Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.     

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays.     

Development and characterization of antibodies directed against the mouse D4 dopamine receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H(+)-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H(+)-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H(+)-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H(+)-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H(+)-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H(+)-ATPase antiserum and with an anti-14-3-3 antiserum indicated an FC-induced association of FCBP with the PM H(+)-ATPase. Analysis of the activation state of PM H(+)-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.     

Characterization of two monoclonal antibodies against the RON tyrosine kinase receptor

An anthropometric assessment was conducted at 238 !Kung San hunter-gatherers aged between 18 and 65 years (mean = 30.8 years), 156 Kavango horticultural pastoralists aged between 18 and 61 years (mean = 29.2 years) and for 87 urbanized Kavango people aged between 18 and 61 years (mean = 29.3 years) living as wage earning employees in northern Namibia. Weight status was estimated by using body mass index categories according to the recommendations of the WHO. As is typical for human populations, men were taller and heavier than women within the same ethnic groups. An interethnic comparison showed that both !Kung San women and men were lighter than Kavango women and men. The mean BMI of !Kung San women was 19.1 and of !Kung San men 19.4 kg/m2. Kavango people exhibited higher average BMI values, 19.4 for women, 20.3 kg/m2 for men. With the exception of the male urban Kavango people a high percentage (more than 30%) of the subjects were thin and underweight, as shown by a BMI of < 18.5 kg/m2. This was especially true of the !Kung San of both sexes and the rural Kavango men. Nearly 25% of !Kung San women met the criterion of weight depletion (BMI < 17.0). The cultural transition from nomadic hunter gatherer subsistence to a more sedentary life style over the last 20 years can be interpreted as an environmental stress which affected male as well as female nutritional status. The hard economic situation of the rural Kavango people may also be a stress factor which negatively influenced their nutritional status, especially of the men. The significantly better nutritional status of the urban Kavango men may be the result of the opportunities for work as wage earners or as soldiers.     

Characterization of haemagglutinin-esterase protein (HE) of murine corona virus DVIM by monoclonal antibodies

We analyzed the characteristics of seven monoclonal antibodies (mAbs) raised against purified HE (hemagglutinin-esterase) glycoprotein of the murine coronavirus DVIM (diarrhea virus of infant mice). Immunocrossreaction of these mAbs with JHM and/or MHV-S suggest that antigenic epitopes of HE of DVIM are similar to those of JHM and/or MHV-S. Four mAbs (1b4, 3a28, 4c19, 10b7), designated as group A mAbs, strongly inhibited both HA and AE activities. On the other hand, three mAbs (5a3, 6a6, 13a4), referred to as group B, had a comparatively weak HA inhibition activity. These results indicate that the antigenic epitopes of this glycoprotein can be classified into at least two groups and that the functional sites of HA and AE activities are similar but not identical. Neutralizing activity was shown in group A mAbs exclusively, suggesting that the ratio of HA and/or AE activities may play important roles in the cell fusion activity of DVIM-infected cells.     

Role of immunoglobulin A monoclonal antibodies against P23 in controlling murine Cryptosporidium parvum infection

Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvum infection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer's patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvum oocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvum infection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72. 7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murine C. parvum infection.     

Three monoclonal antibodies differentiate human from murine epidermis

In order to establish a set of epidermal species markers, normal human skin, murine skin and human skin transplanted to nude mice were stained with monoclonal antibodies directed to cell membrane-bound carbohydrates, a basement membrane component and a structure in the cell nucleus. Three epidermal species markers were identified. Two markers stained exclusively human epidermis: LH7.2 detects type VII collagen and stained the basement membrane of human epidermis in unfixed frozen sections, while LP4N stained cell nuclei in human epidermis in methanol-acetone-fixed frozen sections. The third marker, HH14, stained exclusively murine epidermis. HH14 defines the histo-blood group H carbohydrate antigen and stained spinous cell membranes of murine epidermis in both frozen and formalin-fixed sections.     

