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1.
《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
2.
A method is suggested for the preparation of chelating polymers containing a single terminal reactive group capable of interaction with proteins. These polymers were synthesized from N-CBZ-polylysine and DTPA and contain a terminal SH or pyridyldisulfide group. A polymer molecule with MW 13,500 is able to carry up to 40 DTPA residues. Polymers easily and quantitatively bind with antibodies (Fab fragments of antimyosin antibodies R11D10) with minimal effect on antibody immunoreactivity as revealed in ELISA assay and in direct immunoanalysis. Conjugates prepared can chelate radioactive metal ions reaching very high specific radioactivity (greater than 1 mCi 111In/10 micrograms of protein). Perspectives for their application are discussed. 相似文献
3.
《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
4.
J W Ryan A R Day D R Schultz U S Ryan A Chung D I Marlborough F E Dorer 《Tissue & cell》1976,8(1):111-124
Antibodies to pig lung angiotensin converting enzyme (kininase II) were conjugated to a heme-octapeptide (8-microperoxidase, 8-MP) derived from cytochrome c. 8-MP, which has only one reactive amine, was coupled to antibody in a two-step procedure using a bifunctional active ester, bis-succinyl succinate. In the first-step, 8-MP-succinyl succinate, a stable compound which can be stored. In a second step, the remaining active ester was used for coupling to reactive amines of the antibody. The conjugate consists of 1.6-2.3 8-MP moieties per antibody. Using these procedures, the formation of complex polymers is avoided. Each molecule of conjugate possesses both immunoreactivity and peroxidatic activity. The conjugate has been used to localize angiotensin converting enzyme along the plasma membrane and associated caveolae of pig aortic endothelial cells in culture. 相似文献
5.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1990,17(2):247-254
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
6.
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
7.
Radioimmunotherapy using antibodies with favorable tumor targeting properties and high binding affinity is increasingly applied in cancer therapy. The potential of this valuable cancer treatment modality could be further improved by increasing the specific activity of the labeled proteins. This can be done either by coupling a large number of chelators which leads to a decreased immunoreactivity or by conjugating a small number of multimeric chelators. In order to systematically investigate the influence of conjugations on immunoreactivity with respect to size and number of the conjugates, the anti-EGFR antibody hMAb425 was reacted with PAMAM dendrimers of different size containing up to 128 chelating agents per conjugation site. An improved dendrimer synthesis protocol was established to obtain compounds of high homogeneity suitable for the formation of defined protein conjugates. The quantitative derivatization of the PAMAM dendrimers with DOTA moieties and the characterization of the products by isotopic dilution titration using (111)In/(nat)In are shown. The DOTA-containing dendrimers were conjugated with high efficiency to hMAb425 by applying Sulfo-SMCC as cross-linking agent and a 10- to 25-fold excess of the thiol-containing dendrimers. The determination of the immunoreactivities of the antibody-dendrimer conjugates by FACS analysis revealed a median retained immunoreactivity of 62.3% for 1.7 derivatization sites per antibody molecule, 55.4% for 2.8, 27.9% for 5.3, and 17.1% for 10.0 derivatization sites per antibody but no significant differences in immunoreactivity for different dendrimer sizes. These results show that the dendrimer size does not influence the immunoreactivity of the derivatized antibody significantly over a wide molecular weight range, whereas the number of derivatization sites has a crucial effect. 相似文献
8.
Decorporation efficacy of prompt pulmonary delivery of DTPA dry powder was assessed following lung contamination with plutonium nitrate and compared to an intravenous injection of DTPA solution and a combined administration of both DTPA compounds. In addition, efficacy of a delayed treatment was assessed. In case of either early or late administration, insufflated DTPA was more efficient than intravenously injected DTPA in reducing the plutonium lung burden due to its high local concentration. Prompt treatment with DTPA powder was also more effective in limiting extrapulmonary deposits by removing the early transportable fraction of plutonium from lungs prior its absorption into blood. Translocation of DTPA from lungs to blood may also contribute to the decrease in extrapulmonary retention, as shown by reduced liver deposit after delayed pulmonary administration of DTPA. Efficacy of DTPA dry powder was further increased by the combined intravenous administration of DTPA solution for reducing extrapulmonary deposits of plutonium and promoting its urinary excretion. According to our results, the most effective treatment protocol for plutonium decorporation was the early pulmonary delivery of DTPA powder supplemented by an intravenous injection of DTPA solution. Following inhalation of plutonium as nitrate chemical form, this combined chelation therapy should provide a more effective method of treatment than conventional intravenous injection alone. At later stages following lung contamination, pulmonary administration of DTPA should also be considered as the treatment of choice for decreasing the lung burden. 相似文献
9.
