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1.
In Dictyostelium discoideum, the lysosomal enzyme alpha-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (t 1/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with lysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.  相似文献   

2.
In Dictyostelium discoideum the lysosomal enzyme alpha-mannosidase is initially synthesized in vivo as a 140,000 Mr protein which is subsequently processed into two mature acidic glycoproteins of 60,000 and 58,000 Mr. To investigate the initial events involved in the synthesis of this protein, mRNA isolated from growing cells was translated in vitro and the resulting protein products were immunoprecipitated with antibodies prepared against the purified enzyme. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of an immunoprecipitable 120K protein that was identified as the alpha-mannosidase primary translation product by a variety of criteria. Translation in vitro in the presence of dog pancreas microsomes resulted in the conversion of the 120K primary translation product to a 140K form. This 140K species was not accessible to added trypsin under conditions preserving membrane integrity, suggesting it is sequestered in the lumen of the endoplasmic reticulum following synthesis. Treatment of either the in vitro modified or cellular 140K alpha-mannosidase precursors with endoglycosidase H resulted in the appearance of proteins 2K larger than the primary translation product. The pulse-labeled cellular precursor and the in vitro processed form have similar isoelectric points as revealed by two-dimensional gel electrophoresis. These results imply that the precursor is N-glycosylated in the endoplasmic reticulum possibly without removal of the signal sequence and that the majority of acidic modifications are added late in the post-translational pathway.  相似文献   

3.
The cellular specific activity of lysosomal alpha-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. alpha-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t1/2 less than 10 min). This indicates that the developmental accumulation of alpha-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional alpha-mannosidase-1 mRNA during development.  相似文献   

4.
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   

5.
A mouse monoclonal antibody, FMC55 (an IgG1), to alkaline phosphatase was prepared and evaluated in immunostaining. Clones producing antibody to alkaline phosphatase were selected using a micro-ELISA which identified antibodies forming active soluble complexes (APAAP) with the enzyme. Conditions that influenced the formation of the complex were investigated by using a quantitative assay in which the complex was captured by a bridging anti-mouse antibody. The ratio of FMC55 to enzyme had a major influence on the activity of the complex. Although all complexes had some activity, those that contained excess antibody had reduced ability to bind to anti-mouse antibody because of competition with excess unlabeled antibody. The optimal complex was formed with 3 micrograms of FMC55 per unit of enzyme. This complex contained neither free enzyme nor free antibody. The molecular weight by gel permeation chromatography was 600,000, giving a composition of two enzyme and two antibody molecules or one enzyme and three antibody molecules. The size of the complex was not altered by adding excess antibody or excess enzyme. Immunoblotting showed that FMC55 bound only to the Mr 140,000 homodimeric form of alkaline phosphatase. The APAAP complex was used in combination with biotin-streptavidin-peroxidase reagent to detect two antigens labeled with two different mouse monoclonal antibodies in the same tissue preparation.  相似文献   

6.
The structural characteristics and glycoprotein nature of the human growth hormone (hGH) receptor in cultured lymphocytes (IM-9 cell line) were studied with the use of a bifunctional reagent (disuccinimidyl suberate) to couple 125I-hGH covalently to intact cells. After cross-linking, the hormone-receptor complexes were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A single band of Mr 140,000 was identified under reducing conditions. The labelling of this band was blocked by unlabelled hGH but not by insulin, ovine prolactin, bovine or ovine growth hormones. The Mr 140,000 band was immunoprecipitated by either anti-hGH antibody or by a monoclonal antibody against rat liver growth hormone receptor. In the absence of reductant two major bands of Mr 270,000 and 140,000 were found. On two-dimensional gel electrophoresis, with the first dimension in the absence of reductant and the second in its presence, the Mr 270,000 complex generated the Mr 140,000 band. The nature of the oligosaccharide chains of the receptor was studied by treatment with different glycosidases. The electrophoretic mobility of the Mr 140,000 receptor complex was markedly increased after digestion with endoglycosidase F but showed no or little change after digestion with endoglycosidase H. The Mr 140,000 band was also sensitive to neuraminidase treatment. In addition the 125I-hGH-receptor complex was adsorbed by immobilized wheat germ agglutinin and to a smaller extent by immobilized concanavalin A, lentil lectin, ricin I and ricin II. In conclusion, taking into account that hGH is a Mr 22,000 polypeptide, the binding subunit of the GH receptor in human IM-9 lymphocytes has an Mr of approx. 120,000. The native receptor may exist as a homodimer of the binding subunit formed by disulphide bonds. Furthermore, the GH receptor subunit contains asparagine N-linked type of oligosaccharide chains. Most, if not all, of these chains are of the complex type and appear to be sialylated whereas no high-mannose type chains are detectable in the mature form of the receptor.  相似文献   

