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K Nustad

Publications and source records attributed to K Nustad.

At least 55 records · Page 3Linked to original sources
PubMed1990

Functional properties of CD19+ B lymphocytes positively selected from buffy coats by immunomagnetic separation.

Here we report that human B lymphocytes can be positively selected directly from buffy coats applying the anti-CD19 antibody AB1 coupled to magnetic beads. This isolation protocol is highly efficient and the isolated cell population is of very high purity and viability. As judged by cell cycle analysis and various parameters for cell activation, the cells are still in a resting state after isolation. Furthermore, different functional assays have shown that the isolation procedure does not interfere with either activation or proliferation/differentiation of CD19 selected cells as compared to negatively isolated cells. As a consequence of cross-linking during the isolation process, the CD19 antigen is temporarily down-regulated as measured by AB1 binding. Despite this decreased expression, monoclonal antibodies to the CD19 antigen nevertheless inhibited anti-mu plus B cell growth factor induced B cell activation as reported also for negatively isolated cells. Taken together, the presented data strongly suggest that B cells isolated through the CD19 antigen can be used in critical functional assays.

Antibodies, Anti-Idiotypic↗
PubMed1990

Radioimmunotargeting of human tumour cells in immunocompetent animals.

A tumour model system is reported that for many purposes may be an alternative to xenografted nude mice. The model allows immunotargeting of human tumour cells in immunocompetent animals. The target cells are contained in i.p. diffusion chambers (DC) with micropore membrane walls that are permeable to molecules, including the cell specific monoclonal antibodies (MoAb), but impermeable to cells. Thus, the tumour cells are protected from the host immunocompetent cells. In the work here presented the model was tested in immunocompetent mice and pigs, with tumour cells and antibody preparations that had demonstrated specific targeting in the nude mouse xenograft model. Hence, the DC were filled with cells from the human cell lines Hep-2 (expressing placental alkaline phosphatase, PLALP), or OHS (a sarcoma cell line), and the MoAb preparations injected i.v. were a 125I-labelled Fab fragment of the PLALP specific antibody H7, or a 125I-labelled F(ab')2 fragment of the sarcoma specific antibody TP-1. Specific targeting of the human tumour cells was demonstrated in both mice and pigs. The target: blood ratios were comparable in the two species, reaching a maximum of about 15 after 24 h with the Fab preparation, and a ratio of 25 after 72 h with the F(ab')2. The target uptake relative to injected dose was lower in pigs than in mice, but the difference between the two species was smaller than expected, presumably due to a slower antibody clearance in the pigs than in the mice. An artificial cell targeting system like this has several advantages in the search for solutions to many of the fundamental problems experienced in immunotargeting. Firstly, parallel binding experiments can be carried out in vitro with the same target. Because in vitro results are only influenced by the diffusion into the DC and the immunological binding characteristics of the antibodies, targeting differences between antibody preparations due to these factors can then be distinguished from differences due to pharmacokinetical properties. Secondly, the animals can be implanted with any type and number of target cells, or with antigen negative control cells. Thirdly, and perhaps most important, the system opens a possibility for evaluation of the murine MoAb in xenogenic species, and this may predict the clinical targeting potential better than experiments on mice.

Animals↗
PubMed1989

Elimination of malignant clonogenic breast cancer cells from human bone marrow.