We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La(3+)) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells. 相似文献
10.
Diethylenetriaminepentaacetic acid (DTPA), a strong chelating agent, was covalently linked to murine monoclonal anti-HLA IgG1 antibody (H-1) with the use of poly(L-lysine) (Mr 14,000) as a multivalent, intermediary carrier, via thiol-disulfide exchange reaction. The conjugates contained up to 42.5 mol DTPA per mol antibody, and retained over 90% of their antibody activity in vitro. The conjugates incorporated gadolinium (Gd) through an exchange reaction with Gd-EDTA, used to prevent colloid formation and nonspecific binding of the free metal. The IgG-poly(L-lysine)-DTPA-Gd had a greater effect per mol on proton relaxation rates than DTPA-Gd itself. Use of poly(L-lysine) as an intermediary carrier for attachment of chelating agents to IgG thus offers great potential for achieving high-specific-activity conjugates, particularly for use as biologically specific contrast agents in nuclear magnetic resonance imaging. 相似文献
11.
Khosraviani M Blake RC Pavlov AR Lorbach SC Yu H Delehanty JB Brechbiel MW Blake DA 《Bioconjugate chemistry》2000,11(2):267-277
A monoclonal antibody (2C12) that recognizes a Pb(II)-cyclohexyldiethylenetriamine pentaacetic acid complex was produced by the injection of BALB/c mice with a Pb(II)-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to interact with a variety of metal-free chelators and metal-chelate complexes was assessed by measuring equilibrium dissociation constants. The antibody bound to metal-free trans-cyclohexyldiethylenetriamine pentaacetic acid (CHXDTPA) with an equilibrium dissociation constant of 2.3 x 10(-)(7) M. Addition of Pb(II) increased the affinity of the antibody for the complex by 25-fold; Pb(II) was the only metal cation (of 15 different di-, tri-, and hexavalent metals tested) that increased the affinity of the antibody for CHXDTPA. The increased affinity was due primarily to an increase in the association rate constant. The antibody also had the ability to interact with ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), and structurally related derivatives, but with affinities from 50- to 10000-fold less than that determined for CHXDTPA. Addition of metals to EDTA-based chelators reduced the affinity of the antibody for these ligands. However, when DTPA was used as the chelator, addition of Pb(II) increased the affinity of the antibody for the complex by 200-fold. The sensitivity of prototype immunoassays for Pb(II) could be modulated by changing the structure of the immobilized metal-chelate complex and/or the soluble chelator used to complex Pb(II) in the test solution. 相似文献
12.
Metal-chelating polymers (MCPs) are important reagents for multiplexed immunoassays based on mass cytometry. The role of the polymer is to carry multiple copies of individual metal isotopes, typically as lanthanide ions, and to provide a reactive functionality for convenient attachment to a monoclonal antibody (mAb). For this application, the optimum combination of chain length, backbone structure, end group, pendant groups, and synthesis strategy has yet to be determined. Here we describe the synthesis of a new type of MCP based on anionic ring-opening polymerization of an activated cyclopropane (the diallyl ester of 1,1-cyclopropane dicarboxylic acid) using a combination of 2-furanmethanethiol and a phosphazene base as the initiator. This reaction takes place with rigorous control over molecular weight, yielding a polymer with a narrow molecular weight distribution, reactive pendant groups for introducing a metal chelator, and a functional end group with orthogonal reactivity for attaching the polymer to the mAbs. Following the ring-opening polymerization, a two-step transformation introduced diethylenetriaminepentaacetic acid (DTPA) chelating groups on each pendant group. The polymers were characterized by NMR, size exclusion chromatography (SEC), and thermogravimetric analysis (TGA). The binding properties toward Gd(3+) as a prototypical lanthanide (Ln) ion were also studied by isothermal titration calorimetry (ITC). Attachment to a mAb involves a Diels-Alder reaction of the terminal furan with a bismaleimide, followed by a Michael addition of a thiol on the mAb, generated by mild reduction of a disulfide bond in the hinge region. Polymer samples with a number average degree of polymerization of 35, with a binding capacity of 49.5 ± 6 Ln(3+) ions per chain, were loaded with 10 different types of Ln ions and conjugated to 10 different mAbs. A suite of metal-tagged Abs was tested by mass cytometry in a 10-plex single cell analysis of human adult peripheral blood, allowing us to quantify the antibody binding capacity of 10 different cell surface antigens associated with specific cell types. 相似文献
13.
