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K Nustad

Publications and source records attributed to K Nustad.

At least 37 records · Page 2Linked to original sources
PubMed1994

[Tumor marker analyses--valuable tools if used correctly].

The tumour marker assays in routine use today are not cancer-specific. Most markers are normal products of epithelial tissues, usually excreted from the intact organ. The infiltrating malignant growth allows release directly into lymph and blood, where the product is measured as a "tumour marker". Many tumour markers are glycoproteins depending on the liver function for their catabolism. These mechanisms explain the slight to moderate increases in tumour marker values found in various non-malignant diseases. Therefore, in general, such assays are of no use for screening healthy populations, and a normal value does not exclude cancer. Their main use is in the primary staging of patients known to have cancer, to evaluate the completeness of primary surgery, to follow-up patients for early detection of relapses, and to monitor the effect of cytotoxic or radiation therapy of advanced disease. In all these applications, good clinical judgement is the necessary basis for using these assays.

Biomarkers, Tumor↗
PubMed1994

Demonstration and minimization of serum interference in flow cytometric two-site immunoassays.

The ability of serum factors to cross-link labeled mouse monoclonal antibody (mAb) of irrelevant specificity (mAb FN61, subclass IgG1) to different particle types coated with sheep IgG, bovine gamma-globulin, or mAb FN61 was measured simultaneously by flow cytometry. Significant interference with mAb FN61-coated particles was detected in 53 of 101 sera. Of the 30 sera showing the most pronounced interference, 23 were characterized by an even stronger cross-linking to particles coated with bovine gamma-globulin. These were designated type 1 sera. Seven sera, designated type 2, displayed a dominant interference with the mAb FN61-coated particles. The interference reaction in the two serum types was characterized by different kinetics, dependence on particle concentration, and response to blocking agents. The interference was minimized by addition of 500 micrograms of bovine gamma-globulin and 50 micrograms of mAb HH1 (IgG1) of irrelevant specificity per 10 microL of serum sample in a final assay volume of 100 microL.

Antibodies, Monoclonal↗
PubMed1993

Amplification of c-erbB-2, int-2 and c-myc genes in node-negative breast carcinomas. Relationship to prognosis.

Primary tumours from 100 Norwegian node-negative breast carcinoma patients were examined for c-erbB-2, int-2, and c-myc proto-oncogene amplification. c-erbB-2, int-2, and c-myc amplification was found in 12.1% (12 of 99), 8.6% (8 of 93), and 1.1% (1 of 89) of the samples respectively. All the c-erbB-2 amplified tumours were of the ductal type, and all the int-2 amplified tumours were oestrogen receptor positive. No other significant or borderline significant associations between gene amplification and clinical or histopathological parameters were found. Relapse occurred more frequently in patients with c-erbB-2 gene amplification (relapse in 33.3% of the patients with c-erbB-2 amplification compared to 20.7% in the non-amplified group), but the difference was not statistically significant, int-2 amplification was not associated with increased risk of relapse, whereas the prognostic value of the c-myc amplification could not be evaluated.

Adult↗
PubMed1993

Immunoconjugate stability in vivo measured by label release from 125I or 99mTc-antibody coated cells kept in intraperitoneal diffusion chambers in mice.

The present work demonstrates how intraperitoneal (i.p.) diffusion chambers (DC) can be used to investigate the in vivo stability of the bond between an antibody and its radioactive label. A monoclonal antibody (MoAb) was labelled with 125I or 99mTc. The 125I-labelled preparation showed high stability in vitro, since little radioactivity eluted from incubated DC containing 125I-MoAb bound to specific, fixed target cells. Similarly, when we evaluated the 125I-MoAb in vivo by using the i.p. DC the stability was intact. The 99mTc-MoAb was also stable in vitro, with only about 10% of the radioactivity lost after 48 h. However, when tested in vivo, about 50% of the 99mTc label was lost after 1 day, increasing to 60% after 2 days. Hence, by carrying out preclinical in vivo stability testing with i.p. DC method we discovered that an immunoconjugate with high stability as tested in vitro, in fact was unstable in vivo and probably unsuited for clinical use.

Animals↗
PubMed1993

Homogeneous immunofluorometric assays of alpha-fetoprotein with macroporous, monosized particles and flow cytometry.

