Production and characterisation of anti-theophylline monoclonal antibodies suitable for immunoassay

Spleen cells from BALB/c mice immunised with KLH-theophylline conjugate were fused with a mouse myeloma cell line P3-X63-Ag8.653, and antibody-producing hybrids were identified by enzyme immunoassay. Three cell clones were obtained, each capable of producing a unique monoclonal antibody to theophylline. Using these monoclonal antibodies, an immunoassay system for theophylline was developed.     

Chromosomal locus for staphylococcal enterotoxin B

The genetic locus of staphylococcal enterotoxin B (SEB) was investigated in the Staphylococcus aureus food-poisoning isolates, strains S6 and 277. Direct neutral sucrose gradient centrifugation analysis of sodium dodecyl sulfate-sodium chloride-mediated cleared lysates demonstrated that strain S6 contained a single 37S plasmid. Transductional analysis revealed that the 37S plasmid in S6 encoded for cadmium resistance (Cad) but not SEB. Additionally, elimination of cadmium resistance in S6 provided a plasmid-negative derivative that produced SEB at the same level as the parent. Examination of strain 277 showed two plasmids, a 37S species encoding for penicillin resistance (Penr) and a 21S species containing the gene(s) responsible for tetracycline resistance (Tetr). Elimination of the 37S, penr plasmid in 277 had no effect on SEB production, whereas introduction of the 21S tetr plasmid via transformation into strain 8325 (SEB--) did not confer enterotoxigenesis upon the transformants. The data obtained in this investigation suggest that the SEB gene(s) in these food-poisoning isolates of S. aureus is chromosomal.     

Production and characterization of monoclonal antibodies to Clostridium perfringens enterotoxin.

Four hybridoma cell lines producing monoclonal antibodies to Clostridium perfringens enterotoxin were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with the enterotoxin and its toxoid. An enzyme-linked immunosorbent assay indicated that the two antibodies, 2-B-4 and 3-G-10, bound to those regions that were located close each other; the others, 3-B-2 and 2-H-2, bound to other independent regions on the enterotoxin. Release of 51Cr from Vero cells with the enterotoxin was inhibited by either 2-B-4 or 3-G-10, both of which inhibited the binding of 125I-labeled enterotoxin to the cells. Neither binding nor cytotoxicity of the enterotoxin was affected by 2-H-2; 3-B-2 only barely inhibited the binding but neutralized the enterotoxin shown by 51Cr release. It seems justified to conclude that 3-B-2 blocks the toxic action after the enterotoxin has bound to Vero cells.     

Production of neutralizing monoclonal antibodies to Escherichia coli heat-stable enterotoxin.

In an effort to develop new approaches to the study and control of infectious diarrhea, we prepared murine monoclonal antibodies to the Escherichia coli heat-stable enterotoxin (STa). The toxin was purified from E. coli culture media and conjugated to bovine serum albumin. The STa-bovine serum albumin conjugate was used to immunize BALB/c mice, and the immune spleen cells from these mice were fused with SP2/0 myeloma cells. Resultant hybridomas were screened in an enzyme-linked immunosorbent assay protocol against 500 ng of STa-bovine serum albumin bound to microtiter wells as the solid-phase antigen. Five stable clones were selected and grown further in ascites fluid, which demonstrated anti-STa activity at dilutions of up to 1:500,000 in the enzyme-linked immunosorbent assay for heat-stable enterotoxin. In a competitive enzyme-linked immunosorbent assay format, the antibodies recognized several human and porcine strains of STa to various extents, but did not recognize E. coli heat-labile toxin, cholera toxin, or staphylococcal enterotoxin B. The antibodies were all able to bind lactoperoxidase-labeled [125I]STa, and antibody 20B3 was also able to dissociate [125I]STa bound to toxin receptors on rat jejunal villous cells. Preincubation of STa with antibodies 20B3 or 20F5 led to a concentration-dependent neutralization of toxin activity in a suckling mouse intestinal secretion assay. These antibodies are likely to provide new tools for the continued study of STa structure-function relationships and may lead to improved diagnosis and treatment of E. coli-induced infectious diarrhea.     

