Monoclonal antibody AE-2 modulates carbamate and organophosphate inhibition of fetal bovine serum acetylcholinesterase

The monoclonal antibody AE-2, raised against the human erythrocyte acetylcholinesterase (AChE) dimer (acetylcholine acetylhydrolase, EC 3.1.1.7), binds to other mammalian AChEs, including the tetramer that occurs in fetal bovine serum (FBS). AE-2 partially inhibited the rate of hydrolysis of the charged substrate acetylthiocholine by FBS AChE, whereas it increased the rate of hydrolysis of the neutral substrate indophenyl acetate. Present results show that AE-2 decreases the rate of inhibition of FBS AChE by the positively charged organophosphate amiton-p-toluene sulfonate and the positively charged carbamates pyridostigmine and neostigmine but accelerates inhibition of FBS AChE by the neutral organophosphates paraoxon and diisopropylfluorophosphate. Results suggest that AE-2 may allosterically modulate an anionic site in the catalytic center of FBS AChE.     

Monoclonal antibody against CD45RB for the therapy of rejection and autoimmune diseases

Rejection continues to be the single largest impediment to successful organ transplantation. Current therapy, which must be taken for a lifetime is nonspecific and has significant side effects including infection and cancer. There is a need to develop improved means of immunosuppression. The current goal of transplantation immunology is to induce a prolonged state of nonreactivity to the allograft but preserving an otherwise intact immune system (tolerance). We have recently reported that a monoclonal antibody against CD45RB is a potent immunosuppressive agent, and that it induces donor specific tolerance in the mouse. In this contribution we briefly review our understanding of the molecular basis for the activity of this therapy and update results in various transplant and autoimmune disease animal models. The clinical relevance and future development of this novel therapy is also discussed.     

Monoclonal antibody specific to bovine lens epithelial cell membrane: preparation of a lens epithelial cell-selective immunotoxin

Dithranol has been used successfully in the treatment of psoriasis for more than 75 years, and much in vitro and in vivo research has been done on the elucidation of the mode of action of this potent and safe antipsoriatic therapy. In vivo research has revealed major effects of dithranol on epidermal proliferation and inflammation. Information on the in vivo effects on epidermal differentiation is limited. Therefore, the dynamics of a set of differentiation markers (keratin 16, filaggrin, keratinocyte transglutaminase, involucrin) and markers for proliferation and inflammation (Ki-67, T lymphocytes, polymorphonuclear leucocytes) were studied in skin biopsies of six patients with psoriasis during 4 weeks of dithranol therapy. The treatment regimen involved a short contact protocol at our out-patient day treatment centre with an easily washed off cream. Treatment resulted in a decrease of the PASI score of 48% in 4 weeks. Immunohistochemically, a major decrease of keratin 16 content and virtually complete restoration of the filaggrin positive cell layer were seen. These changes proved to be significant by comparison of the markers over the group of six patients. Although many other topical treatments for psoriasis (occlusive therapy and vitamin D3 analogues) result in a prominent reduction in the amount of transglutaminase and involucrin positive cell layers, the effect of dithranol on these markers is minimal.     

Monoclonal antibody GL1 and its possible involvement in the morphogenesis of the otic vesicle

In a previous study, a monoclonal antibody (MAB) named GL1 was identified that is expressed in a precise pattern during gastrulation and early neurulation stages in chick embryos. In this article we have further investigated the expression pattern of this MAB in the chick embryo. GL1 antigen is present in several organs that seem not to be related developmentally. Among them, GL1 is present during the early steps of the otic placode formation, in the pharyngeal endoderm, in some neural crest cells, in the somites, and in the ventricular surface of the nervous system. The distribution in the nervous system is well patterned with two broad lines of expression in the ventricular side of the metencephalic region, a unique and centered expression in the border between the metencephalon and the myelencephalon and again in two lines running along the myelencephalon and the rostral spinal cord. Additionally, GL1 can be induced by members of the FGF family, and we have used this system to elucidate its role in otic placode formation. The results obtained reveal that GL1 can be a useful marker for the study of developmental processes in the endoderm, the otic anlage, and the apical surface of the nervous system. Biochemical analysis of the antigen recognized by this MAB must be carried out to elucidate the molecular nature of the antigen.     

