共查询到20条相似文献,搜索用时 31 毫秒
1.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1992,19(5):517-522
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.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1988,15(3):285-292
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.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1988,15(6):707-711
Monoclonal antibody CO17-1A was radiolabeled with 90Y using the cyclic DTPA anhydride technique and administered intravenously to athymic nude mice bearing SW 948 human colorectal carcinomas. The tumor specificity of 90Y-CO17-1A was improved by coadministration of 100 μg of the unlabeled antibody per animal and by purification using HPLC instead of column gel filtration chromatography. Absorbed radiation dose estimates for 90Y-CO17-1A were calculated. The radiation dose to the bone marrow will limit the amount of 90Y-CO17-1A that can be administered for cancer therapy. 相似文献
4.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1988,15(6):717-720
Our laboratory investigated the use of a rapid miniaturized chromatography system, ITLC-SG with 0.9% NaCl, to assess the radiochemical purity of 111In labeled monoclonal antibodies (MoAbs). Radiochemical analysis was performed on numerous 111In labeled antibody preparations with labeling efficiencies ranging from 40 to greater than 95% and the results compared to those obtained with size exclusion high performance liquid chromatography (HPLC). The chromatographic procedure involved challenging radiolabeled antibodies with 0.05 M DTPA to chelate unbound and/or non-specific bound 111In, spotting on miniaturized instant thin layer-silica gel chromatography strips, developing in 0.9% NaCl, and counting appropriate segments for radioactivity. Results of the study demonstrated that the miniaturized chromatography procedure was rapid, taking less than 4 min to complete, and accurate in assessing the amount of unbound or non-specific bound 111In in 111In labeled monoclonal antibodies, when compared to size exclusion HPLC. 相似文献
5.
A novel bifunctional maleimido CHX-A' DTPA chelator 5 was developed and conjugated to the monoclonal antibody trastuzumab (Herceptin) and subsequently radiolabeled with (111)In. The resulting (111)In labeled immunoconjugate 2 was demonstrated to bind to SKOV-3 ovarian cancer cells comparably to an isothiocyanato CHX-A' DTPA modified native trastuzumab, 1. Through efficient thiol-maleimide chemistry, antibodies, peptides or other targeting vectors can now be modified with an established radioactive metal chelating agent CHX-A' DTPA for imaging and/or therapies of cancer. 相似文献
6.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1986,13(4):345-346
DTPA N-hydroxysuccinimide pentaester (DHSE) can be used as an alternative to bicyclic anhydride for coupling proteins with DTPA.(1) However, in-vitro analysis of labeled proteins coupled with DHSE compared to bicyclic anhydride showed more high molecular weight product and less serum stability. We studied the magnitude of these differences in vivo by coupling HSA and IgG with DTPA using both methods, labeling with 111In, and analyzing the biodistribution in mice. The results of this analysis showed comparable activity in the liver, kidneys and blood. 相似文献
7.
F A Liberatore R D Comeau J M McKearin D A Pearson B Q Belonga S J Brocchini J Kath T Phillips K Oswell R G Lawton 《Bioconjugate chemistry》1990,1(1):36-50
A new, more reactive group of protein cross-linkers in the class of equilibrium transfer alkylating cross-link (ETAC) reagents has been synthesized. These compounds include alpha,alpha-bis[(p-chlorophenyl)methyl]- and alpha,alpha-bis[(p-tolylsulfonyl)methyl]acetophenones substituted in the acetophenone ring with chloro, nitro, amino, and carboxyl groups and derivatives. Included are an 125I-labeled ETAC reagent and a 111In-labeled DTPA (diethylenetriaminepentaacetic acid) ETAC for site direction and biodistribution studies. These ETAC compounds were reacted with unreduced and partially reduced antibody under mild pH (pH 4-8) and room temperature conditions to give cross-linked structures. Examination of resultant cross-linked antibody via size-exclusion HPLC, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and an enzyme linked immunosorbent assay revealed that (1) both interantibody as well as intraantibody cross-linking had occurred; (2) the level of inter- and intraantibody cross-linking varied with the substituent on the ETAC; (3) the stability of the cross-links on the reducing SDS gels varied with substituents on the ETAC; (4) little if any immunoreactivity was lost after reaction with one of the more effective ETAC cross-linking compounds; (5) the 125I-labeled ETAC sulfhydryl cross-linking in partially reduced antibody increased with pH whereas amine cross-linking with the unreduced antibody decreased with pH; (6) the optimum pH for sulfhydryl site direction was pH 5.0; (7) the 111In DTPA ETAC labeled antibody had a biodistribution in CD1 mice similar to that of the 111In bis cyclic anhydride DTPA labeled antibody. 相似文献