Autologous bone marrow transplantation is a promising approach to the treatment of breast cancer but is at present limited to patients without bone marrow metastases. To eliminate malignant clonogenic breast cancer cells from normal human bone marrow, immunomagnetic separation has been combined with chemoseparation using 4-hydroperoxycyclophosphamide. Breast cancer cell lines have been mixed with a 10-fold excess of irradiated human bone marrow from normal donors. Mixtures have been incubated with a combination of five different monoclonal antibodies which bind to epithelial cell surface antigens of Mr 42,000, 55,000, 72,000, 200,000, and greater than 200,000. Antiglobulin coated microspheres which contained magnetite were added, and tumor cells were trapped in a magnetic field. Elimination of tumor cells from the decanted marrow was measured in a limiting dilution assay. Two treatments with antibody and microspheres permitted elimination of 2-4 logs of clonogenic breast cancer cells, depending upon the cell line studied. Similar treatment of nonirradiated normal marrow failed to affect levels of colony forming units-granulocyte-macrophage significantly. Use of immunomagnetic purging in combination with 4-hydroperoxycyclophosphamide eliminated up to 5 logs of tumor cells but reduced the recovery of colony forming units-granulocyte-macrophage. If prompt engraftment is observed following reinfusion of similarly treated marrow in phase I trials, these techniques should permit extension of autologous bone marrow transplantation to a larger population of breast cancer patients.

Antibodies, Monoclonal↗
PubMed1989

Immunoradiometric assay for alpha gamma- and gamma gamma-enolase (neuron-specific enolase), with use of monoclonal antibodies and magnetizable polymer particles.

Monoclonal antibodies were raised against neuron-specific enolase, gamma gamma-enolase, and used in an immunoradiometric assay (IRMA), with mono-disperse magnetizable particles as the solid phase. The assay's sensitivity was 0.4 microgram/L and the interassay coefficient of variation was less than 5% in the working range from 0.4 to 170 micrograms/L. Compared with our radioimmunoassay based on polyclonal antibodies, the incubation time is shorter, and precision and sensitivity are improved. The IRMA also improved detection of neuron-specific enolase in sera from patients with lung cancer without a concomitant change in measured enolase in the reference population. The better sensitivity of the IRMA results from its ability to measure alpha gamma- and gamma gamma-enolase with equal response. Ninety percent of the small-cell lung carcinoma patients (36 of 40) had increased values before treatment, compared with 7% of non-small-cell lung carcinoma patients (8 of 114).

Animals↗
PubMed1989

Immunomagnetic removal of B-lymphoma cells using a novel mono-sized magnetizable polymer bead, M-280, in conjunction with primary IgM and IgG antibodies.

The efficacy of a novel monosized and magnetizable polymer bead, denoted M-280, in immunomagnetic removal of Rael B-lymphoma cells was compared with that of M-450 beads, previously used in bone marrow purging. The M-280 beads which are smaller (diameter 2.8 microns) and contain less iron than the M-450 beads were coated with polyclonal IgG sheep antimouse (SAM) antibody. The two types of immunobeads were equally efficacious when used together with the mouse monoclonal IgG antibodies HH1 or FN1, giving tumor cell depletions of about 3 logs in one cycle of operation. However, when used together with the primary IgM monoclonal antibodies (MoAbs) AB1 or HH2, the new immunobeads were significantly more efficacious than the M-450 immunobeads. To elucidate the underlying mechanism flow cytometric studies and measurements of the binding of the labeled primary MoAbs to the cellular antigens as well as to the immunobeads were carried out. Competition experiments showed that in the case of IgG MoAbs, the SAM beads bind predominantly to the Fc portion, whereas in the case of the IgM MoAbs, the Fab part plays a relatively greater role in the binding. The results indicate that if M-280 immunobeads are used, IgM MoAbs may profitably be included in antibody cocktails together with IgG antibodies in immunomagnetic purging of B-lymphoma cells. They suggest that in the case of cell bound MoAbs, the epitopes on IgG are more accessible to SAM beads than those of surface bound IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1988

In vivo evaluation of radiolabelled antibodies with antigen-coated polymer particles in diffusion chambers.