We evaluated two homogeneous immunofluorometric assays (IFMAs) of alpha-fetoprotein (AFP) based on new macroporous acrylate particles combined with flow cytometry. The standard IFMA, requiring 1 h of incubation, provided a working range from 1.8 to > 900 kIU/L (CV < 10%) and a detection limit of 0.6 kIU/L. Use of overnight incubation and a lower particle concentration extended the working range by 1 decade in the lower end. Analytical recoveries for the standard IFMA varied between 97% and 108%. The slope and y-intercept of the regression line correlating measurements by the standard IFMA and a routine immunoradiometric assay were not significantly different from 1 and 0, respectively (P > 0.5), and the correlation coefficient was 0.996. High precision and warning of spuriously high measurements were obtained by including in each sample separate particle types for detecting instrument instability and measuring nonspecific binding only.

Acrylates↗
PubMed1992

Elevated levels of thrombin-heparin cofactor II complex in plasma from patients with disseminated intravascular coagulation.

An ELISA assay for quantitation of the thrombin-heparin cofactor II complex (T-HC II) in plasma was developed. Plasma was incubated with immobilized, specific antibodies to human thrombin. The second, biotinylated antibody was directed against human HC II. The assay was insensitive to thrombin-antithrombin complex (TAT) and to uncomplexed HC II. In plasma samples from 31 normal individuals (aged 21-68, mean 43.3 years), the T-HC II ranged 0.3-6.1 ng/ml; median 1.5, mean 2.0, and SD 1.6 ng/ml. In plasma samples from 13 patients with disseminated intravascular coagulation (DIC), T-HC II ranged 0.4-30.0 (median 13.5) ng/ml. In plasma samples from 6 patients in which the clinical suspicion of DIC was not verified, T-HC II complex ranged 1.4-14.3 (median 3.6) ng/ml. In plasma samples with elevated T-HC II levels, TAT was usually elevated, and on the average more than was T-HC II. These results indicate that HC II contributes significantly to the inactivation of in vivo generated thrombin.

Adolescent↗
PubMed1992

The binding parameters of radiolabelled monoclonal F (ab')2 and Fab' fragments relative to immunoglobulin G in reactions with surface-bound antigens.

The binding parameters of iodine-125-labelled intact monoclonal immunoglobulin G (IgG), F(ab')2 and Fab' fragments were compared. The study was carried out with the two monoclonal antibodies (MoAbs) K13 and K16 specific for human Ig light chains kappa and lambda, respectively. When testing the 125I-MoAbs against monodisperse polymer particles coated with the specific antigens, the Ka for the F(ab')2 fragments were similar to that for IgG, while the Ka for the Fab' fragments were reduced to 10%-20% of that for IgG. The number N of effective target sites revealed with Fab' was higher than with F(ab')2 and IgG, presumably because less surface area is occupied by the small Fab' molecules. The immunoreactive fraction F ranged according to IgG greater than F(ab')2 greater than Fab'. The explanation of the moderate difference between the Ka of the monoclonal Fab' and the divalent IgG and F(ab')2 was that the divalent molecules were not divalently attached to the particles. When testing the same antibody preparations against human lymphoma cells producing Ig with light chains kappa or lambda, the binding results were less reliable than when particles were utilised, presumably due to antigen shedding. Different MoAbs vary in their loss of immunoreactivity due to enzymatic degradation and the radiolabelling procedure. The preparation of the radiolabelled fragments should therefore be optimized for each MoAb, and evaluation is necessary before injection. Artificial targets with a low leakage of antigen, like the monodisperse polymer particles here applied, are recommended for the in vitro evaluation of the immunoreactivity of labelled MoAb preparations.

Animals↗
PubMed1992

Distribution of intraperitoneally injected microspheres labeled with the alpha-emitter astatine (211At) compared with phosphorus (32P) and yttrium (90Y) colloids in mice.

The alpha-emitter 211At was bound to polymer microspheres with a diameter of 1.8 microns. The distributions in mice of intraperitoneally injected 211At microspheres, 90Y silicate colloid, and 32P chromic phosphate colloid were compared. The microspheres with 211At spread rapidly in the peritoneal cavity and remained mainly on the intraperitoneal surfaces. Intraperitoneal injection of 90Y colloid resulted in high levels in intraperitoneal fat and the diaphragm, but 1 day after injection 8.5% of the injected dose per gram was found in blood and after 6 days 2.5% was observed in bone. The highest accumulation of 32P was found in liver and spleen. The injection of additional nonradioactive chromic phosphate colloid resulted in an even higher accumulation of 32P in spleen and liver. The same phenomenon was not observed with 211At microspheres. It is suggested that it is not only the particle size which is important in the distribution of intraperitoneally injected colloid, but the amount of colloid, the type of colloid, the addition or presence of other substances such as ascites, and the animal species might also influence the distribution. In conclusion, the intraperitoneal distribution of 211At-labeled microspheres in mice was favorable compared with 90Y and 32P colloid. These data must be viewed cautiously since the distribution might be different in other animal species or humans.