Production and characterization of highly specific anti-methotrexate monoclonal antibodies

Spleen cells of a Biozzi HR mouse immunized with a bovine serum albumin-methotrexate conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-three monoclonal antibodies (MAbs), selected by indirect ELISA, were produced and partially characterized. Using methotrexate (MTX) and eight structurally related compounds, binding specificities of the MAbs were assessed by inhibition enzyme immunoassay. All the MAbs had very low affinity for folic acid and its analogs and for the major MTX metabolite 7-hydroxymethotrexate. Using a computer cluster analysis program based on the binding specificities, the MAbs were divided into three groups. The thirteen MAbs in group I recognized primarily the pteridine portion of the MTX molecule; the eight group II MAbs recognized the benzene ring as well as the pteridine structure. The two MAbs in group III poorly distinguished between the different parts of the MTX molecule. The apparent equilibrium association constants of the anti-MTX MAbs in groups I, II, and III ranged from 7 x 10(9) to 3 x 10(8) M-1 (except for 1 MAb), from 5 x 10(7) to 6 x 10(6) M-1 (except for 2 MAbs), and from 1 x 10(6) to 3.5 x 10(5) M-1, respectively.     

Production of monoclonal antibodies specific to theophylline-treated lymphocytes

The T11 molecule is reported to play a key role in T lymphocytes activation. Moreover, theophylline is known to modify the functional properties of T lymphocytes probably inducing early changes in T11 molecule during T cell activation. Aim of our work was to clarify the effect on T lymphocyte surface after in vitro treatment with theophylline. In this paper, we describe the production and the functional properties of several monoclonal antibodies obtained by immunizing Balb/c mice with theophylline treated cells. Some of the monoclonal antibodies reacted only with the theophylline-treated lymphocytes and showed a promitogenic activity, enhancing the expression of T activated cell antigen. These monoclonal antibodies seem to demonstrate the existence of a membrane molecule which appears on lymphocytes surface after a trigger signal occurring in the early stages of T cell activation likely related to the T11 dependent alternative pathway.     

Mapping of staphylococcal enterotoxin A functional binding sites and presentation by monoclonal antibodies and fusion proteins

Staphylococal enterotoxins (SE) bind with high affinity to major histocompatibility complex (MHC) class II proteins and stimulate large number of T cells via the Vbeta region of the T-cell receptor (TCR). To map the epitopes of SE type A (SEA) involved in MHC binding and cell proliferation, 20 specific anti-SEA monoclonal antibodies (MAbs) and two large glutathione S-transferase fusion proteins corresponding to the amino and carboxy termini, respectively, of SEA were used. The functionality of these antibodies was tested, by MHC binding inhibition, interleukin-2 production, and T-cell proliferation assays. Moreover, I studied the ability of the MAbs to present SEA in vitro to human and murine cells and their reactivity with the two fusion proteins. This study showed that all of the MAbs have a defined effect on one or both immunological properties of SEA and were able to present SEA to human and murine cells. However, one MAb (4H8) recognized SEA but without any interference with its biological activities. When the MAbs were tested to react with the two fusion proteins representing the SEA molecule, all of the MAbs were negative except for two. These results confirmed the presence of two functionally different binding sites of SEA with MHC class II molecules and the importance of the disulfide loop for the mitogenic activity of SEA. I further demonstrated that MAbs can present SEA to immune cells independent of the site recognized by the antibody and that the integrity of the SEA molecule is very important for its functions.     

Regulation of staphylococcal enterotoxin B.

The effect of glucose and the glucose analogues 2-deoxyglucose and alpha-methyl-glucoside on the synthesis and regulation of staphylococcal enterotoxin B was examined. The attenuating effect of glucose on staphylococcal enterotoxin B synthesis was observed. However, when this effect was examined with analogues of glucose, contradictory responses were seen. alpha-Methylglucoside had a slight stimulatory effect on enterotoxin production and other extracellular proteins, whereas 2-deoxyglucose markedly inhibited enterotoxin production. beta-hemolysin and staphylococcal nuclease were also inhibited by 2-deoxy glucose but the synthesis of nuclease could be rescued by the addition of glucose to 2-deoxyglucose-containing cultures. Enterotoxin and beta-hemolysin synthesis were not subject to glucose rescue. The cells used in this study were permeable to cyclic 3',5'-adenosine monophosphate, but the addition of this compound did not reverse glucose repression or 2-deoxyglucose inhibition of enterotoxin B synthesis. We conclude from these data that the regulation of enterotoxin is not under catabolite control as previously reported.     