A method for the evaluation of in vivo immunolocalization of labelled monoclonal antibodies (MoAb) is presented. The technique is an alternative to the nude mouse xenograft system. The antigen reservoir is an intraperitoneal diffusion chamber (DC) filled with a suspension of antigen-coated polymer particles. Intravenously injected 125I-labelled MoAb are allowed to specifically bind to this artificial abdominal 'tumour', which can be removed and measured for radioactivity after animal killing. The model can be used as a preclinical in vivo method for the evaluation of labelled MoAbs prepared for immunodiagnostics or therapy. The DC system permits the amounts of both antibody and antigen to be controlled and antibody access to the antigen within the DC is presumably the same in every animal. The model permits a systematic comparison of different antibodies and antibody fragments, labels and labelling procedures, as well as routes of administration in immunocompetent animals.

Alkaline Phosphatase↗
PubMed1988

Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro.

By employing the principles of "activated swelling", monosized, superparamagnetic polymer particles have been prepared ranging in size from 1-100 microns. Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes. Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies. So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood. These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas. High speed and specificity are the most conspicuous features of immunomagnetic cell separation. These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens. Both higher sensitivity and specificity have been obtained. The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions. The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision. The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail. Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process. Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.

Antibodies, Monoclonal↗
PubMed1988

High frequency of incomplete human chorionic gonadotropin in patients with testicular seminoma.

We measured human chorionic gonadotropin by 2 immunometric assays that require that the entire human chorionic gonadotropin molecule is intact, and by a competitive radioimmunoassay that measures intact human chorionic gonadotropin and its free beta-chain. The sera tested were samples from male cancer patients with elevated human chorionic gonadotropin levels obtained by the radioimmunoassay method. Elevated levels were confirmed in 67 of 92 samples (72 per cent) with the immunometric methods. However, in 25 of 97 patients (28 per cent) elevated human chorionic gonadotropin was found with the radioimmunoassay, whereas the values were in the normal range when measured with the immunometric assays. These discrepancies were found in 22 patients with seminomatous tumors, including 2 extragonadal germ cell tumors, which constitutes 42 per cent of all seminoma patients tested. Of the remaining 3 discrepant patients 2 had lung cancer and 1 had metastases from an unknown primary cancer.

Biomarkers, Tumor↗
PubMed1988

Immunomagnetic removal of B-lymphoma cells from human bone marrow: a procedure for clinical use.

B-lymphoma cells were purged from human bone marrow by incubating the cell suspension with a cocktail of three different pan-B cell mouse IgG1 monoclonal antibodies, and then with immunobeads charged with sheep anti-mouse antibody, followed by magnetic separation. The primary antibodies used, HD37 (CD19), HD6 (CD22), and HH1 (CD37), bind to a very high percentage of the cells in non-Hodgkin's lymphomas of poor prognosis. The secondary antibody is directed against the Fc portion of the IgG antibodies. In model experiments Burkitt's lymphoma cells (Rael) were admixed to mononuclear bone marrow cells in the ratio 1/9. With a ratio of immunobeads/total antibody-binding B cells of 50/1 in a first treatment cycle and repeating the procedure with the same number of beads in a second cycle, a tumor cell depletion of more than 5 logs was achieved, as judged by a clonogenic assay. The concomitant reduction of CFU-GM and CFU-GEMM was about 20%. The purging procedure has been scaled up to clinical use. Equipment suitable for purging patients' marrow specimens, employing standard transfusion facilities, is described. With this equipment the efficacy of tumor cell removal was the same as in the model experiments, and the whole magnetic separation could be completed in 2 hours.

Animals↗
PubMed1987

Excess antibody immunoassay for the measurement of tonin in rat tissues and plasma.