8.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1992,19(6):669-677
The in vitro labeling and stability of 99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the 99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The 99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the 99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between 99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N2S2) compound. The Fab′ with IgG2a isotype displayed tighter binding to 99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N2S2 challenge and incubation with human plasma. The in vivo biodistribution of five 99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable 99mTc labeling of antibody fragments from three of the major murine isotypes. 相似文献
9.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1990,17(3):321-329
A new approach for covalent coupling diethylenetriaminepentaacetic acid (DTPA) molecules to a partially reduced monoclonal antibody utilizes a malemide modified copolymer of hydroxyethyl methylacrylate and methyl methacrylate (DTPA copolymer) prepared by the group transfer polymerization (GTP) method. An average of 6 DTPA molecules were incorporated per mol maleimeide DTPA copolymer and 1.5 mol maleimide DTPA copolymer per mol antibody. Maleimide DTPA copolymer modified antibody was intramolecularly cross-linked, reduced immunoactivity and had a high in vivo liver uptake. 相似文献
10.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1986,13(4):453-456
Monoclonal antibody 17-1A, which has specificity for colorectal carcinoma, was labeled with 90Y (10–20% radiolabeling yield). Tissue distribution studies in tumor-bearing nude mice were carried out. 90Y-labeled 17-1A showed good uptake in the SW 948 colon carcinoma cell line. However, 90Y-labeled A5C3, a monoclonal antihepatitis virus antibody studied as a control, showed similar uptake in this tumor. Neither antibody was taken up well by a WM-9 melanoma. It is believed that the loss of specificity observed is due to the low specific activity of the 90Y-labeled monoclonal antibody preparations used. This hypothesis is supported by radioimmunoassay data. 相似文献
11.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1992,19(2):239-244
To simplify the synthesis of macrocyclic chelators, commercially available macrocyclic amines were condensed with halogenated acetic acid to prepare the five chelators 12N4 (DOTA), 14N4 (TETA), 15N4, 9N3 and 12N3. Only 12N4 and 9N3 showed efficient labeling of the free chelator with 111In and 90Y. Serum stability studies at 37 °C with In-labeled DTPA, 12N4 and 9N3 showed no loss of label over 2 days whereas, with 90Y, only 12N4 showed stabilities comparable to DTPA. The 12N4 chelator was derivatized by attaching biotin on one N-acetate group to simulate the attachment to protein. The serum stability for both 111In and 90Y was identical to that of biotin derivatized DTPA and lower than that of the free chelators. Biodistribution studies in normal mice of a model protein (avidin) labeled with 90Y via biotinylated 12N4 and biotinylated DTPA showed identical distribution at 1 day except in bone where the %ID/g for the macrocyclic-conjugated protein (3.4 ± 0.5, N = 8) was significantly (P < 0.001) lower than that of the DTPA-conjugated protein (9.4 ± 0.9, N = 7). In conclusion, macrocycles may be readily synthesized from the macrocyclic amines and several show useful stabilities with In and Y. When N-linked to a protein, the Y biodistribution was found to be superior to that of the corresponding DTPA-coupled protein. 相似文献
12.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1990,17(4):381-387
A monoclonal antibody against the murine T-cell antigen Thy 1.1 was radioiodinated using N-succin-imidyl p-iodobenzoate (PIP) in an attempt to decrease deiodination of the labeled antibody. The biodistribution of the PIP labeled antibody was compared to Iodogen labeled antibody in Thy 1.1+ lymphoma bearing AKR/Cum mice, where the antibody was tumor specific, and AKR/J mice where the antibody reacted with both tumor and normal T-cells. PIP labeling resulted in decreased iodine concentrations in stomach and salivary gland as compared to Iodogen labeling. There was little difference in radioiodine concentrations between the two preparations in tumor, lymphoid tissues or other organs. These results suggest deiodination of intact antibody plays little role in the clearance of radioiodinated anti-Thy 1.1 antibody from tissues. 相似文献
13.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1991,18(2):227-233