Production and characterisation of monoclonal antibodies to parathyroid hormone-related protein.

The production and characterisation of 17 monoclonal antibodies to human parathyroid hormone-related protein (PTH-rP) 1-34 is described. Five of the antibodies were shown to be of high avidity (Ka 4 X 10(10)-1.9 X 10(11) L/M) and able to detect 15-100 pg PTH-rP 1-34 per tube by RIA. None cross-reacted with PTH 1-34, and inhibition studies with peptide subfragments of PTH-rP 1-34 indicated that all recognise a central region extending from residues 9-18 to between residues 23 and 34. All antibodies tested cross-reacted with native PTH-rP in culture fluids from keratinocytes and squamous cancer cell lines and in human and bovine milk. The concentrations of PTH-rP 1-34 (ng/ml) in these fluids as determined by RIA were: keratinocytes 1-3, squamous cancer 0.2-2.5, human milk, up to 80. Selected antibodies coupled to Sepharose 4B were used to extract PTH-rP from biological fluids with high yields.     

Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin.

Hybridomas secreting monoclonal antibodies (MABs) specific for Clostridium perfringens type A enterotoxin were produced by fusion of P3X63Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with purified enterotoxin. Wells containing hybridomas secreting immunoglobulin G (IgG) antibodies against enterotoxin were specifically identified by an indirect enzyme-linked immunosorbent assay (ELISA), and 10 ELISA-positive hybridomas were selected and cloned twice by limiting dilution. All 10 hybridomas produced MABs containing immunoglobulin G1 heavy chains and kappa (kappa) light chains. These hybridomas were then grown as ascitic tumors in mice, and MABs were purified from the ascites fluids with DEAE Affi-gel blue. The specificity of the MABs for enterotoxin was demonstrated by immunoblotting and ELISA. Competitive radioimmunoassay with 125I-MABs suggests that these MABs recognized at least four epitopes on the enterotoxin molecule. The enterotoxin-neutralizing ability of MABs from both hybridoma culture supernatants and ascites fluids was assessed by using a 3H-nucleotide-release Vero (African green monkey kidney) cell assay. Only 2 of the 10 hybridomas produced MABs which completely (greater than 90%) neutralized the biologic activity of enterotoxin. Preincubation of 125I-enterotoxin with MABs demonstrated that MAB neutralizing ability correlated with MAB-specific inhibition of specific binding of enterotoxin to intestinal brush border membranes.     

Production and characterization of monoclonal antibodies specific for human mast cell tryptase

Human mast cell tryptase was purified from lung tissue by high salt extraction, ammonium sulphate precipitation, octyl Sepharose and heparin-agarose chromatography. The tryptase isolated was a tetramer with a molecular weight of 132 kD on gel filtration, and on SDS-polyacrylamide gel electrophoresis was reduced to a single diffuse band with a mean molecular weight of 32.5 kD. Purified tryptase catalysed the cleavage of the tryptic substrates tosyl L-arginine methyl ester and benzoyl DL-arginine p-nitroanilide; enzymatic activity was enhanced in the presence of heparin but markedly decreased in the presence of 2 M sodium chloride. Rabbit antisera and three new monoclonal antibodies (AA1, AA3 and AA5) were produced which were specific for tryptase in indirect ELISAs, immunoenzymatic overlay in crossed immunoelectrophoresis and by Western blotting. Additive and competitive ELISA experiments suggested that the three monoclonal antibodies all recognized epitopes within a single highly immunogenic area of the tryptase molecule, and enzyme assays indicated that this site was distant from the active site. Binding of monoclonal antibodies to tryptase was not affected by the presence of heparin, or by periodate treatment of the antigen suggesting that carbohydrate epitopes were not recognized. Western blotting indicated that some heterogeneity in molecular weight for monomeric tryptase was not reflected in antigenic differences. An immunofluorescence procedure with cytocentrifuge preparations of enzymatically dispersed lung, colon and skin revealed highly specific localization of tryptase to the granules of all mast cells, but there was no binding to other cells in these preparations, to cultured keratinocytes, to basophils or to any other blood leucocyte.     