7.
We describe the development of five murine monoclonal antibodies (14A12, 39A1, 53A9, 73A7, and 128A6) specific to human apolipoprotein[a] (Mr approximately 570,000), and their characterization by a number of procedures including cotitration, competition and inhibition enzyme-linked immunosorbent assays (ELISA), immunoblotting of native lipoproteins and of SDS-solubilized apolipoproteins electrophoresed in polyacrylamide gels, and dot immunobinding assays. The patterns of immunoreactivity of these antibodies were similar. Each reacted in ELISA assays and upon electroimmunoblotting with purified apo[a], with apo[a] liberated by reduction of Lp[a], and with delipidated Lp[a] solubilized in SDS, but by contrast, they reacted with native Lp[a] to a significant degree only upon electroimmunoblotting. No reactivity was seen with LDL-apoB-100 or with other apolipoproteins. The cross-reactivity of these antibodies with the homologous protein, plasminogen, was examined by comparison of the amount of plasminogen or apo[a] required for 50% inhibition of antibody binding to apo[a], and by an ELISA assay. The inhibition assay showed reactivity with plasminogen to be 37- to 50-fold lower than with apo[a], while dot immunobinding showed the lower limit of detection of plasminogen and of apo[a] to be approximately 320 and 31 micrograms, respectively. In an ELISA sandwich assay based on monoclonal antibodies LHLP-1, 14A12, and 53A9, the lower limit of Lp[a] detection (approximately 1 ng/ml protein) was about 100-fold less than that of plasminogen. Chemical modification of apo[a] revealed a significant contribution of arginine residues to the epitopes of 14A12, 39A1, and 53A9. Modification of cysteine residues with iodoacetamide was without effect, thereby distinguishing these antibodies from LHLP-1. Each antibody reacted with the six major size forms of apo[a] (Mr approximately 450,000-750,000) in immunoblots of human sera electrophoresed in SDS-polyacrylamide gels. Marked heterogeneity in apo[a] phenotype was detected and both single and double band phenotypes were observed in a randomized study. Cotitration and competition binding studies showed varying degrees of interaction between all five epitopes, with the exception of 128A6 which appeared to be independent of 39A1 and 53A9 (and vice versa). These data suggest that our five monoclonal antibodies recognize epitopes on apolipoprotein[a] that are exposed and accessible on the native Lp[a] particle. We conclude that our monoclonal antibodies recognize a specific region of apo[a], and that this region undergoes a conformational change upon adsorption of Lp[a] to plastic thereby diminishing epitope recognition.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

8.
Three monoclonal antibodies to avian lipoprotein lipase have been isolated by fusing spleen cells from immunized BALB/c mice with myeloma P3X-63 Ag 8. The antibodies were detected by their ability to bind immobilized lipoprotein lipase in enzyme-linked immunosorbent assay (ELISA) and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibodies. Two of these antibodies, CAL1-7 and CAL1-11, inhibited catalytic activity, whereas with CAL1-2 interaction with lipoprotein lipase could be demonstrated only in ELISA and in Western blot assays following denaturation of the enzyme with sodium dodecyl sulfate. An immunoadsorbent column was prepared by coupling immunopurified CAL1-11 to Sepharose-4B. When acetone powder extracts of adipose tissue were applied on the column, 70% of the catalytic activity bound to the matrix. Effective elution was achieved with 1.8 M NaCl, 40% glycerol, 5% acetone, 20 mM Chaps (3[(3-cholamidopropyl)dimethylammonio]propanesulfonate), 0.5 mM EDTA, 1 mM phosphate (pH 6.5). After concentration of the active fractions on a heparin-Sepharose 4B column, the purified enzyme was obtained with an overall recovery of 25%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrates that the preparation is homogeneous with a major band at Mr 60900. Thus, avian adipose lipoprotein lipase has been purified by a one-step immunoaffinity followed by a concentrating step on heparin-Sepharose 4B.  相似文献   

9.
To facilitate selection of monoclonal antibodies for immunoaffinity chromatography, an ELISA screening procedure was developed. The assay is based on the avidin-biotin system and provides a profile of the monoclonal antibody which is based on the binding characteristics of the antigen binding site when exposed to different elution reagents. The elution profiles of 5 monoclonal antibodies to soy bean trypsin inhibitor (SBTI) were determined and for 2 of the antibodies the results obtained in the ELISA were verified using column experiments. The affinity constants were determined for the same 5 monoclonal antibodies and no correlation was seen with the ease of elution. The elution profiles presented here are easily obtained and the results indicate that a general screening procedure for suitable combinations of antibodies and elution conditions can be carried out using an elution ELISA assay when modified as described herein.  相似文献   