Animals↗
PubMed1992

Therapeutic efficacy of the alpha-emitter 211At bound on microspheres compared with 90Y and 32P colloids in a murine intraperitoneal tumor model.

alpha-Emitting radionuclides such as 211At have a number of physical characteristics which make them attractive for the treatment of micrometastases. 211At was bound to polymer microspheres and its efficacy was compared with the beta-emitting 32P and 90Y colloids for the treatment of intraperitoneally growing K13 hybridoma tumors in mice. Single graded doses of 0.1-2.5 MBq 211At microspheres injected intraperitoneally 24 hr after inoculation of the hybridoma cells improved survival and produced higher cure rates than 32P colloid, 90Y colloid, or no treatment. One of the most striking contrasts between 211At microspheres and 90Y or 32P colloids was the ability of relatively low doses 211At to affect cures. When comparing the groups with the highest survival rate for each radionuclide (0.1-1 MBq 211At, 2.5 MBq 90Y, and 2.5 MBq 32P), 211At treatment resulted in an improved survival over that with 32P therapy, but the difference was not significant between 211At and 90Y. Toxicity studies with 211At microspheres showed that dosages up to 17 MBq per mouse were not lethal. In conclusion, the present study suggests that the high-energy transfer and the short-range cytotoxicity of the alpha-emitter 211At might be of benefit for intracavitary radiotherapy.

Animals↗
PubMed1992

In vivo measurement of the association constant of a radio-labelled monoclonal antibody in experimental immunotargeting.

Exploring the fundamental mechanisms behind the low tumour uptake of labelled monoclonal antibodies (MoAbs) during in vivo immunotargeting, experiments were performed to estimate the in vivo value of the association constant (Ka) in an experimental targeting reaction. An artificial tumour model was utilised, based on diffusion chambers (DC) filled with antigen-coated polymer particles, implanted i.p. in normal, immunocompetent mice (NMRI/BOM). The MoAb H7 with specificity for placental alkaline phosphatase (PLALP) was chosen for this experiment. Each mouse carried two DC, one target DC filled with PLALP-coated particles, and a second control DC with the same amount of uncoated particles. The DC contained escalating doses of particles, ranging from 0.1 mg to 16 mg per DC, with groups of 6-12 animals per dose level. The next day after the implantation, a constant dose of 125I-labelled Fab fragments of H7 was injected i.v. in each mouse. The association constant Ka as measured from the binding data obtained in vivo was not significantly different from the value measured in vitro when the same target DC were incubated with the 125I-Fab in test tubes. This indicates that in vivo impairment of the antibody avidity is not the reason why a relatively low tumour uptake is generally experienced in immunotargeting studies.

Alkaline Phosphatase↗
PubMed1992

B-cell hybridoma as intraperitoneal tumor model: correlation between tumor growth and monoclonal antibody production.

Murine B-cell hybridoma cells producing an immunoglobulin G1 (K13), specific for human immunoglobulin kappa chains were inoculated intraperitoneally in mice. After intraperitoneal injection of 10(6) K13 hybridoma cells, superficial intraperitoneal implants and ascites developed, resulting in death after 10 +/- 3 days (mean +/- SD). An immunoradiometric assay was developed to measure K13 in murine blood, ascites and culture supernatant. The assay utilized polymer beads coated with human immunoglobulin G. The amount K13 bound to the particles was measured with a 125I-labelled monoclonal rat antibody (LO-MG1-13) specific for mouse IgG1. The assay could be used over a wide working range (2-500 micrograms/l). Kinetic studies suggested that about 10(5) secreting cells were required for detection of K13 in blood. After injection of 10(6) cells, K13 was measurable in blood 1 day later in all animals. Nine of 33 mice injected with 10(5) or less cells survived, and initially showed rising K13 blood levels followed by decreasing blood levels. In conclusion, a close relationship was established between i.p. growth of the hybridoma K13 cell line and the MAb blood levels. The basic concepts of this assay can readily be adopted for other clones with the limitation that pure antigen is needed for solid phase extraction of the MAb from mouse blood.

Animals↗
PubMed1992

Radio-immunotargeting in experimental animal models of intraperitoneal cancer.

