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1.
Anti-human serum albumin antibody (Ab) was used as a model antibody. Ab was conjugated with DTPA using cyclic DTPA dianhydride reaction and radiolabeled with 111In. The labeled Ab was purified by affinity chromatography. Size exclusion HPLC of this product showed 62% of 111In bound to monomeric Ab and 38% of the activity bound to antibody oligomers with molecular weights ranging from 300,000 to 450,000. The labeled antibody preparation was injected into the tail vein of rats. The radioactive substances in serum and the supernatant from liver homogenates were analyzed for molecular weight and immunoreactivity. Size exclusion HPLC of the serum samples indicated that the monomeric and dimeric Abs disappeared from the serum at a similar rate over a 48 h period. In addition, a new radioactive substance with an estimated molecular weight of 35,000 appeared in the serum. The immunoreactive fraction of the circulating 111In substances decreased slowly, somewhat proportional to the appearance of the metabolite. On the other hand, the immunoreactivity of the 111In substances in the supernatant from the liver homogenate decreased rapidly and no appreciable immunoreactivity was observed after 48 h. The labeled antibody was catabolized very rapidly in the liver and the major activity in the supernatant was associated with a small molecular weight metabolite which had a HPLC retention time identical to that of DTPA-111In. The second metabolite had an estimated molecular weight of 35,000. No radioactivity was associated with transferrin.  相似文献   

2.
We have developed a simple, rapid, and reproducible method for conjugating the bifunctional metal chelator diethylenetriaminepentaacetic acid (DTPA) to an IgM monoclonal antibody (MoAb) without first isolating the MoAb from the ascites fluid. Treatment of the protein mixture in the ascites fluid with cyclic DTPA anhydride (cDTPAA) followed by HPLC purification on a size exclusion column allowed isolation of the DTPA-IgM conjugate which could then be labeled with 111 In ⩾80% yield. Over the range of total protein concentrations used (11–44 mg/mL), the number of DTPA molecules per molecule of IgM was approximately one-half the molar ratio of cDTPAA to total protein. We have used this method to prepare an 111In labeled anti-Thy 1.2 IgM, a MoAb with specificity for a murine cell-surface antigen found on normal and malignant T cells and neuroectodertmal tissues. Analysis of the DTPA-IgM conjugate prior to and after 111In labeling using indirect immunofluorescence flow cytometry and a target-cell binding assay showed that the antigen specificity of this anti-Thy 1.2 MoAb is not substantially altered by the presence of up to 8 DTPA molecules per IgM molecule.  相似文献   

3.
Site-specific modification of monoclonal antibodies at their oligosaccharide had previously been demonstrated to produce excellent 111In imaging in a xenograft model using a Brown Norway (BN) rat lymphoma and a rat anti-BN MHC monoclonal antibody [Lee C. et al. Fed. Proc. Abstr. 43 3014 (1984)]. These results are due, in part, to lack of liver uptake, so we wanted to evaluate the extent of hepatic uptake observed with different monoclonal antibodies in normal mice. Biodistribution data were obtained for four monoclonal antibodies by first modifying each antibody at its carbohydrate with a diethylenetriaminepentaacetic acid (DTPA) derivative. The antibodies were then labelled with 111In and injected into normal mice. Images were obtained 24 h post-injection, and at 48 h the mice were dissected and the tissue-to-blood (T:B) ratios determined. T:B ratios were approximately 1 (or less) for every organ evaluated, indicating minimal non-specific uptake into these organs. Data is also presented for the BN-rat system which shows excellent localization into the tumor xenograft and low non-specific organ uptake. These data indicate that modification of antibodies site-specifically at their oligosaccharide results in minimal non-specific uptake into non-target tissues and enhanced localization into the tumor target, and that this may represent a preferred method for production of 111In labelled antibodies.  相似文献   

4.
Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.  相似文献   

5.
Rat liver cytosolic glucocorticoid receptor labelled with [3H] dexamethasone and stabilized with molybdate was bound to heparin-ultrogel and eluted with NaCl or heparin as a single peak of radioactivity. After heat exposure of cytosol, two steroid receptor complexes could be separated by NaCl or heparin. Characterization of the two forms was performed by means of affinity towards isolated nuclei, ssucrose gradient centrigugation and gel exclusion high performance liquid chromatography. The results presented here suggest that the two forms eluted from heparin-agarose correspond to the untransformed and transformed states of the glucocorticoid receptor complex. Taken together, these observations argue in favor of heparin-ultrogel as a suitable procedure to study the mechanism of glucocorticoid-receptor transformation.  相似文献   