Monoclonal antibodies against human collagenase and stromelysin

Mouse monoclonal antibodies against recombinant human fibroblast procollagenase and prostromelysin have been generated and characterized. The epitope-containing domains for the antibodies have been assigned based on their immunoreactivities against recombinant proenzymes, mature enzymes, truncated collagenases, proteolytic fragments of stromelysin, and chimeric molecules constructed from different domains of the two enzymes. These antibodies can be divided into four groups: (1) antibodies that recognize the truncated 19-kDa NH2-terminal collagenase, (2) antibodies that recognize the C-terminal domain of collagenase and stromelysin, (3) antibodies that recognize a 31-kDa NH2-terminal collagenase fragment, and (4) antibodies that recognize the 19-kDa NH2-fragment of stromelysin. The prostromelysin-specific antibody 11N13 is of particular interest; it neutralizes stromelysin activity in a stromelysin peptide substrate assay, with an IC50 value of 75 nM. MAb 11N13 may be useful for in vivo and in vitro studies to validate the roles of stromelysin in tumor cell invasion, metastasis, and connective tissue disorders.     

Monoclonal antibody production with on-line harvesting and process monitoring

A semi-automated system has been designed for on-line harvesting and monitoring of monoclonal antibody (mAb) production. [The antibody was directed against the peptide AGPAGTGKTTKDL.] Analytical and purification units were interfaced to the fermenter via a hollow fiber cartridge in which fermentation broth was continuously circulated through the lumen of the hollow fiber system. Permeate from the hollow fiber cartridge was pumped through either an analytical sampling loop or a preparative Protein G column where antibody species were captured. Switching between monitoring and harvesting was achieved by two 3-way toggle valves. Samples from the analytical sampling loop were transported to an analytical Protein G chromatography column for quantitation of all immunoglobulin G species in the fermenter. Data acquisition and processing was performed by the data system of the liquid chromatograph. All valves in the system except the two toggle valves were controlled by the liquid chromatograph. Antibody biosynthesis was monitored for the first 60 h of fermentation. Harvesting was initiated when mAb accumulated in the fermenter. Complete harvesting took approximately 90 h.     

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross-reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand -Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.     

Reactivity of native conglutinin in bovine serum with rabbit antibody against recombinant bovine conglutinin with deletion of the N-terminal and collagen-like regions

The reactivity of native bovine conglutinin (Kg) with antibody against recombinant Kg (rKg), with deletion of the N-terminal and collagen-like regions of the native Kg molecule, was studied by sandwich enzyme-linked immunosorbent assay. With anti-recombinant Kg antibody as the coating antibody, rKg reacted with biotinylated homologous anti-rKg and heterologous anti-Kg antibodies as probing antibodies, while native Kg did not. With anti-native Kg antibody as coating antibody, native Kg reacted with biotinylated homologous antibody as probing antibody, while recombinant Kg reacted weakly with both biotinylated homologous and heterologous antibodies. Consequently the N-terminal and collagen-like regions of native Kg molecule are essential to express the complete immunogenicity and/or antigenicity of the native Kg molecule.     

抗沙丁胺醇单克隆抗体制备与鉴定

[目的]制备特异性抗沙丁胺醇单克隆抗体,为其免疫学检测方法的建立奠定基础.[方法]用SAL-BSA 抗原免疫BALB/c小鼠;使用细胞融合技术建立抗SAL 的单克隆抗体(SAL mAb)杂交瘤细胞株;体内诱生腹水法制备SAL mAb,并鉴定其免疫学特性.[结果]筛选出2D9和IC4两株杂交瘤细胞.其细胞培养上清效价达1:10<'4>,腹水效价达1:10<'6>;免疫球蛋白亚型鉴定为IgG<,1>;抗体对SAL 的半教抑制浓度(IC<,50>)为1.14ng/ml;与沙丁胺醇和盐酸克伦特罗的交叉反应率(CR)分别为100.00%和26.09%,与其他化合物无交叉反应.[结论]获得了高效价、敏感且异性的抗SAL 抗体,为沙丁胺醇残留的免疫学检测方法的建立奠定了基础.     

Liposome-induced conformational changes of an epitopic peptide and its palmitoylated derivative of influenza virus hemagglutinin

Meningococcal sodC encodes periplasmic copper- and zinc-cofactored superoxide dismutase (Cu,Zn SOD) which catalyzes the conversion of the superoxide radical anion to hydrogen peroxide, preventing a sequence of reactions leading to production of toxic hydroxyl free radicals. From its periplasmic location, Cu,Zn SOD was inferred to acquire its substrate from outside the bacterial cell and was speculated to play a role in preserving meningococci from the action of microbicidal oxygen free radicals produced in the context of host defense. A sodC mutant was constructed by allelic exchange and was used to investigate the role of Cu,Zn SOD in pathogenicity. Wild-type and mutant meningococci grew at comparable rates and survived equally long in aerobic liquid culture. The mutant showed no increased sensitivity to paraquat, which generates superoxide within the cytosol, but was approximately 1,000-fold more sensitive to the toxicity of superoxide generated in solution by the xanthine/xanthine oxidase system. These data support a role for meningococcal Cu,Zn SOD in protection against exogenous superoxide. In experiments to translate this into a role in pathogenicity, wild-type and mutant organisms were used in an intraperitoneal mouse infection model. The sodC mutant was significantly less virulent. We conclude that periplasmic Cu,Zn SOD contributes to the virulence of Neisseria meningitidis, most likely by reducing the effectiveness of toxic oxygen host defenses.     

