphotyrosine and similar analogs have been used to elicit antibodies that have found widespread use in the study of cellular tyrosine phosphorylation. In order to better understand the anti-phosphotyrosine immune response and to elucidate the details of the specific association between a tyrosine phosphate and an antibody combining site, we have undertaken a detailed comparison of antibody stability, specificity, apparent affinity, and primary structure for eight different anti-phosphotyrosine antibodies derived from immunizations with three different antigens. Two of these, 2G8 and 1G2, were derived from an immunization using azobenzylphosphonate conjugated to carrier, and five others, Py2, Py20, Py42, Py54, and Py69, were the products of an immunization with phosphotyrosine conjugated to carrier. Each of these anti-hapten antibodies was an IgG. One antibody, 129, an IgM, was the result of an immunization with a mixture of tyrosine-phosphorylated proteins which had been purified from growth factor treated cells. We found that antibody binding was significantly inhibited by millimolar levels of divalent cations or high concentrations of monovalent salt, with the exception of the antibody 129 where binding was significantly enhanced by both. Under optimal conditions, the highest apparent affinities for phosphotyrosine were observed for antibodies Py69 and Py20 (10(-6)-10(-7) M) and the lowest for 129 and 1G2 (10(-3)-10(-4). The heavy and light chain variable regions of seven of these antibodies were cloned and sequenced and a predominant anti-phosphotyrosine response was observed. The light chains of these antibodies could be assigned to one of two major VK groups, VK10 and VK19, with sequence identity between the different light chains of each class ranging from 65 to 100% at the amino acid level. Similar sequence identity was found among the heavy chain sequences (89-98% identity at the amino acid level) with the exception of one antibody, 2G8, which was only distantly related to the others (61-64% amino acid identity). These heavy chains belong to the same heavy chain family, J558. Two of the antibodies, Py20 and Py69, were clearly derived from the same progenitor cell since both share a highly unusual apparent V-D-D-JH organization. However, a significant level of somatic mutation has occurred between the two antibodies resulting in subtle changes in their apparent affinity and specificity.     

Antigenic determinants of the HLA-B7 molecule; Bw6- and B7-specific determinants are spatially separate

Monoclonal antibodies that bind HLA-B7 were used to show that the B7-specific determinant is at a topologically different site from that of the broad polymorphic, Bw6 determinant. The relationship to other antigenic determinants defined by monoclonal antibodies was also assessed. These results were independently obtained in four ways: (1) by cellular blocking assays, in which there was no inhibition of 125I-B7 antibody binding in the presence of Bw6 antibody and no inhibition of 125I-Bw6 antibody binding in the presence of B7 antibody; (2) cellular binding assays under conditions of antibody saturation showed the binding of B7-specific and Bw6 antibodies were additive; (3) solid-phase radioimmune assays demonstrated enhancement between B7-specific and Bw6 antibodies; (4) analysis of antigen antibody complexes by size-exclusion high pressure liquid chromatography showed Bw6 and B7 antibodies could form tetramolecular complexes with papain-solubilized HLA-B7. Limitations were encountered in using cellular blocking assays to map antigenic determinants of HLA-B7. These assays can produce blocking in cases where two antibodies are not competing for an antigenic determinant. Mapping antigenic determinants with assays using purified HLA-B7 as the antigenic target, in addition to cell-based assays, provided a more accurate picture.     

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.     

Differential regulation of the immune response to SRBC by monoclonal antibodies to interferon-gamma

Three monoclonal antibodies against different epitopes of interferon-gamma (IFN-gamma) were used to assess its role as a normal immunomodulatory molecule. Two of these antibodies were able to reduce significantly the primary antibody response to sheep erythrocytes in an in vitro culture system. One of these two antibodies has been reported to suppress both the antiviral and macrophage activation factor activities of IFN-gamma by binding to its carboxyl terminus. These findings indicate that IFN-gamma is an important lymphokine for the maximum expression of the immune response and that it acts via the carboxyl terminus of the molecule. This antibody suppressed the immune response only when added at the initiation of culture, suggesting that the action of IFN-gamma is on an early component of the response. The third monoclonal antibody, which binds to the amino end of IFN-gamma, did not suppress the in vitro response. However, it was able to block the effects exerted by an immunosuppressive dosage of exogenous IFN-gamma on in vitro antibody production. These results indicate that the immunosuppression vitro antibody production. These results indicate that the immunosuppression induced by the addition of IFN-gamma to a primary antibody response and the role that it plays in that response are mediated through different sites on the molecule and, therefore, probably by different mechanisms.     

