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At least 631 records · Page 35Linked to original sources
PubMed1989

Preparation of antibodies against xanthine oxidase from human milk.

1. Human xanthine oxidase [XO; EC 1.2.3.2.] was isolated by a non-proteolytic method from fresh human milk. Final purification of the protein was achieved by hydroxyapatite chromatography. Most (less than 95%) of the enzyme was released in the 0.40 M phosphate fraction at pH 6.8. 2. The specific activity of this preparation was found to be 0.047 microM min-1 mg-1 with xanthine as substrate. 3. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) separated two subunits, each with a mol. wt approximately 122 kDa. 4. On non-denaturing acrylamide gels both of these subunits exhibited oxidase-like activity with xanthine as substrate in the presence of nitroblue tetrazolium and molecular oxygen. 5. Immunoconjugates of XO were prepared by the keyhole limpet hemocyanin (KLH)- and glutaraldehyde-crosslinking techniques. 6. Polyclonal antibodies to XO were raised by i.m. injection of these conjugates into female New Zealand rabbits. 7. Western blot analysis using the semi-dry technique was employed to confirm the specificity of the antibody.

Animals↗
PubMed1995

Radioimmunodetection of human cervical carcinoma xenograft by 111In-labeled monoclonal antibody MAb Cx-99.

Radioimmunodetection (RAID) is more sensitive and specific than conventional diagnostic methods. In this study, a monoclonal antibody against cervical carcinoma antigen, MAb Cx-99, was labeled with 111Indium (111In). This immunoconjugate was intravenously injected into athymic nude mice bearing cervical squamous cell carcinoma (SCC) xenografts. The tissue distribution study showed that the xenograft tumor had higher binding activity than most other tissues after 48 h from injection, demonstrated by localization ratio of tumor of tissues (c.p.m./g) against blood (c.p.m./g). However, this localization ratio was also high in the liver, spleen and kidney. The imaging study by immunoscintigraphy also showed that the tumor and liver were distinct from other background tissues 2 days after injection. This preliminary study showed that 111In-labeled MAb Cx-99 may have potential for RAID of cervical cancer, especially for tumors in the pelvis.

Animals↗
PubMed1991

Investigation and prevention of artifactual staining in flow cytometric analyses of whole blood samples from patients treated with H65-RTA, an anti-CD5 monoclonal antibody conjugated to ricin A chain.

To assess the biological effect of therapeutic monoclonal antibodies (mAbs) immunoconjugates, flow cytometric assays are often performed on patients' blood samples. We report here that, using standard protocols, staining whole blood samples with mAb-fluorochromes can result in erroneous immune cell phenotyping. Blood samples were obtained from patients treated with H65-RTA, a murine IgG1 anti-CD5 mAb conjugated to ricin A chain. While a transient decrease in CD4+ and CD8+ cells was observed during the 5 days of therapy, alterations in lymphocyte phenotype were also noted, beginning around days 15-30. The most prominent effect was an apparent loss of CD4+ cells. However, if the cells were separated from plasma prior to staining, the patients' samples demonstrated their pre-treatment lymphocytic phenotypes. Co-incubation of post-treatment (day greater than or equal to 15) patients serum with lymphocytes from normal donors also resulted in artifactual staining. The effects of the post-treatment serum could be correlated with the onset of a human immune response directed against H65-RTA. Moreover, co-incubation of normal PBMC with goat anti-mouse immunoglobulin could replicate the artifactual staining patterns. Even though the human immune response was generated against a murine IgG1 mAb, there were sufficient human antibodies crossreactive with other murine IgG isotypes to induce artifactual staining with all mAb-fluorochromes tested. To minimize artifacts, cells should be separated from plasma prior to flow cytometric analysis as well as for other immunoassays involving murine mAbs.

Animals↗
PubMed1995

Optimising the radioimmunotherapy of malignant disease: the broadening choice of carrier and effector moieties.

The treatment of cancer by radioimmunotherapy remains an experimental approach successful only in a limited number of selected disease conditions. One ground for optimism over the future of radioimmunotherapy lies in the fact that where cures have been obtained it has been despite the design of the immunoconjugate rather than because of it. As the choice of available functional components for conjugate construction increases, the process of evaluation and optimisation is underway. The replacement of the commonly used 131I with radionuclides possessing radiation characteristics more suited to particular disease states, and tailored to the behaviour of the carrier vehicle, should bring improved energy deposition within tumour while reducing whole body radiation burden. Similarly, the introduction of chemically or genetically engineered targeting molecules in place of conventional antibodies may bring improved pharmacokinetic characteristics and higher tumour accumulation. Optimisation of the therapeutic and carrier moieties employed in radioimmunotherapy should bring distinct improvements in clinical efficacy.