6.
HDP-1 monoclonal antibody was labeled with 111In using deferoxamine, diethylenetriaminepentaacetic acid or 1-(para-bromoacetamidobenzyl)-EDTA as chelating agents or with 125I. The in vitro binding capacity and stability of the labeled molecules were evaluated using affinity chromatography. The biodistribution and imaging capabilities were compared using an animal model system that does not involve the use of tumors. Similar studies were done using the corresponding labeled F(ab′)2 and Fab′ fragments. All labeled molecules, except those treated with deferoxamine, were stable in vitro. When tested in vivo, all retained their capacity to localize in the target tissue (lung). The lung %ID/g levels for the 111In-labeled molecules were, however, slightly lower than those observed for the corresponding 125I-labeled molecules. High uptake was also observed in the liver or kidneys when the 111In-labeled molecules were used; no such results were obtained with the 125I-labeled molecules. More work appears to be necessary before the use of bifunctional chelates becomes the optimal method for radiolabeling monoclonal antibodies for use in tumor imaging.  相似文献   

7.
Indium-111-labeled antibodies, though providing superior photon flux to iodine-labeled antibodies, can exhibit high levels of accumulation in some non-target organs. In an effort to understand the nature of this non-target uptake we have evaluated the molecular weight of 111In species retained in several tissues by radio-FPLC (sizing chromatography) following injection of [111In]DTPA 5G6.4, a murine monoclonal antibody, into normal mice. Blood, liver and kidneys were removed, and liver and kidneys were homogenized at several time points after antibody injection. The proportion of 111In-containing species was found to vary with the tissue and with time. Analysis of blood showed only radiolabeled antibody. In the liver, several 111In species were identified with molecular weights compatible with intact antibody, [111In]transferrin, and low molecular weight complexes, with an increase in the proportion of [111In]transferrin and low molecular weight species occurring over time. While the same molecular weight species were also identified in the kidneys, the kidneys contained the largest percentage of low molecular weight species which increased over time. When 125I-labeled 5G6.4 was injected and the tissues similarly analyzed, only radioactive material with the molecular weight of intact antibody was detected. Comparison of two methods of purification of [111In]labeled antibody after labeling revealed a significant difference in the organ uptake of radiolabeled products for 111In. Although dialysis was sufficient for the removal of labile 111In, as determined by TLC, subsequent sizing chromatography on Bio-Gel P-60 dramatically dropped the hepatic and renal uptake of 111In relative to blood and diminished the proportion of the low molecular weight species present on sizing FPLC of extracts from tissues. These data indicate that low and intermediate molecular weight 111In compounds are accreted in the liver and kidneys following the i.p. injection of 111In-labeled monoclonal antibodies and that their uptake can be diminished by more stringent radioantibody purification. This knowledge may be valuable in developing methods for reducing non-target 111In uptake.  相似文献   

8.
Employment of radiolabeled antibodies in biological studies, allows their specific accumulation in organs and tissues to be accurately detected. However, stability of such radiolabeled antibodies depends on both the method of labeling and particular experimental conditions. Therefore, stability of labeled antibodies should be determined in every particular experiment. In this study we have investigated stability of the 125I-labeled monoclonal antibodies to gliofibrillary acidic protein (GFAP), endothelial antigen AMVB1, and non-specific mouse IgG at the stage of their synthesis and after their intravenous administration to rats with experimental C6 glioma. Stability of labeled antibodies was determined in blood samples and homogenates of organs by the method of their precipitation with trichloroacetic acid. The 125I-radiolabeled antibodies were characterized by high radiochemical putiry, immunochemical activity and stability of the resultant preparations in blood and tissues, and the brain after administration in vivo. Electrophoretic analysis, thin-layer chromatography, and immunohistochemical tests have demonstrated the radiochemical purity, immunochemical competence, and stability of the labeled antibodies in vivo.  相似文献   

9.
Angiotensins I, II, and III (AI, AII, AIII) and Saralasin (Sar1-Ala8-AII) were labeled with 125I and separated from the nonlabeled forms on minicolumns (a Pasteur pipet) of chromatofocusing medium. At low ionic strength, 125I-labeled angiotensins could be eluted with Polybuffer or a piperazine-histidine buffer at their approximate isoelectric points, while nonlabeled angiotensins remained adsorbed to the column and required 1 mol · liter?1 NaCl for elution. The 125I-labeled angiotensins prepared by this method were bound by antibodies (AI) and adrenal receptors (AII, Saralasin) to an extent similar to angiotensins prepared by DEAE-Sephadex A-25 chromatography. This new method of preparing radioiodinated angiotensins is rapid (15 min), inexpensive, and requires no fraction-collecting equipment.  相似文献   

10.
Abscisic acid (ABA) was purified from soybean (Glycine max [L.]) seed extract using a preparative high performance liquid chromatography (HPLC) procedure. The preparative procedure was rapid (70 minutes per sample), required no prior partitioning for purification and was quantitative as demonstrated with an internal standard of [2-14C]ABA, of which 98.9% was recovered.  相似文献   