A two-step method was developed to homogeneously insert carbonic anhydrase (CA, E.C. 4.2.1.1) into Michael-adduct-based coatings. CA was first covalently coupled to an N-vinylformamide-based water-soluble polymer. Unlike native CA, the resulting polymer/CA system could be dispersed within a film matrix. The enzyme-containing coating (ECC) hydrolyzes p-nitrophenyl propionate in buffered media at high rates retaining approximately 7% apparent activity. In comparison, other two-step techniques for the chemical coupling of CA to the coating surface were less efficient and led to coatings with significantly less activity. A three-step immobilization process coupling the enzyme to the surface of a partially hydrolyzed coating also raised retention of activity after coating synthesis. CA-ECC is stable under ambient conditions retaining 45% activity after 90 days of storage at room temperature. 相似文献
14.
Using a new method, rabbit IgG and a monoclonal antibody have been conjugated with the chelating agent DTPA. This was accomplished with reaction conditions that should entail lower antibody damage than existing methods. Gel filtration of the 111In-labelled antibody conjugate indicated minimal damage to the antibody and radioimmunoassay showed no significant change in its immunological activity. 相似文献
15.
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
16.
To develop a general method for the detection of histidine-tagged proteins, the interactions of the histidine epitope tag of MutH and MutL proteins with the epitope specific monoclonal anti-His6 antibody were monitored by a label-free direct method using impedance spectroscopy. The immunosensor was fabricated by covalent coupling of the antibody on a conducting polymer coated electrode surface. The impedance of the antibody modified electrode was decreased after binding to the histidine-tagged proteins. The specificity of the sensor was demonstrated by showing that no impedance change was occurred when the sensor was exposed to both of non-tagged MutH and MutL proteins. The specific interaction was further characterized using quartz crystal microbalance studies. Based on impedance measurements, the linear ranges were obtained from 50.0 to 125.0 and 50.0 to 250.0 micorg/ml, for His-tag MutH and His-tag MutL proteins, respectively. The detection limits were determined to be 37.8 and 59.1 microg/ml, for His-tag MutH and His-tag MutL proteins, respectively. 相似文献
17.
Synthesis of oligonucleotides with sequences identical with or analogous to the 3''-end of 16S ribosomal RNA of Escherichia coli: preparation of A-C-C-U-C-C via the modified phosphotriester method. 总被引:6,自引:6,他引:0
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J H van Boom P M Burgers G van der Marel C H Verdegaal G Wille 《Nucleic acids research》1977,4(4):1047-1063
A combination of two different methods for the synthesis of oligoribonucleotides, i.e. the two-step phosphotriester method with 2-chlorophenyl phosphate as bifunctional phosphate source and the modified triester method with 2,2,2-trichloroethyl 2-chlorophenyl phosphorochloridate as monofunctional phosphate source, is applied for the synthesis of the fully-protected hexaribonucleotide A-C-C-U-C-C. The two-step method is used for the synthesis of the required dinucleotide monophosphates 9, 10 and 11. Application of the modified triester method for the coupling of the oligonucleotide blocks results in the formation of the fully-protected hexamer 15. Furthermore, attention is paid to 2,4,6-triisopropylbenzenesulphonyl 4-nitroimidazolide as a new condensing agent for the coupling of larger oligonucleotide blocks. 相似文献
18.