Animals↗
PubMed1986

Tissue distribution and serum kinetics of T101 monoclonal antibody during passive anti-cancer therapy.

We have administered fifty-six 24 hr infusions of the anti-human T-cell monoclonal antibody T101 to 10 patients with cutaneous T-cell lymphoma (CTCL) and 6 patients with chronic lymphocytic leukemia (CLL) in doses of 10, 50, 100, 150, and 500 mg. The larger doses of T101 resulted in higher, more persistent serum T101 concentrations, and CTCL patients generally developed higher serum T101 levels than CLL patients given equivalent doses. The presence of host anti-mIgG antibodies prior to infusion was associated with decreased serum concentrations of T101. Treatments that demonstrated measurable serum T101 levels were also associated with in vivo T101 binding and cytodestruction of circulating target cells. Immunofluorescence analysis of bone marrow and lymph node biopsies in CLL, and skin biopsies in CTCL, suggested that T101 had reached extravascular tumor sites. Infusion of 111In-conjugated T101 showed uptake in the liver, spleen, lymph nodes, and (in CTCL) skin infiltrates. Our data demonstrate the tissue distribution of T101 and suggest that immunoconjugates of T101 with toxins, drugs, or radioisotopes may result in better therapeutic responses.

Antibodies, Monoclonal↗
PubMed1988

Mechanisms of human CD5 modulation and capping induced by murine monoclonal antibody T101.

We have previously demonstrated that the murine monoclonal antibody T101 induces antigenic modulation when infused into patients with chronic lymphocytic leukemia and cutaneous T-cell lymphoma. In this paper, we extend our studies of T101-induced modulation and compare it to T101-induced capping. We found that, in contrast to antigenic modulation, capping occurred only in the presence of secondary anti-mouse IgG antisera and was altered by drugs that affect the cellular cytoskeleton or energy metabolism. F(ab')2 fragments of T101 induced antigenic modulation with kinetics similar to those of intact T101, but Fab-induced modulation proceeded more slowly and required the continual presence of Fab throughout the incubation. Experiments with radioiodinated T101 demonstrated that initial internalization of the antibody is followed by rapid efflux of intact, immunoreactive T101 from the cells. These data indicate important differences between capping and modulation and suggest that these two phenomena proceed by different mechanisms. More importantly, the data have implications for the potential therapeutic use of monoclonal antibody immunoconjugates.

Antibodies, Monoclonal↗
PubMed1989

Performance of multistep immunochemical reactions by counterflow isotachophoresis on nitrocellulose membranes--I. Immunoblotting.

Urine with trace amounts of different proteins from healthy people or B-lymphoma patients was concentrated and separated simultaneously by counterflow isotachophoresis on cellulose acetate membranes (CAM). The protein zones were blotted onto nitrocellulose membrane (NCM) by direct contact of CAM and NCM. NCM-blots were exposed to second isotachophoresis with the leading electrolyte 0.06 M Tris-HCl and the terminating one, 0.012 M Tris-beta-alanine. Under these conditions the moving boundary formed by Cl-/beta-alanine- migrated towards the anode with decreasing velocity. At a certain point the rate of migration of the moving boundary became completely compensated by the electroendosmotic counterflow. In this steady state position the boundary stopped on the NCM support, while the electroendosmotic rate in the area before the boundary was much higher than the rate of the opposite migration of any protein to the anode. Under these conditions electroendosmosis served as a "conveyer belt" which transferred consecutively the immunoreagents, antibodies, immunoconjugates, or antiperoxidase-peroxidase system through the protein blots "printed" on NCM. The immunoblots obtained in this way were developed by the substrate for the immunoenzyme complex used in the experiment. The technique could be used to characterize light chains present in the urine of normal donors and monoclonal light chains in the urine of patients with B-cell malignancies.

B-Lymphocytes↗
PubMed1986

Serological diagnosis of influenza A infections in the horse by enzyme immunoassay. Comparison with the complement fixation test.

An enzyme immunoassay (EIA) using horseradish peroxidase and a type-specific antigen is described for the detection and quantitation of anti-influenza antibodies in the horse. Compared with the complement fixation (CF) test (using the same antigen), EIA proved to be superior with respect to sensitivity and reliability. The internal variation of EIA was low and thus small titres in EIA can be considered of diagnostic significance. However, no strict correlation with CF was observed. The use of an immunoconjugate against equine IgM in parallel with IgG would certainly improve the sensitivity of the test, especially in early stages of infection.