11.
Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the first step, was purified by specific elution with NAD+/sulphite (22.5-fold purification, 55% step-yield). The procedure afforded mMDH preparation of specific activity approx. 1,300?u/mg (25?°C) at 45% overall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase. The LDH activity, which, bound to the anion-exchanger during the first step, was recovered from the adsorbent in 200?mM KCl, and finally purified by biomimetic-dye affinity chromatography (NAD+/sulphite elution) and a second ion-exchange chromatography step (elution with 200?mM KCl). The LDH preparation exhibited specific activity approx. 500?u/mg at 25?°C (content of impurities: pyruvate kinase and GOT were not detected; MDH, 0.01%).  相似文献   

12.
The use of internal-surface reversed-phase (ISRP) chromatography of unprocessed plasma samples was investigated as an alternative method of quantitation of the arterial plasma metabolite time course of [18F]N-methylspiperone. The ISRP method was directly compared to standard solid phase extraction/HPLC (SPE/HPLC) methods currently in wide use. Results indicate that: (1) the ISRP method is rapid and minimizes sample preparation; (2) recovery of radioactivity from the ISRP column is > 90%; (3) no radioactivity remains associated with chromatographically size excluded proteins and (4) the quantitative results are well correlated with conventional SPE/HPLC methods.  相似文献   

13.
A procedure to determine the absolute weight-average molecular weight (Mw) and molecular weight distribution (MWD) of guar by aqueous size exclusion chromatography coupled with low angle laser scattering is described. It is shown that for a rigorously purified sample of guar solution the values for Mw and MWD are 2·2×106 and 1·9 respectively. The effect of sample preparation and purification on these molecular parameters are discussed. Limitations and challenges in the aqueous size exclusion chromatography of complex water soluble polymers such as guar are also explored.  相似文献   

14.
A monoclonal antibody to carcinoembryonic antigen showing minimal cross-reactivity with blood cells and normal tissues was derivatized with benzylisothiocyanate derivatives of EDTA and DTPA. Seven chelators per immunoglobulin could be incorporated without loss of immunoreactivity. The resulting conjugates, labeled with indium-111, showed low liver uptake in animals. A cold kit, comprising the DTPA conjugate at a molarity of antibody bound chelator exceeding 1 x 10−4M, gave radiochemical yields of indium labeled antibody of ⩾95% and was stable for 1 yr.  相似文献   

15.
Summary Xanthan concentration and molecular weight distribution are determined by size exclusion chromatography in the fermentation medium after dilution and cell removal by centrifugation. The analysis is rapid enough for process control. During a batch fermentation, the average molecular weight is found to be in the range of 7.2–9.3·106 g/mole and to run through a maximum.  相似文献   

16.
The use of radiolabeled monoclonal antibodies as a diagnostic tool in nuclear medicine has grown rapidly over the past several years. Early studies utilized antibodies labeled with radionuclides of iodine (i.e. 125I and 131I) although these radionuclides are not ideal for use in tumor imaging. Recent advances and the development of new chemical methods has made it possible to label antibodies with other radionuclides (i.e. 77Br, 111In and 99mTc). The advantages and disadvantages associated with all of the different radionuclides and labeling methods will be discussed.  相似文献   

17.
Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of N-α-tosyl-L-glycyl-L-prolyl-L-arginine-p-nitroanilide hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.  相似文献   

18.
Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.  相似文献   

19.
Site-specific DNA-affinity chromatography of the lac repressor.   总被引:4,自引:1,他引:3       下载免费PDF全文
To test the feasibility of site-specific DNA-affinity chromatography, E. coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column. Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity.  相似文献   

20.
Aqueous two‐phase systems (ATPSs) as separation technique have regained substantial interest from the biotech industry. Biopharmaceutical companies faced with increasing product titers and stiffening economic competition reconsider ATPS as an alternative to chromatography. As the implementation of an ATPS is material, time, and labor intensive, a miniaturized and automated screening process would be beneficial. In this article such a method, its statistical evaluation, and its application to a biopharmaceutical separation task are shown. To speed up early stage ATPS profiling an automated application of the cloud‐point method for binodal determination was developed. PEG4000–PO4 binodals were measured automatically and manually and were found to be identical within the experimental error. The ATPS screening procedure was applied to a model system and an industrial separation task. PEG4000–PO4 systems at a protein concentration of 0.75 mg/mL were used. The influence of pH, NaCl addition, and tie line length was investigated. Lysozyme as model protein, two monoclonal antibodies, and a host cell protein pool were used. The method was found to yield partition coefficients identical to manually determined values for lysozyme. The monoclonal antibodies were shifted from the bottom into the upper phase by addition of NaCl. This shift occurred at lower NaCl concentration when the pH of the system was closer to the pI of the distributed protein. Addition of NaCl, increase in PEG4000 concentration and pH led to significant loss of the mAb due to precipitation. Capacity limitations of these systems were thus demonstrated. The chosen model systems allowed a reduction of up to 50% HCP with a recovery of greater than 95% of the target proteins. As these values might not be industrially relevant when compared to current chromatographic procedures, the developed screening procedure allows a fast evaluation of more suitable and optimized ATPS system for a given task. Biotechnol. Bioeng. 2011; 108:69–81. © 2010 Wiley Periodicals, Inc.  相似文献   

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