10.
Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin.  相似文献   

11.
单克隆抗体与多克隆抗体配对ELISA方法比较   总被引:2,自引:0,他引:2  
以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.  相似文献   

12.
Göran Key  Elmar W. Weiler 《Planta》1988,176(4):472-481
Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis  相似文献   

13.
Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme.  相似文献   

14.
Four monoclonal antibodies were raised against polypeptides present in a high-salt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunofluorescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antibodies labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with variable intensities. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,000 Mr (two to three bands) polypeptides and a diffuse band around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypeptides putative plant intermediate filament proteins.  相似文献   

15.
The polypeptides recognized by a monoclonal antibody against calf thymus DNA polymerase alpha (secreted from a hybridoma CL22 -2- 42B , Nucleic Acids Res. (1982) 10, 4703-4713) were identified by the immunoblot method as the large polypeptides of the partially-purified 10S DNA polymerase alpha fraction. Using an immunoprecipitation technique with the monoclonal antibody, a rapid immunological isolation of the polypeptides has been achieved. By this method, the large polypeptides with Mr = 140,000, 145,000, and 150,000 were isolated from a partially-purified preparation of 10S DNA polymerase alpha. On the other hand, the polypeptides with Mr = 150,000, 180,000, and 240,000 were obtained from a crude extract of calf thymus. Tryptic peptide maps showed that the large polypeptides with Mr = 150,000, and 180,000 were very similar in primary structure and that the structures of Mr = 180,000 and 240,000 polypeptides contained partially common sequences. Among these polypeptides, the Mr = 150,000 polypeptide was shown to correlate with the enzyme activity. These results suggest that the large polypeptide of 10S DNA polymerase alpha is initially synthesized as Mr = 180,000 or larger polypeptide, then converted to the form with Mr = 150,000. The Mr = 140,000 and 145,000 polypeptides in the purified preparation may be artificial products formed during purification.  相似文献   

16.
Sandwich ELISA experiment requires two mutually compatible affinity reagents, typically antibodies, that are highly sensitive and specific to the target analyte in its native conformation, and whose epitopes do not overlap each other’s. Finding or developing a pair of antibodies that meet these requirements can be a challenge and many otherwise useful antibodies fail in sandwich ELISA-format analysis. In order to discover sandwich immunoassay-compatible antibodies for epidermal growth factor receptor (EGFR), we first immobilized cetuximab, a high-affinity monoclonal antibody against EGFR, on a plastic surface and affinity-captured EGFR in A431 cell lysate. Panning of a phage antibody library on the surface-captured antigen yielded a couple of antibody clones that can be paired with cetuximab and also with each other in the sandwich-format immunoassay. This provides a potentially useful strategy for the development of sandwich ELISA reagents or antibodies against antigens that are hard to purify.  相似文献   

17.
Using a panel of monoclonal antibodies against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture ELISA can be significantly increased by simultaneous immobilization of two cooperating capture monoclonal antibodies on a solid phase. This method ("triple-site ELISA") uses three monoclonal antibodies to different epitopes of the same antigen (two capture/one tracer) unlike the traditional double-site assay using one capture and one tracer monoclonal antibody. We developed double- and triple-site ELISA for Mb by varying the capture and tracer monoclonal antibodies. Triple-site assays showed 4-6-fold increase in sensitivity compared to the double-site assays. A model for this effect is suggested; according to the model, in triple-site ELISA, high-affinity cyclic configurations can be formed by an antigen, two capture monoclonal antibodies, and the surface of the solid phase.  相似文献   

18.
Summary Purified lactate dehydrogenase (LDH) isoenzyme 1 (H or B subunits) and isoenzyme 5 (M or A subunits) were used to prepare monoclonal antibodies (MAb) suitable for immunohistochemical detection on formalin fixed paraffin-embedded tissue sections. In the initial fusions, screening of the antibodies was based on enzyme linked immunosorbent assay (ELISA) against the immunogens. None of the antibodies obtained was satisfactory. There were various problems related to specificity, crossreactivity, affinity and also the properties of the monoclonal antibody itself. Using a combined system involving more than one method for screening, two suitable monoclonal antibodies, MAb65 (to H-type LDH) and MAb25 (to M-type LDH) were selected. Both antibodies reacted specifically with corresponding LDH isoenzymes as shown in a series of tests. Their reactivity in sections of formalin fixed paraffin-embedded tissue indicated that both antibodies are suitable reagents for immunohistochemical studies.  相似文献   

19.
Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.  相似文献   

20.
Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of Mr 14 kilodaltons (kDa) and a smear of bands of Mr 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - Mr relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis  相似文献   

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