Animals↗
PubMed1989

Targeted cancer treatment.

The fourth international conference on monoclonal antibody immunoconjugates for cancer heard of great progress in the area of radioimmunoconjugates, and of continued research into the potential of immunotoxins, chemoimmunoconjugates and anti-immunoglobulin therapy as potential cancer therapies.

Antibodies, Monoclonal↗
PubMed1987

The pharmacokinetics and toxicity of murine monoclonal antibodies and of gelonin conjugates of these antibodies.

We studied in mice the in vivo pharmacokinetics and toxicity of murine monoclonal antibodies (MCA) and of disulfide-linked MCA conjugates of gelonin, a ribosomal inhibitor prepared from the seeds of Gelonium multiflorum. Iodinated MCA with specificity for human determinants and of gamma 1 or gamma 2a isotype had a circulatory half life (T 1/2) in the mouse of 4 days, which is consistent with previously published estimates of the circulatory T 1/2 of heterogeneous murine IgG. Iodinated murine MCA with specificity for murine determinants had a much shorter T 1/2, probably reflecting antigen binding. This effect could be partially overcome by the simultaneous injection of unlabeled MCA of identical specificity. Clearance of MCA-gelonin conjugates was characterized by an initial rapid phase lasting 8-12 h with a T 1/2 or from 4 to 7 h, followed by a slower clearance phase with T 1/2 approaching that of MCA. Moreover, the presence of significant amounts of intact conjugate in the murine circulation was demonstrable, by SDS gel electrophoresis, for up to 48 h post injection. Intraperitoneal injection of MCA-gelonin conjugate resulted in circulating levels identical to those achieved after i.v. administration after an initial 4 h equilibration. The LD50 of MCA-gelonin conjugates was approximately 25 mg/kg (i.v.) while that of gelonin was approximately 75 mg/kg (i.v.) MCA alone showed no toxicity in doses in excess of 150 mg/kg. At doses below the LD50 immunoconjugates caused a dose-dependent reversible weight loss. The main site of toxicity of MCA-gelonin conjugates was the liver; histopathological examination revealed dose-dependent foci of necrosis and acute inflammation. No pathology was observed in lung, spleen, kidney, gut or brain. The relationship to previous work in this area is discussed.

Animals↗
PubMed1993

Comparison of conventional adjuvants and 'adjuvant-free' monoclonal antibody targeting for stimulating antibody responses against a conjugate of luteinizing hormone releasing hormone and avidin.

A conjugate of luteinizing hormone releasing hormone (LHRH) and avidin was used for immunizing mice and sheep. In mice, a range of adjuvants co-administered with the conjugate was shown to affect antibody titre and isotype. The commercially available Montanide and Ribi adjuvant emulsions, polyadenylic-polyuridylic acid, Quil A and Freund's complete adjuvant all produced high antibody titres to LHRH (reciprocal titre range 8000-20,000) and avidin (range 9000-35,000). Alhydrogel, Pluronic gel, Immunostim, DEAE-Dextran and muramyl dipeptide were weak to moderately effective (reciprocal titre ranges of 1300-5000 for LHRH and 3000-6000 for avidin, respectively). With Quil A, the response to avidin in mice was found to consist of roughly equal proportions of the IgG1, IgG2a and IgG2b isotypes. In contrast, the response to avidin using the other adjuvants and to LHRH consisted predominantly of the IgG1 isotype. An 'adjuvant-free' immunization strategy was attempted by targeting the LHRH-avidin conjugate to class II major histocompatibility complex (MHC) determinants on cells of the immune system. Using an immunoconjugate formed by linking LHRH-avidin to an antibody specific for mouse and sheep class II molecules, a twofold augmentation of titres relative to controls was obtained in mice. These antibodies were almost exclusively of the IgG1 isotype. In contrast, monoclonal antibody targeting in sheep resulted in a significantly enhanced immune response (reciprocal titres of 15,000 and 20,000 for LHRH and avidin, respectively) which was comparable to that achieved by immunizing with Quil A as adjuvant. Both the monoclonal antibody targeting and Quil A treatments tended to favour the production of antibody isotype IgG1 over IgG2.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1990

Immunotargeting approach utilizing production of oxygen free radicals.