Monoclonal antibodies against human endothelin-converting enzyme-1

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease which specifically converts the inactive precursor big-endothelin-1 (big ET-1) to the vasoactive endothelin-1 (ET-1). Six different mouse hybridoma cell lines have been generated secreting monoclonal antibodies specific to human ECE-1. These antibodies have been proven useful in a fast and efficient one-step purification of membrane-bound ECE-1 as well as of artificial soluble ECE-1 by immunoaffinity chromatography. The antibodies are suitable for a quantification of ECE-1 in solution by a sandwich-ELISA and for the immunohistochemical detection of ECE-1 in the cell membrane.     

Monoclonal antibody binding affinity determined by microchip-based capillary electrophoresis

The affinity constant of a monoclonal antibody to fluorescently labeled bovine serum albumin (BSA) was measured in diluted mouse ascites fluid using a microfluidic chip to perform affinity capillary electrophoresis. Borofloat glass-based devices could be used repeatedly with samples for many months. On-chip separations were performed in less than 60 s, and 30-60 s was required for manual sample exchange. The change in peak height for BSA with increasing BSA/anti-BSA concentration ratio was used to determine concentration changes in bound and free BSA. A Scatchard plot analysis gave an affinity constant (more exactly the intrinsic association constant) of 3.5+/-0.6 x 10(7) M(-1) for a 1:1 stoichiometric ratio. Two affinity complexes were separated. One complex was identified by the Scatchard method as having a 1:1 stoichiometric ratio. The other complex is proposed to have a stoichiometry with an excess of anti-BSA to BSA, most likely (anti-BSA)2-BSA, on the basis of a faster migration time than the 1:1 complex, a decrease in the amount of this complex with increasing [BSA], and predictions of theoretical models for multi-valent antigens. Potential applications of microchip-based devices in affinity measurements are discussed.     

Monoclonal anti-idiotypic antibody to a potent thromboxane A2 receptor antagonist and its interaction with thromboxane A2 receptor

A monoclonal anti-idiotypic antibody that interacts with thromboxane A2 receptor was generated using an anti-idiotypic approach. Idiotypic antibodies against a potent receptor antagonist, HS-145, were generated in rabbit. The idiotypic antibodies were then selected by an affinity procedure using SQ29,548-Affi-Gel-102 matrix. The selected idiotypic antibodies were used as surrogate receptor for anti-idiotypic antibody generation. A mouse monoclonal antibody, 3D-9E-12, was generated. It was shown to displace 125I-HS-145 from affinity-purified idiotypic antibodies. It also inhibits 125I-IS-145 binding to thromboxane A2 receptor in human platelet membranes in a dose-dependent manner. Furthermore, it attenuated U46,619-induced increase in [35S]guanosine 5'-O-(thiotriphosphate) binding and GTPase activity in human platelet membranes. Finally, it inhibited U46,619- but not PAF-induced platelet aggregation. These results indicate that 3D-9E-12 acts as a specific antagonist in the thromboxane A2 receptor.     

Monoclonal antibodies against Schistosoma mekongi surface tegumental antigens

Monoclonal antibodies were produced from naturally infected BALB/c mice. Thirteen hybridomas which were found to produce monoclonal antibodies against surface tegumental antigens of Schistosoma mekongi by ELISA assay were used in this study. The antigen specificities of hybridomas reactive with surface tegumental antigens were characterized and localized by immunoblotting analysis and Avidin-Biotin method. Of the 13 hybridomas, only three produced monoclonal antibodies to the single epitopes in the surface tegumental antigens. These epitopes (125 kDa, 97 kDa and 38 kDa) have been found to be the major antigenic components of the surface tegument of S. mekongi. The 38 kDa antigen was found to associate with the surface tegumental layers, the muscular layers lying just beneath the tegument, as well as in the gut surface. The 97 and 125 kDa antigens were detectable only in the surface tegumental area. The biochemical identity of these proteins or glycoproteins is unknown. However, these antigens have also been described in S. japonicum and S. mansoni.     

