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1.
Guinea pig C1q was purified, in a highly active hemolytic form, by a combination of precipitation with chelating agents, CM-cellulose and Sepharose 6B. Yields ranged from 30 to 35% protein, and the activity of final preparations was in the range of 2 × 1013– 3 × 1013 C1q effective molecules/mg. The molecular weight of C1q was approximately 430,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). C1q was shown to be composed of two non-covalently linked subunits of approximate molecular weights 46,500 and 45,000 in a molar ratio 2 : 1. On reduction, the higher molecular weight subunit gave two chains having approximate molecular weights of 24,500 and 23,000 in equimolar ratio, and the lower weight subunit gave one chain with a molecular weight of approximately 22,300. C1q contained hydroxyproline, hydroxylysine and a high percentage of glycine. Thus, the overall molecular structure of guinea pig C1q appears similar to that of human C1q.The antiserum against the purified C1q showed only one precipitation band with guinea pig whole serum of purified C1q on immunodiffusion analyses and was found to be monospecific. 相似文献
2.
C. SAND PETERSEN N. STRANDBERG PEDERSEN N. H. AXELSEN 《Scandinavian journal of immunology》1982,15(5):459-465
TR-c is a Reiter treponemal antigen that cross-reacts with an antigen in Treponema pallidum (Nichols pathogenic strain). Sera from patients with secondary syphilis contain precipitating antibodies against TR-c. The isolation of TR-c from a crude bacterial sonicate involves five fractionation steps: anion exchange chromatography (DE-52 Whatman), gel filtration (Ac-A-22. Ultrogel), and affinity chromatography respectively on phenyl-Sepharose CL 4B, iminodiacetic acid-Sepharose CL 4B. and lysine-Sepharose 4B. The purified TR-c was enriched 320 times compared with the starting material. and the recovery was 22%, TR-c was shown to be a protein, it did not bind to a series of lectins. and by gel filtration and polyacrylamide gel electrophoresis (PAGE) the mol, wt was determined to be in the range of 630, 000–730.000. It was found by SDS-PAGE to be composed of identical subunits, each having a mol. wt of 48, 000. The isolated TR-c was immunochemically pure when tested in crossed immunoelectrophoresis against polyspecific anti-Reiter Ig. The purified TR-c antigen was used for production of a monospecific rabbit antiserum. Monospecific rabbit anti-TR-c gave strong fluorescence with both the Reiter treponeme and T.pallidum . 相似文献
3.
Purification and characterisation of ovine C4: evidence for two molecular forms in ovine plasma 总被引:3,自引:0,他引:3
A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the alpha chain--108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated alpha' chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the alpha and beta chains were lowered following digestion of ovine C4 with neuraminidase. 相似文献
4.
Three glycoproteins of intact hepatitis B surface antigen (HBsAg) with mol. wt. of 32 000, 30 000 and 28 000 respectively were identified by reaction with 125I-concanavalin A (Con A) after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen was effectively disrupted with Triton X-100 to produce a structure with a sedimentation coefficient of 3.9S. Affinity chromatography of disrupted HBsAg using concanavalin A-Sepharose 4B (Con A-Sepharose) resulted in two fractions. The first contained material which did not bind to the lectin and consisted of a single polypeptide of mol. wt. 64 000. Further studies revealed this component to be serologically identical to serum albumin and to lack any affinity for antibody to HBsAg. A comparison of the tryptic peptide map of this polypeptide with that of purified serum albumin demonstrated identical amino-acid sequences. The second fraction contained material which bound to Con A and contained two polypeptides with mol. wt. of 28000 and 23000 respectively. HBsAg reactivity was associated with this fraction. This procedure allows the prepartion of HBsAg sub-units in milligram quantities for further immunological studies. 相似文献
5.