Rapid verification of the identity of questionable specimens using immunohistochemistry with monoclonal antibodies directed against HLA-class I antigens

PURPOSE: To evaluate the effect of substituting topical cyclosporin A 0.5% for topical corticosteroids in patients with postkeratoplasty glaucoma. METHODS: Topical cyclosporin A 0.5% was prospectively substituted for topical corticosteroids to treat 25 patients with postkeratoplasty glaucoma. RESULTS: Twenty-one (84%) of 25 patients showed a reduction in intraocular pressure (IOP) (range, 1-22 mm Hg; mean, 8.7 mm Hg). Follow-up ranged from 3 to 12 months (mean, 5.8). Graft clarity was maintained in all patients, with one allograft rejection episode. Thirteen patients were able to discontinue one or more glaucoma medication(s). CONCLUSION: Topical cyclosporin A 0.5% may be substituted for topical corticosteroids to aid in the management of postkeratoplasty patients with glaucoma. However, the resultant decrease in IOP may be associated with an increased risk for immune rejections.     

Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.     

Characterization and regional binding of human sperm monoclonal antibodies

Therapeutic effect of superoxide dismutase (SOD) and three derivatives: a conjugate with polyethylene glycol (SOD-PEG2), a cationized derivative (cSOD), and a mannosylated derivative (Man-SOD), on acute renal failure induced by ischemia/reperfusion was studied in rats. SOD and derivatives were administered intravenously to the rat after nephrectomy of the right kidney and before and after 60 min occlusion of the left renal artery. At 48 hr after reperfusion, the renal function was evaluated by determining the urinary excretion rate of 14C-inulin injected intravenously. No therapeutic effect on the impaired renal function was shown in the case of low dose SOD (2600 unit/kg) treatment. In contrast, administration of cSOD which was shown to be taken up by the isolated perfused kidney from its capillary side and SOD-PEG2 which maintained high plasma concentration exhibited significant therapeutic effect, as did SOD at ten-fold higher dose (26,000 unit/kg). On the other hand, renal damage was promoted by Man-SOD. Thus, the present study demonstrated that chemical modification may improve the therapeutic effect of SOD on the ischemic acute renal failure and increased SOD concentration in the renal vascular space is an important factor for the improved effect.     

Characterization of murine monoclonal anti-endothelial cell antibodies (AECA) produced by idiotypic manipulation with human AECA

The IgG fraction of human anti-endothelial cell antibodies (AECA) obtained from a patient with Wegener's granulomatosis was used as immunogen to raise AECA mAb in mice selected among those which developed vasculitis-like lesions after immunization. Three mAb (BGM, 3C8 and 7G2), selected by cyto-ELISA and flow cytometry analyses, featured a specific reactivity with human umbilical vein endothelial cells (HUVEC) and the mouse endothelial cell line H5V; on the contrary, HEp2 cells, the murine melanoma B16 cell line, the extracellular matrix as well as several other antigens tested were not recognized. BGM mAb, an IgG3 precipitating a 70 kDa structure from HUVEC, was able to induce endothelial cells to secrete amounts of IL-6 significantly higher than irrelevant controls or mAb binding different endothelial antigens (i.e. CD31, CD29, ICAM-1 and HLA class I). BGM mAb induced significant levels of antibody-dependent cell cytotoxicity (13 +/- 2.5 versus 0.6 +/- 0.03%). To the best of our knowledge, BGM is the first murine mAb specific for human endothelial cells generated by idiotypic manipulation; secondly, its biological properties further support the notion of a pathogenic role for AECA in autoimmune-mediated diseases.     

Mechanism of cryoprecipitation of anti-blood group B murine monoclonal antibodies

In our experience, some examples of mouse monoclonal antibodies of anti-B blood group specificity develop a precipitate when stored at 4 degrees C. This poses problems during the preparation of blood grouping reagents containing anti-B, and in the storage and use of such reagents. Here we show that this problem can be circumvented by alteration of the glycan moiety of the secreted immunoglobulin, either by glycosidase treatment of the partially purified immunoglobulin, or by the addition of glycan processing inhibitors to the hybridoma cell cultures. These findings have importance for the manufacture of monoclonal antibodies, and highlight a possible new role for carbohydrate in immunoglobulin interaction and immune complex formation.     