用硫氰酸盐洗脱法直接测定噬菌体抗体的相对亲和力

抗体与相应抗原的结合可以被硫氰酸盐洗脱而解离,抗体的亲和力越高则解离所需要的硫氰酸盐浓度就越大,这一原理在传统的免疫学实验中被用来测定单克隆抗体或多克隆抗体的相对亲和力。如果证明该原理同样适用于噬菌体抗体库技术,则可以建立一种直接测定噬菌体抗体相对亲和力的简便方法。首先将噬菌体抗体与工作浓度的硫氰酸盐共孵育,以证明这一过程并不影响其后的ELISA反应,然后参照硫氰酸盐洗脱法测定完整抗体分子和Fab段相对亲和力的方法,在ELISA实验中以酶标抗M13为二抗检测了5个单克隆噬菌体抗体的相对亲和力,并与相对应的可溶性Fab段的相对亲和力进行了比较。被测抗体中包括3个克隆的抗角蛋白抗体和2个克隆的抗乙型肝炎表面抗原抗体。结果发现,用硫氰酸盐洗脱法测定5个噬菌体抗体所得到的亲和力排序与测定其相应可溶性Fab段所得结果一致,表明硫氰酸盐洗脱法可作为一种简便快速的方法用来直接测定噬菌体抗体的相对亲和力。     

Determination of antibody affinity by ELISA. Theory

New approaches for the measurement of antibody affinity by ELISA are suggested and considered theoretically. It was shown that not only more precise and more convenient in comparison to that suggested earlier, but also more informative graphical representation of the experimental data in the appropriate coordinate could be used for evaluation of antibody affinity. The following cases were considered: (i) determination of antibody affinity for one kind of univalent antibodies, (ii) determination of antibody affinity for one kind of bivalent antibodies, (iii) determination of antibody affinity for two kinds of univalent antibodies, which are in a mixture, and (iv) determination of antibody affinity for two kinds of bivalent antibodies, which are in a mixture. Advantages and disadvantages of the different approaches are discussed.     

Characterization of humanized antibodies secreted by Aspergillus niger

Two different humanized immunoglobulin G1(kappa) antibodies and an Fab' fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex(6)GlcNAc(2) to Hex(15)GlcNAc(2). An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.     

Malignant Glial Neoplasms: Definition of a Humoral Host Response to Tumor-Associated Antigen(s)

There is increasing evidence that human tumors possess tumor-associated neo-antigens. The host mounts an immunological response to these antigens, as evidenced by the detection of circulating humoral antibodies in a variety of human neoplasia.An indirect immunofluorescent antibody technique was employed to detect antibodies to tumor-associated antigens in the sera of patients with malignant gliomas. Viable single cell suspensions were used to demonstrate antibodies to surface contents of tumor cells and cell preparations were snap-frozen at −160° C to demonstrate antibodies to cytoplasmic components of tumor cells. After incubation with serum, the preparations were treated with polyvalent sheep antihuman globulin conjugated to isomer-1-fluorescein isothiocyanate, washed, and examined with a Leitz incident fluorescent microscope.Of the 17 sera from histologically proven malignant glial neoplasm patients, 2 (11%) were positive for an autologous surface antibody reaction. Five (23%) of 21 were positive for an autologus cytoplasmic antibody, however, 10 (47%) of 21 of the sera gave a positive reaction for cross-reacting cytoplasmic antibodies when tested with a battery of tumor cells obtained from different patients with malignant glial tumors.No reaction was observed with normal brain tissue. Absorption studies indicated the presence of a tumor-associated antigen.This study demonstrated that certain patients with malignant gliomas possess circulating antibodies to cytoplasmic components of their own tumor cells. The fact that a number of sera cross-reacted with tumor cells obtained from different patients suggests that antigenic cross-reactivity exists between malignant glioma cells from different patients. It is suggested that with further refinement, immunofluorescent detection of antibodies could evolve as a useful diagnostic adjunct in malignant glioma.     

The kinetics of antibody binding to membrane antigens in solution and at the cell surface.

The reaction kinetics of 125I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+1), dissociation rate constants (k-1) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-1 antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-1 for the univalent interaction, in which case all binding was bivalent. (ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 10(5)--1.1 x 10(6) M-1.s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently. However, the k-1 values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.     

Enhanced neutralization of flavivirus by monoclonal antibodies against Negishi virus

Ten monoclonal antibodies against Negishi virus were classified into 3 groups and 7 types according to the reaction patterns to Negishi virus by the hemagglutination inhibition (HI) test and by several kinds of neutralization tests. When kinetic neutralization was applied to these monoclonal antibodies at the same HI titer of 1:64, the initial slope and the persistent fraction of the kinetic curve was varied in each antibody. In the double-kinetic neutralization test, neutralization did not proceed further when the second antibody was the same type as the first one. When the second antibody was a different type from that of the first one, neutralization proceeded further. The mixtures of 4 monoclonal antibodies classified as different groups and types remarkably enhanced neutralization in the kinetic assay. These results suggested that an assortment of antibodies is needed for effective neutralization of Negishi virus and that a multi-hit model is likely operating in the neutralization of Negishi virus.     