We have evaluated five compounds, stannous chloride (SnCl2), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythritol (DTE), and ascorbic acid (AA) to reduce monoclonal antibody MoAb (disulfide groups and compared their efficacy for labeling MoAbs with 99mTc. The reduction of 99mTc with dithionite at pH 11 was nearly quantitative. The use of AA, at a molar ratio of 3500:1, for three IgG and three IgM antibodies examined, gave a labeling efficiency greater than 95%. Hence no purification was needed. The immunospecificity of AA preparations determined by specific antigen assay was 84 ± 1% for an IgM and 82.6 ± 1.1% for an IgG, highest among all agents tested. The stability of the tracer was evaluated by challenging the product with such 99mTc avid agents as cysteine, DTPA, and human serum albumin. By HPLC analysis, no 99mTc was transchelated using chelating agent to protein molar ratios as high as 500:1. In two separate groups of five mice each, the liver uptake at 4 h post injection averaged 6.8 ± 2.9% per gram for 125I-TNT-1 (IgG) and 6 ± 5.1% per gram for the same MoAb labeled with 99mTc using AA. The AA technique promises to label antibodies with 99mTc and perhaps with 186Re, by a simple “kit” procedure. 相似文献
14.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1991,18(3):313-321
Monoclonal antibody CO17-1A was radiolabeled with 90Y by five bifunctional chelate techniques. Radiation absorbed dose estimates for normal organs and tumor were calculated for each preparation based on timed tissue distribution studies in nude mice bearing SW 948 human colorectal carcinoma xenografts. The cyclic DTPA anhydride technique was inferior to the four other techniques studied. Data for SCN-Bz-DTPA and SCN-Bz-Mx-DTPA, which were conjugated to ϵ-lysyl amino groups, were similar to those for NH2-Bz-DTPA and NH2-Bz-Mx-DTPA, which were conjugated site specifically to oligo-saccharides. 相似文献
15.
C. S. R. Gooden D. E. Snook Agamemnon A. Epenetos 《Cell biochemistry and biophysics》1993,22(1-3):49-59
It is essential in any method for radiolabeling antibody with99mTc that the labeling procedure is rapid and reliable, producing a highly stable99mTc-antibody complex with minimal effect on the immunoreactivity of the antibody. In the present study, analysis of the stability and homogeneity of radiolabeled (99mTc and125I) antibodies (HMFG1 and PR1A3) was carried out by fast protein liquid chromatography (FPLC) using superose-6 and S-200 columns, and by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Superose 6 and S-200 gel filtration analysis showed the radiolabel (99mTc or125I) eluting with a retention time identical to that of native antibody. No peaks of relative molecular size (Mr) corresponding to possible antibody fragments were seen in either the UV or the radioactive FPLC elution profiles. PAGE analysis of99mTc labeled antibody, however, revealed the presence of a number of radiolabeled antibody fragments (Mr<IgG) that were not detected by the same analysis of125I labeled antibody. The stability of the radiolabeled antibodies in serum in vitro was also studied. FPLC (superose-6) analysis after 45 h incubation in normal serum in vitro revealed 3.3% (HMFG1), and 20% (PR1A3) of the99mTc on a molecule or aggregate with a Mr greater than that of IgG. There is also the appearance of small amounts of99mTc-labeled material with a Mr<IgG in the later fractions (2.2% for HMFG1 and 4.9% for PR1A3). Similar results were obtained using radioiodinated antibody, although the small amount of low molecular size material detected as a single peak with a longer retention time than the99mTc equivalent corresponds to free iodide. 相似文献
16.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1986,13(4):359-362
An exclusive labeling of high affinity sites of IgG and its F(ab′)2 fragments with 99mTc was accomplished. Antibody was first labeled in 0.1 M acetate buffer at pH 4.5, using stannous chloride as a reducing agent. Thus, high capacity, low affinity sites and low capacity, high affinity sites were both labeled. These 99mTc complexes were stable at pH 4.5 and 7.0; however, they became destabilized at pH 8.2 and 9.0. Transchelation of 99mTc to DTPA took place at the higher pH values and leveled off at 54% 99mTc-F(ab′)2 and 73% 99mTc-IgG. These results indicate that the majority of 99mTc bound to the low affinity sites was transchelated to the high affinity sites rather than to DTPA since low affinity sites account for 84% of total F(ab′)2 sites and 76% of IgG sites. Biodistribution data in mice at 2.5 h postinjection were consistent with this hypothesis in that tissue concentrations of 111In-DTPA-F(ab′)2 were similar to the reequilibrated 99mTc-F(ab′)2 but were much higher than that of the unequilibrated 99mTc-F(ab′)2. 相似文献
17.