The aim of the present study was to define the physicochemical structure of C3b and C3d receptors of lymphoid cells, C3b and C3b receptors were isolated from KBr lysates of the 20,000 g fraction of human tonsil homogenates by immunoprecipitation with an anti-C3 receptor serum (AC3RS). Sodium dodecyl sulphate (SDS) gel filtration and polyacrylamide gel electrophoresis (PAGE) of unreduced immunoprecipitates revealed a highly predominant component with an apparent molecular weight (mol.wt.) greater than 1 × 104 and a small component with a mol.wt. of 80,000. After reduction, the SDS-PAGE profile was made up of a constant major 38,000 mol.wt. component and an inconstant smaller 18,000 mol.wt. component. The 38,000 (and also the 18,000) component could he isolated only from C3 receptor-active lysates, and not from C3 receptor-negative lysates. Taken together, the results of this study suggest that the active C3 receptor molecule of tonsil cells is a lipoprotein complex with a mol.wt. greater than 1 × 104 ; its protein moiety consists predominantly of disulphide-bridged polypeptide chains with a mol.wt, of 38,000; C3b and C3d receptors are composed of equal-sized polypeptide chains, but the specific binding sites for C3b and C3d are located on different molecules. 相似文献
6.
P Lauzurica N Maruri B Galocha J Gonzalez R Diaz P Palomino D Hernandez R García C Lahoz 《Molecular immunology》1988,25(4):337-344
A dialyzed extract of olive (Olea europea) pollen was fractionated by anion exchange chromatography on DEAE-Sepharose CL-6B using a discontinuous gradient of ammonium bicarbonate. The most important protein allergen was obtained from the 0.3 M fraction after gel filtration on Sephadex G-100 and separation by lentil-lectin Sepharose-4B. The major allergen of olive pollen was contained in the effluent and was designated Olea Antigen I. This material inhibited the RAST activity of 15 patients' sera that were tested. Analytical IEF demonstrated a major band at pH 5.3 and two minor ones at pH 5.6 and 5.0. When these were run into SDS-polyacrylamide gel electrophoresis in a second dimension, all were separated into two bands of mol. wt 17 and 19 K. A second protein, which is the next most important allergen, Olea Antigen II, was obtained from the 0.5 M fraction by chromatofocusing in a 4-7 pH range followed by filtration on Bio-gel P-30. Olea Antigen II had a mol. wt of 8 K as assessed by SDS-PAGE. IEF analysis displayed one main band at pH 3.6 and two minor bands at pH 3.8 and 4.0, respectively. OL-1, an anti-Olea europea monoclonal antibody (MAb) previously reported by us Lauzurica et al. (1988) reacted with the 17 and 19 K antigens from the crude extract and with Olea Antigen I but not with Olea Antigen II. 相似文献
7.
Further purification of the mitochondrial inner membrane autoantigen reacting with primary biliary cirrhosis sera 总被引:6,自引:0,他引:6
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The mitochondrial inner membrane autoantigen reacting with sera from patients with primary biliary cirrhosis depends on lipid and protein moieties for complement-fixing activity, but its chemical analysis requires some degree of solubilization. Attemps to achieve this by citraconylation led to anticomplementary effects and inactivation, but treatment with 8 M urea fragmented the membranes sufficiently to allow gel filtration and estimation of its mol. wt at 180,000–200,000. The antigen was further purified by affinity chromatography using Igs from patients with anti-mitochondrial antibodies (AMA) coupled to Sepharose 4B, as immunosorbent. The 8 M urea eluate was about 100 times more active than crude inner membranes and showed a single band on polyacrylamide electrophoresis. Liver and brown fat gave the same band, brown fat having four times the potency of liver. Electron microscopy of the purified antigen from the two organs showed that it reaggregated into membranous vesicles when urea was removed. The purified antigen may be of use if an automated radioimmunoassay were to prove sensitive and specific for the detection of AMA as this antibody is an important marker for `autoimmune'' chronic liver disorders. 相似文献
8.