Spatially distinct allodeterminants of the H-2Kk molecule as detected by monoclonal anti-H-2 antibodies

In an attempt to delineate spatial relationships between various allodeterminants of cell surface MHC antigens, competitive binding studies were performed using 4 different monoclonal antibodies, each of which reacted with the H-2Kk antigen but with different serologic specificities. Competition was studied by examining the effect of unlabeled antibodies on the binding of each 125I-labeled antibody to spleen cells of the H-2a haplotype. Mutual inhibition was observed between 2 of the antibodies, and a 3rd antibody of lower affinity was inhibited by the first 2 antibodies but did not itself inhibit the binding of these antibodies. The 4th antibody did not block the binding of the other 3 labeled antibodies, and binding of this 4th labeled antibody was only partially inhibitable by the other 3 antibodies. These results indicate the presence of at least 2 spatially distinct allodeterminants on H-2Kk molecules expressed on the cell surface.     

Antibodies specific to modified glyceraldehyde-3-phosphate dehydrogenase induce inactivation of the native enzyme and change its conformation

The antibodies specific to an inactive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus prepared by the treatment of the tetrameric holoenzyme with glutaraldehyde were obtained. They were purified from the pool of polyclonal rabbit antibodies to GAPDH with the use of immobilized GAPDH cross-linked by glutaraldehyde as an affinity sorbent. Such antibodies were capable of interacting with the native enzyme, inducing its time-dependent inactivation; the effect was different with the apo- and holoenzyme forms. Differential scanning calorimetry of the purified [GAPDH].[antibody] complex revealed a large shift of the temperature corresponding to the maximal heat capacity of the holoenzyme towards the lower temperature. Again, the effect appeared to be different with the apoenzyme. Together, the results are consistent with the hypothesis that a specific antibody is able to exercise a certain strain on the target protein, altering its conformation toward the structure of the species which served to select the antibody. The possibility of preparing selective enzyme inhibitors based on the antibodies specific to inactive enzyme conformations is considered.     

Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes

Human immunodeficiency virus-1 (HIV-1) infects cells by membrane fusion that is mediated by the envelope proteins gp120/gp41 and the cellular receptors CD4 and CCR5. During this process, some conserved viral epitopes are temporarily exposed and may induce a neutralizing antibody response when fixed in the fusogenic conformation. These transient structures are conserved and may be effective antigens for use in an anti-HIV-1 vaccine. In this study we tested different conditions of preparation of fusion complexes inducing neutralizing antibodies against both R5 and X4 tropic HIV-1 strains. Cell lines expressing HIV-1 gp120/gp41 and CD4-CCR5 were prepared and conditions for producing fusion complexes were tested. Complexes produced at different temperature and fixative combinations were used to immunize mice. Results indicated that (a) fusion complexes prepared at either 21 degrees C, 30 degrees C or 37 degrees C were immunogenic and induced neutralizing antibodies against both R5 and X4 HIV-1 heterologous isolates; (b) after extensive purification of antibodies there was no cytotoxic effect; (c) complexes prepared at 37 degrees C were more immunogenic and induced higher titers of neutralizing antibodies than complexes prepared at either 21 degrees C or 30 degrees C; (d) the fixative used did not affect the titer of neutralizing antibodies except for glutaraldehyde which was ineffective; (e) the neutralizing activity was retained after CD4-CCR5 antibody removal. The production of higher titers of neutralizing antibody with fusion complexes prepared at 37 degrees C, as compared to lower temperatures, may be related to the induction of antibodies against many different conformation intermediates that subsequently act synergistically at different steps in the fusion process.     

Sequential expansion of antibody heterogeneity during the response to bacterial alpha-amylase

By analyzing antibody heterogeneity during the primary immune response to bacterial a-amylase (B alpha A) in high-responder F1 hybrid mice between C57BL/6 (B6) and C3H/He (C3) mice with the use of isoelectric focusing (IEF), it was shown that the maturation of the primary IgG antibody response consisted of at least two stages. The response of every mouse tested was initiated with the production of specific antibody focused as a limited set of bands in a narrow pH range, and the subsequent rise in antibody titer was associated with the sequential expansion of the spectra involving the appearance of new bands in the pH gradient adjacent to the initial bands. A further rise was accompanied only by intensified staining of the pre-existing bands. These two stages were distinguishable regardless of the antigen dose, although increasing the dose led to widely distributed spectra of focused antibodies and an early shift from the first stage to the second. The sequential expansion of spectra following the appearance of initial antibodies with limited isoelectric point (pI) values was not unique to the anti-BaA antibody response, because similar results were obtained with the antibody response to an immunologically unrelated antigen, Taka-amylase A (TAA). Thus, the appearance of initial antibodies in a limited pH range, overlapping among all F1 hybrids tested, is not a direct reflection of similarity in the determinant specificities of these antibodies among different mice.     

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.