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1.
Monoclonal antibodies against rat platelet phospholipase A2 总被引:2,自引:0,他引:2
Monoclonal antibodies which bind specifically to rat platelet phospholipase A2 have been raised. None of them bound to exocrine phospholipase A2 derived from pancreas or snake venom. All antibodies recognized the conformational structure of rat platelet phospholipase A2 supported by intramolecular disulfide bonds, since the reactivity between the antibodies and the enzyme was lost in the presence of 2-mercaptoethanol. One of them, designed MB5.2, inhibited the activity of the platelet phospholipase A2 in a dose-dependent manner. A kinetic study revealed that antibody MB5.2 apparently competed with the substrate for the active site of the enzyme. The other antibodies, designed MD7.1 and ME6.1, inhibited the binding of the enzyme to heparin. The distribution of phospholipases A2 bearing a similar determinant to rat platelet phospholipase A2 was investigated by immunoprecipitation of the enzyme activity or by an immunoblot technique. Among rat tissues, cross-reactivity was observed with phospholipases A2 from spleen, lung, and bone marrow. Extracellular phospholipase A2 detected in the peritoneal cavity of casein-treated rat was also recognized by these antibodies. Furthermore, antibody MD7.1 cross-reacted with rabbit and guinea pig platelet phospholipases A2. 相似文献
2.
K Takayama I Kudo S Hara M Murakami K Matsuta T Miyamoto K Inoue 《Biochemical and biophysical research communications》1990,167(3):1309-1315
Four monoclonal antibodies (HP-1, HP-2, HP-3 and HP-4) with differing reactivities were raised against human synovial fluid phospholipase A2. None of them bound to exocrine phospholipases A2, such as those from pancreas or snake venom. However, antibodies HP-1 and HP-3 showed cross-reactivity with rabbit and rat platelet secretory phospholipases A2, which share common enzymatic and structural features with the human synovial enzyme. Antibodies HP-1, HP-2 and HP-3 inhibited the activity of human synovial phospholipase A2. The antibodies were used to develop a rapid immunoaffinity column chromatographic procedure for enzyme purification. In some preparations, the recovery of total activity after immunoaffinity column chromatography was more than 100% suggesting the existence of endogenous inhibitory factors of phospholipase A2 in human synovial fluid. 相似文献
3.
I Utsunomiya S Oh-ishi I Hayashi J Maruhashi N Tsuji N Yamamoto S Yamashina 《Journal of biochemistry》1988,103(2):225-230
Monoclonal antibodies to rat T-kininogen were produced and 9 hybridomas were selected. Radioimmunoassay (RIA) was developed using 125I-labeled T-kininogen and cell walls of Staphylococcus aureus (Zysorbin) for the separation of bound from free ligand, when IgG2a and IgG2b were used. In the case of IgG1 monoclonals, a second antibody (goat anti-mouse IgG) and Zysorbin were used. By this RIA, 1-16 ng T-kininogen/tube showed a linear inhibition curve, and cross reactivities to rat purified LMW- and HMW-kininogens were less than 0.5%, respectively. These monoclonal antibodies were also used for the immunohistochemical staining of the liver to detect T-kininogen in hepatocytes. By using the RIA and immunohistochemical staining, the T-kininogen levels in rat plasma and liver following carrageenin-induced inflammation were estimated. At 3-5 h after the carrageenin injection, when the paw swelling was at its peak, the plasma level of T-kininogen and staining of the liver were slightly increased. T-Kininogen levels in plasma and liver peaked on the 2nd day, when the paw swelling had already decreased. The result indicates that the increase of T-kininogen level in the liver and plasma occurs with a time lag and T-kininogen is not directly involved in the increase of vascular permeability in carrageenin paw edema. 相似文献
4.
Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed. 相似文献
5.
Monoclonal antibodies against glutaraldehyde-conjugated histamine: application to immunocytochemistry 总被引:2,自引:2,他引:0
K. Fujiwara Tsunehiro Kitagawa Yasuhisa Inoue Gerard Alonso 《Histochemistry and cell biology》1997,107(1):39-45
We have developed mouse monoclonal antibodies (AHA-1–5, all IgG1 sub-isotype mAbs) against histamine (HA) conjugated to bovine serum albumin using glutaraldehyde-NaBH4. Among these, AHA-1 mAb was found to be the most useful for HA immunocytochemistry (ICC) in terms of specificity and sensitivity
without non-specific immunobinding. AHA-1 was demonstrated to be specific to HA with an enzyme-linked immunosorbent assay
(ELISA) binding test, simulating the ICC of tissue sections, and not reactive to any of the other amino acids and peptides
with N-terminal histidine tested. By use of this antibody, indirect immunoperoxidase staining was observed in rat stomach
fixed with glutaraldehyde (GA) in combination with NaBH4 reduction. In contrast, no immunoreactivity was seen in tissue fixed only with GA. Absorption controls indicated that the
immunostaining could be completely inhibited by GA-conjugated HA, which was consistent with the results of an ELISA inhibition
test. No cross-reactivity occurred with other GA-conjugated amino acids. ICC staining was dense in the cytoplasm of gastric
enterochromaffin-like cells and very weak in mast cells. A new finding was that staining was noticed in some cell bands of
the intermediate layer between the stratum lucidum and the stratum corneum of the stratified squamous epithelium of the gastric
cardia, esophagus, tongue, and skin in rats. The results strongly suggest that the monoclonal antibody allowed highly specific
detection of HA in animal tis- sues.