Physicochemical characterization of C3b receptors isolated from human erythrocytes by immunoprecipitation. 总被引:2,自引:1,他引:1
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A high yield of active C3b receptors was obtained by solubilizing human erythrocyte membranes with 2 M KBr, whereas other solubilization agents yielded no, or significantly less activity. Gel filtration of the KBr lysates revealed that the apparent molecular wieght of biologically active C3b receptor molecules was greater than 1 x 10(6). Immunoprecipitates prepared with radio-iodinated KBr lysates and anti-C3 receptor sera (AC3RS) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or sodium dodecyl gel filtration. Unreduced SDS-PAGE and gel filtration profiles showed three predominant peaks with apparent mol. wts of 1--1.3 x 10(6), 80,000 and 60,000. Whereas the high mol. wt component decreased only slightly after reduction, the 80,000 and 60,000 mol. wt components disappeared and two new peaks with apparent mol. wts of 38,000 and 18,000 appeared in SDS-PAGE profiles. Although the high mol. wt component present in reduced SDS-PAGE profiles was detectable in some of the control experiments, none of the other peaks could be precipitated with control sera, and these components could be demonstrated only when KBr lysates of C3b receptor-positive erythrocytes and AC3RS that were able to inhibit ligand binding of the C3b receptors were used for precipitation. These findings suggest that (a) the C3b receptor of human erythrocytes in its biologically active state is a macromolecule with an apparent mol. wt higher than 1 x 10(6) and (b) the protein moiety consists predominantly of non-covalently linked protein molecules with apparent mol wts of 80,000 and 60,000. These protein molecules are composed of disulphide-bridged polypeptide chains with apparent mol. wts of 38,000 and 18,000. 相似文献
9.
H.-H. Mussel T. Ehlen M. Schmitt M.D. Kazatchkine L. Neyses M.P. Dierich 《Immunology letters》1982,4(1):1-6
Applying 2 M KBr, membranes of Ehu were solubilized. By C3-affinity chromatography an activity could be isolated that inhibited the immune adherence reaction and C3b-dependent rosette formation. Since this material did not agglutinate EAC14oxy 23b we termed it monovalent C3b receptor (mC3bR). PAGE and SDS-PAGE and staining with Coomassie brillant blue and PAS reagent revealed a single glycoprotein band with a mol. wt. of 55,000–60,000 daltons and an electrophoretic mobility comparable to ovalbumin. This mC3bR proved to be antigenetically related to gp 205 [17]. The potential of mC3bR to react with C3b-carrying particles was not destroyed by heat and trypsin treatment but by neuraminidase or periodic acid treatment suggesting that mC3bR reacted by its carbohydrate moiety with C3b. As by mC3bR, immune adherence could be inhibited by D-glucose and D-galactose but not by their optical antipodes, L-glucose and L-galactose. 相似文献
10.
An efficient method is described for the isolation of highly purified, IgG-free and stable guinea-pig serum C1q. The procedure includes the chromatography of EDTA-treated serum (25 mM EDTA) on CM- and DEAE-cellulose followed by gel filtration on ACA 34-Ultrogel whereby ammonium sulfate precipitation was used for concentration. The final product stored in a glycerol containing buffer was purified 700-fold with a yield of approximately 50%. It was judged to be homogeneous by several criteria including SDS-PAGE, analytical ultracentrifugation, gel filtration and immunoprecipitation. The protein has a sedimentation rate of 11.3 S and consists of three distinct polypeptide chains A, B and C with mol. wts of 30,200, 28,200 and 24,000. Amino acid analysis revealed a content of 4.42% hydroxyproline, 1.81% hydroxylysine and 18.7% glycine. In contrast to human serum C1q a very low content of cysteine residues was detected. SDS-PAGE analysis performed in the absence of 2-mercaptoethanol but in the presence of 5-10% SDS revealed clearly that gps-C1q is dissociated in a time-dependent manner into the individual chains. 相似文献
11.
The C4a anaphylatoxin was purified from rat sera activated by heat-aggregated IgG. The anaphylatoxin was isolated by a three-step purification procedure and was judged to be homogeneous based on visualization of a single stained band after electrophoresis on both cellulose acetate membrane strips and on 9% SDS-polyacrylamide gels. Results from Ouchterlony and radioimmunoassay analysis indicated that neither rat C5A nor C3a contaminated the C4a preparation. Rat C4a is a glycoprotein estimated to be 11,000-12,000 mol. wt and contains 76 amino acid residues representing a mol. wt of 8577 and one oligosaccharide unit of 2000-3000 mol. wt. Rat C4a is weakly active in contracting guinea pig ileum at 0.1-1 microM, which is comparable with the activity of human C4a. Both human and bovine C4a are polypeptides free of carbohydrate while rat and presumably mouse C4a are glycoproteins. The complete primary structure of rat C4a anaphylatoxin has been elucidated as follows: (formula; see text) 相似文献
12.