Tumor-specific inhibition of cell proliferation through production of oxygen free radicals was studied by using immunoconjugates. Diethyldithiocarbamate (DDC), an inhibitor of superoxide dismutase activity (SOD), was conjugated with anti-Meth A tumor cell antibodies. The addition of these conjugates in combination with ascorbic acid (AsA) induced marked inhibition of Meth A tumor cell proliferation. Pretreatment of the target cells with these conjugates followed by the addition of AsA was also effective in inhibiting cell proliferation. However, the pretreatment of the target cells with unconjugated anti-Meth A tumor cell antibodies in combination with AsA eliminated the DDC conjugate-mediated antiproliferative effect. These results suggest that the combined use of DDC-anti Meth A tumor cell antibody conjugates with AsA may be a beneficial approach to cancer therapy.

Animals↗
PubMed1987

Human leukemia cell binding and killing by anti-CD5 radioimmunotoxins.

We have synthesized a reagent for antibody directed cell targeting composed of the monoclonal antibody (MoAb) T101 linked to the potent toxin ricin. The immunotoxin (IT) was subsequently radiolabeled by a cyclic anhydride procedure with 90Yttrium (90Y) to construct a radioimmunotoxin (RIT) that may have potential for cancer therapy. We evaluated the reagent for selectivity in binding and protein synthesis inhibition (PSI) assays. The RIT selectively bound antigen positive leukemia T-cell lines, with minimal binding to antigen negative control lines. The IT inhibited 87% or greater protein synthesis activity at 1 microgram/ml and exhibited an IC50 (the dose inhibiting 50% activity) of 0.18 +/- 0.08 microgram/ml in the presence of lactose. RIT and nonlabeled IT showed comparable degrees of PSI at 1 microgram/ml and 10 micrograms/ml, suggesting that labeling had little overall effect on the activity of the immunoconjugate. However, indirect evidence showed that the galactose binding site of ricin was inhibited 10-fold by its exposure to 90Y. Control RIT were minimally inhibitory. IT labeled with 131Iodine (131I) by an iodine monochloride technique also retained its capability to selectively inhibit protein synthesis. When RIT were tested for potency in a clonogenic assay against human leukemia T-cell lines, they inhibited 3.61 logs of tumor cell growth at 10 micrograms/ml. This did not represent an improvement over the log elimination with radiolabeled antibody alone, which showed 4.19 log elimination of tumor cells. Our observation that the 90Y-labeled RIT and labeled antibody can selectively eliminate about four logs of tumor cells in an in vitro clonogenic assay is unique. The ability of RIT to kill several logs of tumor cells in vitro renders RIT interesting anti-tumor reagents.

Antibodies, Monoclonal↗
PubMed1994

A new method for determining dose rate distribution from radioimmuno-therapy using radiochromic media.

PURPOSE: To describe and evaluate a new, simple, inexpensive method for directly measuring the radiation dose and its spatial distribution generated from explanted tissues of animals previously injected with radiolabeled immunoconjugates or other agents. METHODS AND MATERIALS: This technique uses the newly developed radiochromic dye medium (Gafchromic) which responds reproducibly for therapeutic dose exposures, has high spatial resolution, does not require film processing, and is relatively insensitive to ambient light. We have evaluated the dose distribution from LS174T tumors and selected normal tissues in nude mice previously injected with 90Y labeled anti-carcinoembrionic antigen antibodies. Individual tissues from sacrificed animals are halved and the flat section of the tissue is placed onto the dosimetry media and then frozen. The dosimetry medium is exposed to beta and Bremsstrahlung radiation originating from the frozen tissues. The relative darkening of the dosimetry medium depends on the dose deposited in the film. The dosimetry medium is scanned with a commercial flatbed scanner and the image intensity is digitally stored and quantitatively analyzed. Isodose curves are generated and compared to the actual tissue outline. RESULTS: The absorbed dose distribution due to 90Y exposure show only slight gradients in the interior of the tissue, with a markedly decreasing dose near the edges of the tissue. In addition, the isodose curves follow the tissue outline except in regions having radii of curvature smaller than the range of the beta-particle (R90 = 5 mm). These results suggest that the shape of the tumor, and its curvature, are important in determining the minimum dose delivered to the tumor by radiation from 90Y monoclonal antibodies, and hence in evaluating the tumor response to the radiation. The dose and spatial dose distribution were calculated assuming that the total 90Y activity is distributed uniformly throughout a half ellipsoid. The calculated spatial dose distributions for the half ellipsoids were similar to those observed from the dosimetry media that had been exposed to radioactivity contained in the tumors. CONCLUSION: This method provides direct dose evaluation without elaborate summary calculations based on activity measurements from serial slices. The measured radiation dose actually indicates the dose rate at the time of animal sacrifice. Quantitative analysis of radiation emitted from the tissues is relatively fast, making it feasible to examine a number of tissues under a variety of conditions.