Accepted: 27 August 1996 相似文献
6.
Monoclonal antibodies against salt-resistant rat liver lipase. Cross-reactivity with lipases from rat adrenals and ovaries 总被引:1,自引:0,他引:1
To obtain monoclonal antibodies against rat salt-resistant liver lipase, mice were immunized with enzyme purified from heparin-containing rat liver perfusates. Hybridomas were screened for antibody production by means of an enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay. Five hybridoma cell lines secreting antibodies against rat liver lipase indicated as A, B, C, D and E, have been obtained. All antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. The antibodies precipitate salt-resistant lipase from rat post-heparin plasma, are positive in ELISA, inhibit liver lipase activity and bind monospecifically with the enzyme as shown by immunoblotting. The monoclonal antibodies showed no significant reactivity with human liver lipase. The salt-resistant lipases of rat adrenals and ovaries are also precipitated by the monoclonal antibodies directed against the liver enzyme. Therefore, the heparin-releasable lipases of the liver, adrenals and ovaries possess identical epitopes. 相似文献
7.
T Suzuki Y Kishi M Totani H Kagamiyama T Murachi 《Biotechnology and applied biochemistry》1987,9(2):170-180
Monoclonal and polyclonal antibodies against porcine mitochondrial aspartate aminotransferase (m-AST) were prepared in order to study their effect on the kinetics of the enzyme and their possible use as diagnostic reagents. The most stable hybridoma clone, designated MH-1, was selected and cultured for mass production of the monoclonal antibody MA-1. MA-1 was purified by affinity chromatography with m-AST as a ligand. The m-AST activity was inhibited uncompetitively by preincubation with MA-1, but preincubation with the polyclonal antibody uncompetitively by preincubation with MA-1, but preincubation with the polyclonal antibody raised in a rabbit resulted in noncompetitive inhibition of the enzyme. These results suggest the usefulness of a monoclonal antibody for studying the mechanism of catalysis. Sandwich enzyme immunoassay methods for m-AST using both polyclonal and monoclonal antibody-coated polystyrene balls were established and permitted the determination of porcine m-AST on the order of 10(-11) and 10(-10)M, respectively. 相似文献
8.
T Barka E W Gresik H van der Noen 《The journal of histochemistry and cytochemistry》1985,33(3):209-218
Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies. After recloning, one hybridoma (4Cl3) was selected for further studies. The hybridoma (4Cl3) cells were grown as ascites tumors, and the antibodies were purified from the ascitic fluid by diethylaminoethyl Affi-gel Blue chromatography. The purified antibody (MA4), immunoglobulin G1, immunoprecipitated a 39K dalton protein from submandibular saliva, and also reacted with a protein of the same electrophoretic mobility on immunoblots. From extracts of submandibular gland slices, incubated with [3H]leucine, the antibody again immunoprecipitated a 39K protein, indicating that this protein is synthesized in the gland. MA4 was used for immunocytochemical stainings of submandibular glands of rats of different ages. In general, immunostaining was seen only in acinar cells. Thus, there was no staining in the glands of 1-day-old rats that lack differentiated acinar cells. In the glands of 1- to 4-week-old rats the number of immunoreactive cells and the extent of immunostaining paralleled the differentiation of the acinar cells. In the glands of adult rats a uniform staining of the secretory granules of the acinar cells was observed. The immunoreactive 39K protein seemed to be restricted to the acinar cells in the submandibular gland; there was no immunostaining in the parotid, sublingual, or lingual salivary glands, or in the pancreas, colon, and duodenum. Stimulation of saliva secretion by isoproterenol resulted in a virtual depletion of the antigen from the acinar cells. These results indicate the feasibility of producing mouse hybridomas that secrete antibodies against rat saliva components. The monoclonal antibody at hand will be useful in analyzing the differentiation of the acinar cells, and the factors that influence this differentiation process. 相似文献
9.