Beck E. Hofmann M. Bernhardt G. Jäger W. Wildt L. Lang N. 《Cell biochemistry and biophysics》1994,24(1-3):163-173
Cis-diammine dichloro platinum (II) (CDDP), is a highly potent antineoplastic agent that is used in the treatment of ovarian
cancer. However, the clinical use of CDDP is restricted by its severe side effects. In order to reduce these side effects
and to enhance its therapeutic efficacy, we developed specific immunoconjugates consisting of the murine monoclonal antibody
OC125 and CDDP, using diethylene triamine pentaacetic acid (DTPA) as a linker.
The coupling efficiencies of the different preparations synthesized, varied between 1.10±0.42 and 2.65±1.60 mol of CDDP per
mol of antibody protein. Despite the chemical modification of the antibody molecule, specific binding activity of the OC125-CDDP
conjugates toward the CA125 antigen was maintained as was demonstrated by means of immunohisto-/cytochemical staining of frozen
sections of ovarian cancer tissue, amniotic epithelium, and the CA125 positive ovarian cancer cell line NIH:OVCAR 3.
The antiproliferative activity of the immunoconjugates was tested against the human ovarian cancer cell lines NIH:OVCAR3 and
SKOV 3, applying a kinetic crystal violet microassay.
Despite the promising results obtained with the specific immuno-staining of the target cells, no significant antiproliferative
activity of our immunoconjugates against the cell lines tested was observed.
One possible explanation for the lack of antitumor activity could be the fact that CA125 is released in large amounts by the
NIH:OVCAR 3 cells. This may have prevented an efficient immunotargeting of the cancer cells by the formation of soluble immune
complexes. 相似文献
19.
The level of conjugation of diethylenetriaminepentaacetic acid (DTPA) to the polysaccharide sodium hyaluronan (HA) has been measured by a colorimetric assay, isothermal titration calorimetry (ITC), and (1)H NMR spectroscopy. The colorimetric assay is based on the red shift, upon complexation with gadolinium ion (Gd3+), of the wavelength of maximum absorption of the dye arsenazo III. It can be performed in a few minutes using as little as 10 microg of polymer with a detection limit of approximately 0.03 mmol of DTPA (gram of polymer)-1. The ITC measurements yield values of the amount of DTPA linked to HA identical to those obtained by colorimetry. The levels of DTPA conjugation calculated by integration of signals at 3.1-3.2 ppm (DTPA protons) and at 2.0 ppm (HA acetamide protons) in the 1H NMR spectrum of HA-DTPA are consistently overestimated by a factor of approximately 2, compared to the data obtained by ITC and colorimetry. The longer relaxation times of protons of the polymer backbone, compared to those of protons attached to the freely moving DTPA side-chains may account for the discrepancy. 相似文献
20.
Increased expression of inducible nitric oxide synthase and peroxynitrite in Helicobacter pylori gastric ulcer. 总被引:2,自引:0,他引:2
A A Sakaguchi S Miura T Takeuchi R Hokari M Mizumori H Yoshida H Higuchi M Mori H Kimura H Suzuki H Ishii 《Free radical biology & medicine》1999,27(7-8):781-789
The role of nitric oxide in ulcer formation remains unknown. Accordingly, we assessed local expression of inducible nitric oxide synthase (NOS) and nitration of tyrosine as an indicator of peroxynitrite formation in patients with Helicobacter pylori (HP)-associated gastric ulcers compared with HP-negative ulcers. Biopsy specimens were taken from the ulcer margin and from an area remote from the ulcer portion. Inducible NOS, nitrotyrosine, and macrophage immunoreactivity were assessed immunohistochemically using a labeled streptavidin-biotin method. In HP-positive gastric ulcers, inducible NOS and nitrotyrosine immunoreactivity was frequently observed at active ulcer margins, sometimes in surface epithelial cells as well as in the lamina propria. Occasionally, inducible NOS and nitrotyrosine reactivity were found in areas remote from the lesion in cases of HP-positive ulcer and HP-related gastritis. Macrophages accumulated significantly in the margin of HP-positive ulcers. In HP-negative gastric ulcers, inducible NOS and nitrotyrosine immunoreactivity also were frequent at the ulcer margin, but no significant immunoreactivity was observed at a distance. HP eradication caused significant attenuation in inducible NOS and macrophage immunoreactivity. In conclusion, nitric oxide and peroxynitrite formation is increased in HP-infected gastric mucosa, suggesting that HP promotes nitric oxide stress. 相似文献