Animals↗
PubMed1993

Targeted delivery of toxins and enzymes by antibodies and growth factors.

Significant progress has been made in our understanding of some of the important physiological and pharmacological barriers that limit immunoconjugate efficacy. Recent successes obtained employing immunotoxins in the treatment of leukemia and lymphoma in mouse models are suggesting ways of improving clinical strategies to treat these diseases in humans.

Animals↗
PubMed1994

A phase I/II study of the intralesional injection of ricin-monoclonal antibody conjugates in patients with hepatic metastases.

A phase I/II study of the intralesional administration of ricin-labelled monoclonal antibodies was conducted in patients with hepatic metastases of gastrointestinal origin. The anti-carcinoembryonic antigen (CEA) antibody I-1 was conjugated to blocked ricin via a disulphide bridge. After a test dose of antibody, patients were injected with ricin-antibody conjugates under computed tomography (CT) guidance on two occasions 1 week apart. Patients with stable or responding disease would receive a third course. The dose of ricin relative to surface area was increased in a predefined manner in cohorts of 3 patients. A total of 27 patients with hepatic metastases were entered into this study. All patients had metastatic colorectal cancer (26 patients) or adenocarcinoma of unknown primary with elevated CEA levels (1 patient). The presence of malignancy was documented cytologically in 9 of 11 patients tested. Minor responses were seen in 7 patients. However, no major objective responses or changes in the growth rate of injected lesions were observed. Toxicity was generally mild, the most common being hepatic capsular pain 24-48 h after each injection. 6 patients experienced rigors. One patient had anaphylaxis. Human anti-mouse and anti-ricin antibody responses were observed. Although substantial amounts of ricin conjugated to monoclonal antibodies were delivered into single lesions, this therapeutic approach was unsuccessful. Future studies of ricin-labelled antibodies should incorporate the systemic administration of immunoconjugates.

Adenocarcinoma↗
PubMed1995

Conjugation of doxorubicin to monoclonal anti-carcinoembryonic antigen antibody via novel thiol-directed cross-linking reagents.

In order to improve the available methods to produce an immunoconjugate the use of longer heterobifunctional cross-linking reagents were investigated. Two new maleimidobenzoyl spacers have been synthesized in a one step process from 4-maleimidobenzoic acid. The new heterobifunctional cross-linking reagents were fully characterized by their IR, 1H NMR, 13C NMR and mass spectra. These spacers are selectively attached to NH2-3' of the daunosamine moiety of doxorubicin. The spacer-doxorubicin derivatives were also characterized by 1H NMR spectrometry before coupling to thiol groups of thiolated anti-carcinoembryonic antigen (CEA) monoclonal antibody (11-285-14). These conjugates contain 1.51-3.44 molecules of drug for each molecule of monoclonal antibody (MAb).

Antibodies, Monoclonal↗
PubMed1996

pDAP2: a vector for construction of alkaline phosphatase fusion-proteins.

BACKGROUND: Expression of enzymatically active protein fusions in Escherichia coli could facilitate the analysis of proteins and even replace some reagents frequently used in immunology such as chemically produced antibody-enzyme conjugates. For this purpose there is up to now no system of general utility available. OBJECTIVES: The vector pDAP2 has been designed for simplified fusion of single-chain Fv fragments to the N-terminus of E. coli alkaline phosphatase. The resulting immunoconjugates can be produced by expression in E. coli and purified in a single step via metal affinity chromatography. STUDY DESIGN: Several different single-chain Fv genes as well as peptide coding oligonucleotides have been cloned into pDAP2 and tested for expression levels, purification properties and usefulness in ELISA and immunowestern blotting. RESULTS: The fusion proteins from pDAP2 can be prepared at levels of several milligrams per liter culture from the periplasma of the cells. The proteins work well in different immunoassay formats such as ELISA or western blotting. CONCLUSION: pDAP2 is compatible to phage display vectors such as pHEN1, pCOCK and the pCANTAB-series (Pharmacia, Sweden). Genes of single-chain antibody fragments can be simply swapped from these vectors into pDAP2. Furthermore, we demonstrate that it is possible to produce alkaline phosphatase-active peptides with this vector by inserting synthetic coding oligonucleotides. This allows simple analysis of the binding properties of peptides and may have some advantages over other systems such as fusion to glutathione-S-transferase.

Alkaline Phosphatase↗