Monoclonal antibodies against human pulmonary surfactant apoproteins: specificity and application in immunoassay 总被引:4,自引:0,他引:4
Monoclonal antibodies were prepared against pulmonary surfactant apoproteins which were isolated from lung lavages of patients with alveolar proteinosis with the following steps: solubilization of the surface-active fraction by Triton X-100, delipidation with butanol-ethanol extraction followed by column chromatographies on Blue-Sepharose and DEAE-Toyopearl in the presence of dithiothreitol. The fraction including 62 and 36 kDa proteins, i.e., pulmonary surfactant apoproteins, was used for the immunization. Monoclonal antibodies against the pulmonary surfactant apoproteins were prepared using hybridoma technology. The monoclonal antibodies prepared, PC6 and PE10, recognized the same proteins, i.e., 62 and 36 kDa proteins, in the patients' lavages. They also recognized 37 and 34 kDa proteins in human lung lavage and amniotic fluid. Quantitation of the apoproteins by enzyme-immunoassay using the monoclonal antibodies has been developed. A combination of PC6 and PE10 was found to be useful for a two-site sandwich enzyme-linked immunosorbent assay (ELISA), where it gave a good dose response and was capable of measuring 10-1280 ng of the apoprotein/ml. The specificity of the monoclonal antibodies in animal species was tested by this sandwich ELISA. The results indicated that the monoclonal antibodies obtained in this study are specific for the human lung. 相似文献
10.
Characterization of cadmium-binding proteins detected in rat liver by the western blotting technique
Out of the three cadmium-binding proteins (CD-BPs) in rat liver parenchyma (40K, 29K, and 24K CdBPs), the 40K Cd-BP showed the highest affinity for cadmium (Cd), with a dissociation constant (KD) of 1.2 × 10?8M. This is in between the affinity of human serum albumin KD = 3.8 × 10?5M) and metallothionein (KD = < 10?11). These Cd-BPs may be responsible for hepatic sequestration of cadmium. 相似文献
11.
Mouse hybridoma cells, secreting monoclonal antibodies (MCA) against tomato spotted wilt virus, were produced and screened for virus specificity by an indirect triple antibody ELISA, using a rabbit polyclonal antiserum for antigen trapping. A Bulgarian virus isolate from tobacco was used for immunisation of mice and rabbits. One fusion eventually led to 10 stable hybridoma cell lines, all of which produced antibodies of IgG-type though of different subgroups. Since none of the MCAs reacted with TSWV structural proteins after electrophoresis and transfer to nitrocellulose, other methods were chosen to examine their protein specificity. Purified viral cores and detergent-solubilised envelope proteins were used as antigens for ELISA, or, alternatively, glycosylated viral envelope proteins were trapped onto microtitre plates coated with lectins in order to detect MCAs specific for them. Both methods, independently, led to the identification of two MCAs that were specific for envelope proteins of TSWV. Only these two antibodies reacted with intact TSWV particles when examined by immunogold labelling in the electron microscope. The reaction of all MCAs with 11 different TSWV isolates eventually led to the selection of one core- and one envelope-specific antibody for routine use. Core-specific MCAs revealed serological differences between isolates belonging to the common serotype (= lettuce serotype), but did not react with the serotype TSWV-I. When comparing different ELISA procedures, broadest reactivity and highest sensitivity with different isolates were obtained in an indirect test procedure, using goat anti-mouse antibody conjugates. 相似文献
12.
Three monoclonal antibody subclasses (IgG1, IgG2a, and IgM) were raised to the phosphoprotein B2 (Mr 68000, pI6.5-8.2) which has been shown previously to be associated with the nucleosomes of rat liver nuclei. These antibodies do not show any significant cross reactivity with CM-cellulose 'unbound' non-histone chromosomal proteins, bovine serum albumin or histones. Further verification of the specificity of these antibodies to this phosphoprotein was carried out using both 'dot' blot and immunological transfer analysis ('Western blot'). The monoclonal antibodies (IgG1 and IgG2a) could also be used to semi-quantify the phosphoprotein B2 in rat liver nuclei. The high specificity and unlimited availability of this type of probe provides a means to study the role(s) of this phosphoprotein in the overall scheme of actively transcribed chromatin. 相似文献
13.