Monoclonal antibodies directed against lipopolysaccharide of Legionella pneumophila serogroup 1

The monoclonal antibodies (MAbs) against lipopolysaccharide of virulent strain of Legionella pneumophila serogroup 1 were produced. Three most productive hybrid clones (5F4, 5F10 and 2C9) were selected from fusions of mouse myeloma cells with spleen cells from BALB/c mice, immunized with bacterial outer membrane antigens. All generated clones were IgG-secreting. The MAbs had narrow strain specificity and showed no cross-reactions with other unrelated bacterial species. These antibodies were tested in sandwich ELISA. The results suggest that the MAbs could be used for diagnostic purposes.     

Expansion of the nonadherent myeloid cell population by monoclonal antibodies against tenascin-C in murine long-term bone marrow cultures

Tenascin-C, a predominantly mesenchymal extracellular matrix protein, has a restricted distribution in adult tissues. It has previously been shown that this protein is expressed in the bone marrow. In this paper we show that murine myeloid and lymphoid long-term bone marrow cultures differ in their expression of tenascin-C splice variants. In the adherent stromal layer of myeloid cultures, the 260-kDa polypeptide encoded by the 8-kb mRNA was the major splice variant, whereas in the stromal layer of lymphoid cultures both the shorter 210-kDa polypeptide encoded by the 6-kb mRNA and the 260-kDa polypeptide were abundantly expressed. However, in both culture systems the larger 260-kDa tenascin-C polypeptide was the major isoform secreted in the culture supernatant. This finding is in agreement with previous reports indicating that the smaller 210-kDa isoform is preferentially deposited in the stroma, whereas the alternatively spliced segment in the 260-kDa tenascin-C may contain anti-adhesive domains. Glucocorticoids in myeloid long-term bone marrow cultures and in the MC3T3-G2/PA6 cell line downregulated the expression of tenascin-C. In the present study we observed that this was due primarily to downregulation of the 8-kb major splice variant of the tenascin-C mRNA. We also studied the possible role of tenascin-C in the bone marrow by using antibodies against tenascin-C in long-term bone marrow cultures. We found that three monoclonal antibodies against the carboxyterminal type III fibronectin repeats of tenascin-C (TNCfn 7-8) increased the number of the non-adherent myeloid cells in myeloid long-term bone marrow cultures. It has recently been suggested that the TNCfn 6-8 domain of tenascin-C binds to the alpha8beta1 integrin. Using Northern blotting, we found that the integrin alpha8 subunit was expressed in adherent cells in bone marrow cultures, raising the possibility that tenascin-C acts in bone marrow cultures by binding to the alpha8beta1 integrin.     

Characterization of Bangor virus proteins by using monoclonal antibodies

A new virus was isolated from a finch in quarantine in Northern Ireland in 1973. The virus had the morphological characteristics of a paramyxovirus, and was named Bangor virus (BaV). In order to identify the structural proteins of BaV and to investigate the biological characterization of the virus, 28 monoclonal antibodies (mAbs) directed against BaV were prepared. Eight of these mAbs reacted with the nucleocapsid protein (NP), 10 with hemagglutinin-neuraminidase (HN) protein, and 10 with fusion (F) protein. With the aid of these mAbs, the structural proteins of BaV were determined, namely, p52, gp74, gp63, and gp51 were identified as the NP, HN, F0, and F1 proteins, respectively. The biological activities of the mAbs directed against the envelope glycoproteins of BaV were examined. Intriguingly, it was found in the neutralization assay that four mAbs directed against the HN protein of BaV can enhance the fusion of HeLa cells infected with BaV, showing the presence of a potential third function of the HN protein that affects the fusion activity of the F protein. Furthermore, all of the anti-F protein mAbs showed neutralizing activity.     