Samokhin GP Mongayt DA Iakoubov LZ Levchenko TS Torchilin VP 《Analytical biochemistry》2001,292(2):245-249
For many practical applications, monoclonal antibodies must be chemically modified without any significant loss in their immunoreactivity. In some situations, however, the amino acid residue crucial for antibody activity may be highly reactive toward the modifying agent, which results in antibody inactivation. The method to prevent inactivation of a modification-sensitive antinuclear monoclonal antibody by acylating agents was developed. The method is based on the hypothesis that a highly reactive amino group exists within, or in the vicinity of, the binding site of the antibody, providing crucial interaction with negatively charged moieties of DNA. It has been shown that negatively charged polymers, such as dextran sulfate or heparin, may provide temporary protection, presumably interacting noncovalently with this amino group and thus masking it. The protecting molecule can be removed later by chromatography on a protein A column, thus regenerating modified but not inactivated antibody in the free form for use in subsequent applications. In particular, we have modified antibody 2C5 with a chelating agent, diethylenetriaminepentaacetic acid (DTPA) without the loss of activity. Modified antibody was labeled with radioactive isotope, (111)In, via chelation by antibody-attached DTPA. The labeled antibody was shown to demonstrate the same specificity of binding to nucleosomes as the nonmodified antibody, so it may be used in immunoscintigraphy or biodistribution studies. The method might be useful for the modification of other modification-sensitive antibodies with other acylating chemicals, such as crosslinking agents or biotin derivatives. 相似文献
18.
A monoclonal antibody to human insulin receptor 总被引:1,自引:0,他引:1
Frederick C. Kull Steven Jacobs Ying-Fu Su Pedro Cuatrecasas 《Biochemical and biophysical research communications》1982,106(3):1019-1026
A murine hybridoma secreting antibody against human insulin receptor was produced by fusing FO myeloma cells with spleen and lymph node cells from a mouse that had been immunized with insulin receptor purified from human placenta. The secreted antibody was an IgG1 (κ), designated αIR-1. Like the previously described rabbit polyclonal antibody, αIR-1 did not inhibit insulin binding. It specifically immunoprecipitated 125I-insulin-receptor complexes as well as unoccupied receptor previously labeled directly with lactoperoxidase. Thus, αIR-1 interacts with the receptor at a site distinct from the insulin binding site. Unlike previously described anti-insulin receptor antibodies, αIR-1 exhibits strong tissue and species specificity. 相似文献
19.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1989,16(8):839-845
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
20.
Amrita Dutta Matthew Schaller Aime T. Franco Kavitha Sankavaram Bruce J. Grattan Hedley C. Freake 《The Journal of nutritional biochemistry》2010,21(2):162-170
Previous studies in our laboratory have demonstrated that reducing the availability of zinc with the extracellular metal chelator DTPA (diethylenetriaminepentaacetate) enhances, rather than inhibits, the thyroid hormone induction of growth hormone mRNA in GH3 rat anterior pituitary tumor cells. To understand the actions of the chelator on cellular zinc status, we observed the effects of DTPA on 65Zn uptake and retention. DTPA reduced the uptake of 65Zn by GH3 cells from the medium, but when GH3 cells were prelabeled with 65Zn, it resulted in greater retention of the isotope. In primary hepatocytes, DTPA both reduced the uptake of 65Zn from the medium and increased efflux from prelabeled cells. To investigate this difference, we studied the effects of DTPA on radioactive zinc flux in the H4IIE (rat hepatoma), MCF-7 (human breast cancer) and Hs578Bst (nontransformed human mammary) cell lines and in rat primary anterior pituitary cells. DTPA reduced the uptake of 65Zn in all cell lines examined. DTPA increased the retention of 65Zn in prelabeled H4IIE, MCF-7 and Hs578Bst cells but reduced it in primary pituitary cells. Time course experiments showed that 65Zn efflux is shut down rapidly by DTPA in transformed cells, whereas the chelator causes greater efflux from primary hepatocytes over the first 6 h. Experiments with 14C-labeled DTPA confirmed that this chelator does not cross cell membranes, showing that it operates entirely within the medium. Expression of ZnT-1, the efflux transporter, was not affected by DTPA in H4IIE cells. Thus, zinc deprivation enhanced zinc retention in established cell lines but increased efflux from primary cells, perhaps reflecting differing requirements for this mineral. 相似文献