Antigens related to the carcinoembryonic antigen (CEA) were isolated from normal human plasma by perchloric acid extraction, gel permeation chromatography and immunoaffinity chromatography using a monoclonal antibody with broad specificity and high affinity. The antigens were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The binding of five monoclonal anti-CEA antibodies with different epitope specificities to the immobilized antigens was analyzed. Two antigens with mol. wts of greater than 200,000 and 177,000 bound all five antibodies, and two antigens with mol. wts of 114,000 and 85,000 bound three of the five antibodies. The findings reported indicate that even monoclonal antibodies with high specificity for colonic cancer CEA detect CEA-related antigens in normal human plasma. 相似文献
13.
Identification, purification and characterization of a streptococcal protein antigen with a molecular weight of 3800. 总被引:6,自引:0,他引:6
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A small molecular weight streptococcal antigen of about 3800 was isolated from Streptococcus mutans. The peptide was obtained by gel filtration of a predominantly 185,000 mol. wt. antigen preparation, with two major antigenic determinants (I/II), on Sephacryl S-200, in the presence of sodium dodecyl sulphate (SDS). The 185,000 mol. wt. antigen was prepared from the culture supernatant of S. mutans by ammonium sulphate precipitation, DEAE cellulose chromatography and gel filtration on Sepharose 6B. The 3800 mol. wt. material gave a single band on SDS/polyacrylamide gel and reacted with antisera to streptococcal antigen I/II, I and II but not III. Furthermore, it was digested by pronase, contained only traces of carbohydrate and lipids were not detected. It is suggested that SA I/II is either synthesized in a range of molecular sizes from 185,000 to 3800 or the former is broken down by streptococcal proteases into smaller fragments. 相似文献
14.
Highly purified toxin A of Clostridium difficile was obtained by bovine thyroglobulin affinity chromatography followed by two sequential anion-exchange chromatography steps on Q Sepharose FF and Mono Q. After Q Sepharose FF chromatography of a thyroglobulin affinity-purified toxin A preparation, two major peaks of cytotoxicity representing toxins A and B were detected. The homogeneity of the final toxin A preparation obtained from Mono Q anion-exchange fast protein liquid chromatography was ascertained by gel electrophoresis developed by silver stain. The mol. wt of toxin A in non-denaturing conditions was estimated to be 520-540 Kda by native polyacrylamide gel electrophoresis (PAGE) developed by silver stain. In contrast, with sodium dodecyl sulphate (SDS)-PAGE under reducing or non-reducing conditions, a major band of 240 Kda and 10 minor and 27 faint bands (non-reduced conditions), or four minor and 31 faint bands (reduced conditions) were detected after silver staining. In two-dimensional PAGE, the seven minor bands of greater than 240 Kda obtained by non-reducing SDS-PAGE migrated to the 240-Kda position after reduction with beta-mercaptoethanol. 相似文献
15.
Purification of cytotoxic enterotoxin of Aeromonas sobria by use of monoclonal antibodies 总被引:3,自引:0,他引:3
Cytotoxic enterotoxin of Aeromonas sobria was purified by affinity chromatography with monoclonal antibodies. The purified enterotoxin gave a single protein band in polyacrylamide gradient gel electrophoresis and its mol. wt estimated by this technique was 63,000; it had a pI of 6.2. The purified enterotoxin caused fluid accumulation in rat ileal loops and in infant mice, was cytotoxic to cultured cells, was haemolytic to human erythrocytes, and was lethal to mice after intravenous injection. The relative concentrations of enterotoxic, cytotoxic and haemolytic activities were approximately the same in a culture filtrate and in purified, electrophoretically homogeneous enterotoxin. The three activities were also inactivated to the same extent after incubation for 10 min at 56 degrees C. There was no immunological cross-reactivity with cholera toxin (CT) nor did antiserum to CT neutralise the biological effects of the toxin. 相似文献
16.
An allergen fraction of high specific PCA2 activity has been isolated from incubation fluids of Nippostrongylus brasiliensis adult worms by combining Sephadex gel filtration with polyvinyl-chloride preparative electrophoresis. Allergen behaviour during gel filtration and preparative isoelectrofocusing indicated a molecular weight of 12,000 daltons and an isoelectric point (pI) of 6.0, respectively. A single protein component of the same mol. wt. or pI was detectable after SDS-PAGE or IEF analysis of the purified allergen. These results are discussed with reference to previous studies on Nippostrongylus brasiliensis allergen. 相似文献
17.