Tonin, a proteolytic enzyme isolated from the rat submandibular gland, can generate angiotensin II directly from angiotensinogen. To date a method for the measurement of tonin in plasma has not been available and the present paper describes a sensitive and specific excess antibody immunoassay for determination of tonin in tissue homogenates and plasma. Interference from immunologically cross-reacting proteins was evaluated and the assay was found to be specific for tonin. Tonin measured in various tissue homogenates was directly proportional to the amount of sample added, giving a linear dose-response curve. The slope of this curve was determined by the recovery of tonin, which was better than 55% for urine and all tissues tested. The highest concentration of tonin was seen in the submandibular and sublingual gland (69 and 0.7 microgram/mg protein, respectively). The parotid gland, the exorbital lacrimal gland, liver, kidney, pancreas, and lung contained only negligible amounts (less than 4 ng/mg protein). Tonin in plasma was bound to one major inhibitor with a molecular weight of about 650,000-750,000. A partial splitting of the tonin-inhibitor complex was obtained by preincubating plasma with guanidine, allowing tonin to be measured with a recovery of 38 +/- 13% (n = 16) and with a linear dose-response curve. The concentration of immunoreactive tonin in normal arterial plasma from adult male rats was 0.90 +/- 0.53 ng/ml (n = 16). The concentration decreased after removal of the submandibular glands and increased after sympathetic stimulation.

Animals↗
PubMed1987

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies.

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.

Antibodies, Monoclonal↗
PubMed1987

The diagnostic value of human chorionic gonadotrophin in patients with testicular seminoma.

Human chorionic gonadotrophin (HCG) was measured in sera from seminoma patients by an immunoassay which recognised both free beta-chains and intact HCG molecules. This seemed to increase the sensitivity of the assay without affecting its specificity. The HCG values were correlated to the clinical course in 151 patients. Pre-orchiectomy samples were assayed in 57 patients. Elevated pre-operative HCG levels were found in 15 of 38 patients (39%) in stage I and in 13 of 19 patients (68%) in stages II to IV. After orchiectomy the HCG values were normal in 114 of 115 patients in stage I and elevated in 13 of 36 patients (36%) in stages II to IV. Relapses were observed more often in stage II to IV patients who produced HCG at the time of diagnosis than in non-producers (3 of 13 patients versus 1 of 23 patients). In 7 of 11 relapses the serum HCG was elevated. HCG positive relapses were seen in patients both with and without initially elevated serum HCG.

Adult↗
PubMed1987

Placental alkaline phosphatase as a tumor marker in ovarian cancer.

The serum levels of placental alkaline phosphatase were determined with a radioimmunoassay using a polyclonal antibody on 1236 samples from 414 patients with ovarian cancer. The frequencies of elevated enzyme levels for patients with or without evidence of disease were 17.7 and 10.9%, respectively. The true positive rate was highest in serous cystadenocarcinoma, undifferentiated carcinoma, and dysgerminoma. A tendency to an inverse correlation with differentiation was found. Measurement of the enzyme did not give a useful index of stage of disease, tumor burden, or prognosis. The value of the enzyme as an index of successful therapy was limited because half of the patients lost this marker during progression. Further studies of the use of this enzyme as a tumor marker should evaluate the modulation of the placental alkaline phosphatase pattern during the course of the disease and should be based on monoclonal antibodies.

Alkaline Phosphatase↗
PubMed1986

The interaction between cell-surface antigens and antibodies bound to monodisperse polymer particles in normal and malignant cells.

The interaction between polymer particles with an immune ligand on their surfaces and normal (hepatocytes) and malignant (Raji) cells with a corresponding membrane receptor was investigated by scanning and transmission electron microscopy. The influence of time, temperature, and anti-metabolites on this process was studied. The results indicate that when particles with an immunoligand are added to cells with a corresponding membrane receptor, three successive steps are initiated, the first two of which are not dependent on the temperature or the metabolic state of the cells. The first step in this process is the binding of particles to the cell surface through the immunoligand on the particle and the receptor on the cell surface. The initial binding formed by immunoligand and corresponding receptor is followed by a stronger interaction between cell and particle. This second step seems to be governed by a successive antibody-antigen interaction: the zipper mechanism. A third step in the particle-to-cell interaction is characterized by movements of the cellular surface, as revealed by membrane folds, pseudopods, and lamellae extending from the surface and enveloping large parts of the particle.