Monoclonal antibody to endotoxin attenuates hemorrhage-induced lung injury and mortality in rats

OBJECTIVE: To describe a technique of direct revascularisation of the bronchial artery using the left IMA and assess its medium term results in patients undergoing left single lung transplant (SLT). METHODS: Between March 1991 and September 1993, 22 patients who underwent direct bronchial revascularisation at the time of left SLT (20 pedicled IMA, one free IMA, and one direct anastomosis to the aorta) have been followed up for a minimum period of 1 year (mean 30 +/- 12 months). Their mean age was 47.8 +/- 9.6 and the original disease was emphysema in 19, lymphangioleiomyomatosis in two, and pulmonary fibrosis in one. The mean ischaemia time was 269.7 +/- 23.4 min. RESULTS: There was one early death (4.5%) and 3 patients were re-explored for bleeding. The actuarial survival at 1 and 3 years was 91 +/- 0.4% and 82.6 +/- 1%, respectively. Bronchial healing was excellent in all patients and angiographic studies showed patent vascular anastomosis in all 22 patients, with good run off in 20 and poor in two. One patient developed clinical obliterative bronchiolitis at 22 months (4.5%) during a period of follow up varying from 12 to 43 months (mean 30 S.D. 12). At last follow up the mean FEV1 was 1.4 +/- 0.4 and the mean FVC was 2.2 +/- 0.6. On average, each patient developed 1.5 +/- 0.6 infection episodes and 1 +/- 0.2 acute lung rejection. CONCLUSION: It is concluded that the medium term results of direct bronchial revascularisation are good. However the influence of this procedure on long term results needs further investigation.     

Monoclonal antibody to human Trk-A: diagnostic and therapeutic potential in neuroblastoma

5C3 is a murine IgG1 antibody specific for the nerve growth factor (NGF) docking site of the human p140 trk-A receptor, with no cross-reactivity with human trk-B. In vitro, 5C3 and its Fab mimic the effects of NGF, a neurotrophin mediating growth and differentiation of neural crest-derived cells. When labelled with radioisotope, 5C3 images human trk-A positive tumours in vivo. More importantly, 5C3 induces regression of human trk-A positive tumours in rodents. We therefore investigated the value of 5C3 in detecting trk-A expression in human neuroblastoma by immunohistochemistry. 5C3 reactivity was detected in 73 of 113 neuroblastoma specimens and correlated strongly with localised/4s disease (55/60) with either a homogeneous or mixed pattern. Among stage 4 neuroblastoma, only 18/53 had homogeneous or mixed trk-A expression. 5C3 did not react with 46/48 other human malignancies, but was positive in 1 melanoma and 1 Wilms' tumour specimen. The prognostic, imaging and NGF-mimetic properties of antibody 5C3 and its derivatives may offer alternatives for the diagnosis and treatment of neuroblastoma.     

Passive protection of neonatal calves against bovine coronavirus-induced diarrhea by administration of egg yolk or colostrum antibody powder

The protective effect of egg yolk and colostrum powders prepared from hens and cows vaccinated with inactivated bovine coronavirus (BCV) antigen was evaluated in a challenge model with a virulent BCV strain. Twenty three calves from BCV-free herds were randomly divided into control and several treatment groups. All calves were orally challenged with 1 x 10(9) TCID50 of the virulent Kakegawa strain of BCV at 24 to 36 h after birth. Calves in treatment groups received either egg yolk powder or cow colostrum containing BCV specific antibodies. Daily treatment with these antibody preparations started 6 h until 7 days post-challenge. Control calves which received no antibody had severe diarrhea and all died within 6 days after infection. In contrast, calves fed milk containing egg yolk or colostrum with neutralization titers of 1:2560 or 1:10,240 respectively all survived and had positive weight gain unlike the other treatment groups. These results indicate that the orally administered egg yolk and colostrum powders protected against BCV-induced diarrhea in neonatal calves and that the egg yolk used provided a higher degree of protection compared to colostrum powder on a titer basis. Treatment with whole egg yolk from immunized hens therefore provides a more efficacious alternative to the existing methods of specific passive protection against BCV.     

Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems

Voided urine samples from 575 young Japanese under 20 years of age (297 males and 278 females including infants) and from 380 subjects (20-29 years old, 193 males and 187 females) were analyzed for levels of creatinine, selenium, zinc, cadmium and mercury. This investigation presents data regarding the normal urinary levels of these substances in age groups of 0-4, 5-9, 10-14, 15-19, and 20-29 years. Urinary levels of creatinine and cadmium showed remarkable increases with the age of the subjects, whereas that of selenium was constant at all ages under 20. Urinary concentrations of heavy metals were represented by creatinine and selenium ratios. Comparisons between these ratios revealed that selenium is an excellent index for representing the levels of the substances contained in a voided urine sample. Creatinine was not useful as an index for younger subjects, because the urinary concentration of this compound increased almost threefold as the subjects became older, up to 15 years of age.