S. THIEL G. BAATRUP P. FRIIS-CHRISTIANSEN S.-E. SVEHAG J. C. JENSENIUS 《Scandinavian journal of immunology》1987,26(5):461-468
The structural characteristics of a human plasma protein analogous to bovine conglutinin were studied. The protein was previously found to bind to complement-reacted IgG in a calcium-dependent and N-acetyl-D-glucosamine-inhibitable manner and it further shows cross-reactivity with anti-bovine conglutinin antibody. By gel permeation chromatography the conglutinin activity in human plasma was localized to fractions containing proteins of Mr at around 700,000. The conglutinin was localized by one ELISA for antigen determinants and by another for biological activity. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions these fractions were shown to contain proteins of about 300,000. When human conglutinin-like protein, partially purified by affinity chromatography, was analysed unreduced by SDS-PAGE followed by western blotting, the cross-reacting anti-bovine conglutinin antibody bound to a protein with an Mr of 330,000. When the sample was reduced and alkylated before electrophoresis a band of 66,000 was immunostained. The 330,000 and 66,000 proteins were shown to be collagenase sensitive. 125I-iC3b was seen to bind to the 330,000 band when incubated with western blots of partially purified human conglutinin. 相似文献
18.
Isolation and characterization of immunoglobulin of hagfish, Eptatretus burgeri, a primitive vertebrate 总被引:2,自引:0,他引:2
The immunoglobulin of the hagfish, Eptatretus burgeri, one of the most primitive vertebrates extant, was isolated from the serum of non-immune normal adult hagfish in a pure form. Analysis of the immunoglobulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition indicated that the immunoglobulin was composed of heavy (H) and light (L) chains. The mol. wt of the H-chain was 68,000, slightly smaller than that of the human mu-chain. The L-chain of the immunoglobulin appeared as 2 bands on SDS-PAGE, with mol. wts of 25,000 and 22,000. These findings were confirmed by gel filtration of reduced-alkylated immunoglobulin in 5 M guanidine-HCl. The H:L molar ratio of the immunoglobulin was roughly 1:1. Gel filtration of the immunoglobulin in non-dissociating buffer indicated that the mol. wt of the intact immunoglobulin was 150,000-160,000. Thus, the subunit chain composition of the immunoglobulin was assumed to be H2L2, identical with the fundamental structure of immunoglobulins. The instability of the hagfish immunoglobulin was ascertained by the fact that it dissociated into heterogeneous mol. wt components ranging from approx. 90,000 to 160,000 upon SDS-PAGE under non-reducing conditions. However, almost no free or monomeric H- or L-chains were dissociated from the immunoglobulin by this procedure and also by gel filtration in 5 M guanidine-HCl. Theses results indicated that the hagfish immunoglobulin is unusually labile in its tertiary structure but has disulfide binding between at least more than 2 subunit chains. 相似文献
19.
A component of Parietaria judaica pollen extract, previously identified as the major allergen, then reported as Pj10 and hereafter denominated Par j I has been isolated by a combination of 65% ammonium sulphate salt precipitation and gel filtration and an Ultrogel AcA54 column. The purified allergen appeared essentially homogeneous on gel filtration HPLC. The mol. wt of Par j I was estimated by electrophoretic and chromatographic techniques. All results gave values in a range from 13 K to 25 K. Analysis in SDS-PAGE under reducing conditions revealed a broad band corresponding to a mol. wt of 10 K, which retained allergenicity when tested with a patients serum pool. CIE and CRIE patterns of the isolated Par j I displayed the two precipitating lines already reported as those exhibiting the highest IgE-binding ability. Par j I showed a specific allergenic activity about 10-fold higher than that of the whole extract and was demonstrated to be the major allergen of Parietaria judaica as assessed in 25 sensitive human sera. 相似文献
20.
Biosynthetically labelled C1q secreted by guinea pig peritoneal macrophages was analysed by sedimentation through sucrose gradients followed by SDS-PAGE. In addition to the haemolytically active C1q of mol. wt 460,000 Da a low mol. wt (LMW) form of C1q was identified which had no detectable affinity for Fc of aggregated immunoglobulin, but which retained the ability to associate with the C1r2s2-complex. This LMW-C1q was covalently associated with two additional polypeptides of mol. wt 46 and 50 kDa. 相似文献