Production of monoclonal antibodies specific for Haemophilus ducreyi: a screening method to discriminate specific and cross-reacting antibodies

Haemophilus ducreyi is the etiological agent of chancroid. The organism shares extensive immunological cross-reactivity with other Haemophilus species. This presents substantial difficulties for the production of specific monoclonal antibodies (MAbs). A competition ELISA was devised for hybridoma screening which allowed the detection of H. ducreyi-specific antibody-producing hybridoma cultures during the initial screening process. With this screening method, seven MAbs specific for H. ducreyi were obtained in a single cell fusion exercise. The specificities of the 7 MAbs were demonstrated by direct ELISA and dot immunobinding assays against several strains each of H. influenzae, H. parainfluenzae and Neisseria gonorrhoeae. Five of the MAbs reacted against all ten strains of H. ducreyi. These MAbs may permit the development of rapid and efficient immunodiagnostics for chancroid. The principle of the competition ELISA for hybridoma screening should be widely applicable to the development of specific MAbs to other organisms in which immunological cross-reactivity is an impediment to hybridoma screening by conventional methods.     

超抗原金黄色葡萄球菌肠毒素基因B的原核表达

目的构建重组pET-30a-SEB原核表达载体,转化感受态大肠杆菌BL-21(DE3),诱导表达超抗原金黄色葡萄球菌肠毒素B(staphylococcalenterotoxinB,SEB)。方法利用PCR技术从产SEB的金黄色葡萄球菌标准菌株CMCC-26075基因组DNA中克隆SEB全长序列,将其克隆到pGEM-TEasy载体中并进行测序。构建pET-30a-SEB原核表达质粒,转化感受态大肠杆菌BL21(DE3),异丙基硫代β-D半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达,经镍离子螯合亲和层析纯化后免疫鉴定。结果PCR获得超抗原SEB基因片段,与克隆载体连接后经测序与文献报道的基本一致;成功构建了pET-30a-SEB原核表达质粒且成功诱导表达出相对分子质量(Mr)约31×103的蛋白。结论成功克隆了seb基因序列,并进行了原核表达和鉴定,获得了SEB蛋白,为后续对超抗原SEB的进一步研究奠定了基础。     

Production, characterisation and applications of monoclonal antibodies to porcine circovirus 2

Summary.  The production, preliminary characterisation and applications of monoclonal antibodies (mabs) against six porcine circovirus 2 isolates are described. A total of 14 stable hybridomas were produced, of which 7 were characterised. All of the mabs characterised were of IgG isotype. All the mabs tested reacted by IIF with acetone-fixed cell cultures infected with PCV2 isolates from Canada, France, Spain, Denmark, USA and UK. No cross-reactivity with a porcine circovirus 1 field isolate was demonstrated using the panel of mabs tested. In addition, one of the seven mabs tested demonstrated neutralising activity against PCV2 isolates from Canada and France. The use of selected PCV2-specific mabs for the development of virus detection methodologies is described. Received September 13, 2000 Accepted December 19, 2000     

Chemotactic activity generated by staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) has significant toxicity for several mammalian species by both oral and parenteral routes. When highly purified SEB was incubated for 1 hr at 37 C with fresh serum from normal young adult men with little or no antibody activity for SEB, a factor(s) chemotactic for human polymorphonuclear leukocytes (PMN) was formed. Similar experiments using rabbit serum and autologous peritoneal PMN also demonstrated chemotactic activation. All chemotactic activity generated at 37 C was destroyed by heating at 56 C for 30 min. In human studies, precipitating antibody to SEB prevented generation of chemotactic activity by SEB. Based on heat lability and antibody sensitivity, the chemotactic factor(s) generated by SEB differs from that generated by endotoxin, and suggests a mechanism by which the PMN may participate in the pathophysiology of enterotoxemia.     

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.     

Production of syngeneic autoreactive monoclonal antibodies specific for isotypic determinants of IgE

Methods are described for the production of syngeneic mouse anti-IgE monoclonal antibodies (mAb). Hybridomas were prepared by using spleen cells from mice immunized with a conjugate of keyhole limpet hemocyanin with monoclonal IgE. Serum titers varied from approximately 40 to 1000 micrograms/ml. The anti-IgE mAb were isolated by affinity chromatography on columns containing immobilized monoclonal IgE. The mAb are specific for isotypic determinants of IgE and do not react with other immunoglobulin isotypes. One of the mAb, which has a high affinity for IgE (Ka = 4.7 X 10(8) M-1), should be useful for studies of regulation of IgE. The applicability of the mAb to quantitative assays for IgE was demonstrated.