J M De Winter J Korpancova H van den Bosch 《Archives of biochemistry and biophysics》1984,234(1):243-252
A comparative study was made of the metal ion requirement of rat liver mitochondrial phospholipase A2 in purified and membrane-associated forms. Membrane-bound enzyme was assayed using either exogenous or endogenous phosphatidylethanolamine. Although several divalent metal ions caused increased activity of the membrane-associated enzyme, only Ca2+ and Sr2+ activated the purified phospholipase A2. The activity in the presence of Sr2+ amounted to about 25% of that found with Ca2+. When the Ca2+ concentration was varied two activity plateaus were observed. The corresponding dissociation constants varied from 6 to 20 microM Ca2+ and from 1.4 to 12 mM Ca2+ for the high- and low-affinity binding sites, respectively, depending on the assay conditions and whether purified or membrane-bound enzyme was used. A kSr2+ of 60 microM was found for the high-affinity binding site. The effect of calmodulin and its antagonist trifluoperazine was also investigated using purified and membrane-associated enzyme. When membrane-bound enzyme was measured with exogenous phosphatidylethanolamine, small stimulations by calmodulin were found. However, these were not believed to indicate a specific role for calmodulin in the Ca2+ dependency of the phospholipase A2, since trifluoperazine did not lower the activity of the membrane-bound enzyme to levels below those found in the presence of Ca2+ alone. Membrane-bound enzyme in its action toward endogenous phosphatidylethanolamine was neither stimulated by calmodulin nor inhibited by trifluoperazine. Purified enzyme was also not stimulated by calmodulin, while trifluoperazine caused small stimulations, presumably due to interactions at the substrate level. These results indicate that calmodulin involvement in phospholipase A2 activation should not be generalized. 相似文献
14.
Y Hashimoto T Masuko K Itoh H Yagita T Hishinuma M Degawa T Kamataki R Kato 《Biochemical and biophysical research communications》1985,131(2):600-606
Ten monoclonal antibodies reactive with a high spin form of rat cytochrome P-448 (P-448-H) were obtained from hybridoma clones established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells of a BALB/c mouse hyperimmunized with the cytochrome. One monoclonal antibody recognized an epitope characteristic for P-448-H. Five monoclonal antibodies were cross-reactive with a low spin form of rat cytochrome P-448, but not with cytochrome P-450. Reactivity of these monoclonal antibodies with microsomes of rats pretreated with drug metabolizing inducers and Western blots of the microsomal cytochrome P-450 components are also demonstrated. 相似文献
15.
16.
Monoclonal antibodies to monoamine oxidase B and another mitochondrial protein from human liver.
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A monoclonal antibody has been generated to human liver monoamine oxidase (MAO) B by fusion of mouse myeloma cells with spleen cells from a mouse immunized with a mixture of semi-purified MAO A and MAO B. The antibody, 3F12/G10, an immunoglobulin G1, reacts with its antigen in cryostat sections of human liver, showing an intracellular particulate distribution as demonstrated by immunoperoxidase staining. The antibody indirectly precipitates [3H]pargyline-labelled human MAO B both from liver and platelet extracts but fails to precipitate MAO A from liver extracts. The antibody does not recognise rat liver MAO B, showing that the determinant is not universally expressed on MAO B. The antibody has no effect on the catalytic activity of MAO B. Other monoclonal antibodies were generated but they are directed to a protein with a subunit Mr of 54 000, a contaminant of the MAO preparation. One of these antibodies, A8/C2, an IgG2a, reacts with the same protein in both rat and human liver extracts. 相似文献
17.
Iu Iu Skarga L P Dolgacheva N I Fedotcheva G D Mironova 《Ukrainski? biokhimicheski? zhurnal》1987,59(6):54-59
The K+ transport in rat liver mitochondria was studied by the immunochemical method. Antibodies to mitochondrial K+-transporting protein with molecular weight 60 kDa were obtained and used as possible inhibitor or K+ transport. Antibodies depressed the DNP-induced K+ efflux and energy-dependent swelling by 50% without causing changes in respiration and oxidative phosphorylation. 相似文献
18.
Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dot-blot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde- or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu1,7]-eel CT by dot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species. 相似文献
19.
Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions 总被引:1,自引:0,他引:1
This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure. 相似文献
20.
Monoclonal antibodies directed against antigens on rat liver epithelial cell lines were prepared. Three antibodies, 4C3, 19C6, and 3C2, recognized surface antigens present (although in different quantities) on eight epithelial cell lines tested, irrespective of whether they were normal or transformed. For MAb 3C2, the primary antigen common to all but one cell line showed a Mr of 135 kD. In paraffin sections of liver tissue, two antibodies, 40 and 19C6, reacted exclusively with bile duct epithelium, whereas the MAb 3C2 additionally reacted with sinusoidal endothelium and the endothelium of the portal venules. In sections of livers from rats exposed to diethylnitrosamine, the MAb 19C6 selectively stained bile duct-like structures in cholangiomas, while other preneoplastic and neoplastic lesions were not stained. These results demonstrate that the monoclonal antibodies obtained may prove useful for investigating cell lineages related to propagable liver epithelial cell lines and suggest that these cells may be derived from terminal bile ductular cells.Abbreviations ABTS2
2,2azinobis(3-ethylbenzthiazolinesulfonic) acid
- ARL
adult rat liver
- DEN
diethylnitrosamine
- FCS
fetal calf serum
- MAb
monoclonal antibody
- PAP
peroxidase antiperoxidase 相似文献