CNA.42, a new monoclonal antibody directed against a fixative-resistant antigen of follicular dendritic reticulum cells

A new monoclonal antibody (MAb), CNA.42, was generated using the CEM T-cell line. It recognizes a 120-kd formalin-resistant glycosylated antigen that is mainly expressed by follicular dendritic reticulum cells (FDRCs). This antigen is also expressed by a few mononuclear cells in the paracortical area of reactive lymph nodes and by some cortical thymocytes. Two hundred and eighty-nine cases of hematopoietic tumors of various types were tested with this antibody. They showed either intact FDRC networks or FDRC networks dispersed among malignant cells. In follicular lymphomas, the follicular pattern was highlighted by CNA.42 MAb. Expanded FDRC networks were found in angioimmunoblastic T-cell lymphomas. Neoplastic cells were positive in 43.6% (24/55) of T-cell and 4.6% (6/129) of B-cell lymphomas. The highest percentage of cases with positive neoplastic cells was found in anaplastic large-cell lymphomas (62.5%; 15/24). In Hodgkin's disease, FDRC networks, sometimes encasing Hodgkin and Reed-Sternberg (HRS) cells, were found. HRS cells were also stained by this antibody in 23 (21.9%) of the 105 cases examined. A variety of normal nonlymphoid tissues and nonhematopoietic tumors, such as some neurogenic tumors, carcinoma, and occasional sarcomas, were found to be positive. Analysis of the reactivity of CNA.42 antibody with FDRCs of lymphoid tissue from different animal species showed similar reactivity to that observed in humans, suggesting widespread evolutionary conservation of the antigen recognized by this antibody. In daily diagnostic practice, CNA.42 MAb seems to be a suitable FDRC marker and possibly has an auxiliary role in recognizing T-cell lymphomas.     

Characterization of monoclonal antibodies that recognize canine CD34

Using a polyclonal antiserum against canine CD34, we previously found that CD34 is expressed on canine bone marrow progenitor cells in a manner analogous to that found in humans. To further characterize CD34+ cells and to facilitate preclinical canine stem cell transplant studies, monoclonal antibodies (MoAbs) were raised to CD34. A panel of 10 MoAbs was generated that reacted with recombinant CD34 and with CD34+ cell lines and failed to react with CD34- cell lines. Binding properties of five purified MoAbs were determined by BIAcore analysis and flow cytometric staining, and several MoAbs showed high affinity for CD34. Two antibodies, 1H6 and 2E9, were further characterized, and in flow cytometry studies typically 1% to 3% of stained bone marrow cells were CD34+. Purified CD34+ bone marrow cells were 1.8- to 55-fold enriched for colony-forming unit-granulocyte-macrophage and for long-term culture initiating cells as compared with bone marrow mononuclear cells, whereas CD34- cells were depleted of progenitors. Three autologous transplants were performed with CD34+ cell fractions enriched by immunomagnetic separation. After marrow ablative total body irradiation (920 cGy), prompt hematopoietic recovery was seen with transplanted cell doses of 相似文献   

Comparison of several mouse and rat monoclonal antibodies against human fibrinogen

A rare case of desmoplastic melanoma arising from the maxillary gingiva of a 66-year-old woman is reported. This tumour metastasised to the submandibular lymph node 5 years after extirpation, and local recurrence was observed 2 years later. The gingival tumour showed the histopathological characteristics of desmoplastic melanoma and the metastasised tumour cells were immunohistochemically positive for S-100 protein, neuron specific enolase, HMB-45 highly specific for conventional melanoma, and Fontana-Masson staining. The gingival tumour, originally regarded as benign clinically, was actually a desmoplastic melanoma.     

Chicken monoclonal antibodies against synthetic bovine prion protein peptide

A night course taken almost 25 years ago sparked an interest in Leonardo da Vinci that has become a passion for a London, Ont., neurosurgeon. Dr. Rolando Del Maestro now boasts one of the largest collections of da Vinci artifacts in North America.