Antigen-Antibody Reactions↗
PubMed1985

Enzymatic activity of rat submandibular gland kallikrein released into blood.

Enzymatic activity of submandibular gland (SG) kallikrein released into saliva and blood was studied at rest and after autonomic nerve stimulation. Kallikrein was measured by an immunometric assay that allows measurement of immunoreactive kallikrein in complex with inhibitors as well as simultaneous determination of kallikrein enzymatic activity. Measurements using the chromogenic substrate S2266 gave identical results to the natural substrate kininogen. Endogenous SG kallikrein secretory rate was, at rest, 0.9 +/- 0.1 ng/min. Kallikrein secretion into blood in response to autonomic nerve stimulation paralleled that into saliva, and secretion was greatly enhanced by alpha-adrenergic stimulation. In plasma, kallikrein was bound to several inhibitors that completely or partially blocked the enzyme activity. In arterial and SG venous control plasma, 93 +/- 3 and 72 +/- 10% inhibition of kallikrein enzyme activity was observed, respectively. Sympathetic stimulation after administration of a beta-adrenergic blocker increased kallikrein enzyme activity 62 and 11 times in arterial and SG venous plasma, respectively, with a corresponding 78 +/- 8 and 70 +/- 8% inhibition of kallikrein enzyme activity. A fraction containing kallikrein resembling "free kallikrein" was always present in plasma.

Animals↗
PubMed1985

Highly sensitive solid-phase immunoenzymometric assay for placental and placental-like alkaline phosphatases with a monoclonal antibody and monodisperse polymer particles.

We have used a mouse monoclonal antibody (H7) to placental alkaline phosphatase (PLAP, EC 3.1.3.1) in developing an immunoenzymometric assay for PLAP and PLAP-like enzymes. The antibody is bound to sheep anti-mouse IgG (Ab2) covalently coupled to tosylated shell-and-core light (1.07 g/cm3) monodisperse polymer particles. Adding the H7-Ab2-polymer particle suspension to a PLAP-containing sample gives maximal binding of the antigen within 10 min. PLAP and PLAP-like enzymes remain active and bound to the solid-phase throughout all assay manipulations, and can thus be saved for future testing. In testing the enzymes for inhibition by L-Phe, L-Phe-Gly-Gly, L-Leu, and L-homoarginine, the effect of all the inhibitors is fully reversible. The assay is highly versatile, and its sensitivity (routinely 0.05 micrograms/L) can be increased 1000-fold by adjusting the sample volume and incubation time (sample volume is irrelevant between 50 microL and 5 mL). We have measured the basal activities of PLAP in men and women and, by using enzyme inhibitors, have characterized it as corresponding to the PLAP-like phenotypes described in normal human testis.

Alkaline Phosphatase↗
PubMed1984

Excess antibody immunoassay for rat glandular kallikrein. Measurement of kallikrein complexed with inhibitors and in plasma.

We have recently developed an immunoradiometric assay (IRMA) for specific measurement of immunoreactive kallikrein which allows a simultaneous determination of the enzymatic activity of kallikrein. This paper describes the application of this method for measurements of glandular kallikrein complexed with inhibitors. Interference by low molecular weight inhibitors such as benzamidine and Trasylol was easily overcome by increasing the amount of immobilized anti-kallikrein antibody added in the assay, and by prolonging the incubation time of the antigen-binding step. The recovery of kallikrein in complex with plasma inhibitors was complete only when the anti-kallikrein antibody was immunoadsorbed onto a solid-phase sheep anti-rabbit immunoglobulin. The dose-response curve of glandular kallikrein in plasma paralleled that of purified kallikrein in both the immunoradiometric and the immunoenzymometric assays. The concentration of immunoreactive glandular kallikrein in normal rat plasma was 12.8 +/- 4.3 nU/ml. The enzymatic activity of this immunoreactive kallikrein was 86% inhibited.

Animals↗