The field of immunotargeting, and the challenges met when this technique is applied in experimental animals or in patients, are reviewed. Even with highly specific monoclonal antibodies, non-specific uptake in normal tissues and high background level of unbound radioactivity in blood and extravascular body fluids remain significant problems. Further experimental work in animal model systems is needed to bring this technique from the state of being an experimental method, with limited clinical application, to a routine diagnostic or therapeutic method. Different animal models are available, and their potential for elucidation of the various methodological problems in radio-immunotargeting are discussed in the present paper. In our laboratory, two intraperitoneal models were devised, having relevance for gynecologic and other forms of intraperitoneal malignancies. These models were elaborated with special emphasis on the possibility for exact measurement of important parameters in immunotargeting reactions. In the first model, hybridoma cells are inoculated intraperitoneally to mimic intraperitoneal carcinomatosis, and the monoclonal antibody produced by the hybridoma is used as serum tumor marker. In the second model the tumor cells are contained within intraperitoneally implanted micropore chambers, resembling a localized tumor. An artificial tumor like this allows control with the antigen load in the target, and measurement of the concentration of the injected antibody in the fluid within the target.

Animals↗
PubMed1992

CA125 and placental alkaline phosphatase as serum tumor markers in epithelial ovarian carcinoma.

Placental alkaline phosphatase (PLAP) was measured by an immunoradiometric assay using the monoclonal antibody C2 (PLAP-C2). Serum samples of 135 patients with epithelial ovarian cancer were analyzed, and the results were compared with CA125 levels. CA125 and PLAP-C2 were elevated in 85 and 43% of the patients, respectively. Only 1 patient with normal CA125 and evidence of disease at the time of sampling had an elevated PLAP-C2. Fifty-three patients with measurable tumor were followed longitudinally during chemotherapy. Correct correlation with disease evolution was observed in 95% of the patients for CA125 and in 59% for PLAP-C2. The PLAP-C2 assay did not add significantly to the predictive value of CA125 in the diagnosis and follow-up of epithelial ovarian cancer.

Alkaline Phosphatase↗
PubMed1990

A rapid and sensitive immunoassay for tumor necrosis factor using magnetic monodisperse polymer particles.

A sensitive and rapid immunoassay for the detection of tumor necrosis factor (TNF) has been developed. Magnetic monodisperse polymer particles (M-280 Dynabeads) used as solid phase material, were coated with a neutralizing mouse monoclonal antibody to TNF. The coated Dynabeads were shown to have a more rapid binding capacity for recombinant (r) TNF as compared to standard immunowells coated with antibodies to TNF. The amount of TNF bound to the Dynabeads was quantified using either a polyclonal antibody to TNF or a mouse monoclonal antibody to TNF. The antibodies used for detection were either labelled with 125I or peroxidase. The linear assay range for the TNF standard curve was form 62 to 4000 pg/ml, and the assay time was less than 60 min. The sensitivity could be increased 5-8-fold by increasing the sample volume from 0.1 to 2 ml.

Antibodies, Monoclonal↗
PubMed1990

Species-dependent binding of serum albumins to the streptococcal receptor protein G.

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.

Animals↗
PubMed1990

Selection of monoclonal antibodies for use in an immunometric assay for carcinoembryonic antigen.

Six high-affinity mouse monoclonal antibodies against carcinoembryonic antigen produced in our laboratory were evaluated for use in a solid-phase immunoradiometric assay. The antibodies all recognized single peptide epitopes, one being highly conformation dependent. Cross-inhibition studies demonstrated that three antibodies bound to different epitopes, while the remaining three bound to the same or to very closely related epitodes. All antibodies strongly stained colorectal cancer tissue. The three antibodies binding to the same epitode also stained normal granulocytes, indicating a reactivity with the non-specific cross-reacting antigen. On the basis of the results of the characterization, two antibodies were selected for use in a sandwich immunometric assay. One of these was coupled to 2.8 microns magnetizable polymer particles, while the other one was radiolabelled. The assay required 2 h incubation to reach equilibrium, having a working range up to 135 micrograms/l and a sensitivity (Zero + 2 SD) of 0.1 micrograms/l. A comparison of the new assay with two commercial CEA kits that also use monoclonal antibodies was carried out on a small panel of serum samples from colorectal cancer patients and revealed satisfactory correlations but also discrepancies that must be attributed to differences in epitope specificity.

Animals↗
PubMed1990

Immunometric assay by flow cytometry using mixtures of two particle types of different affinity.

An improved dynamic range in a particle based flow cytometric immunoassay for carcinoembryonic antigen (CEA) was obtained using a binary mixture of two distinguishable particle types, namely particles of 7 and 10 microns diameter that were distinguishable by their light scattering characteristics in the flow cytometer. The two particle types were coated with antibody of the same specificity but different affinity. The association constants were 3.2 x 10(10) and 3.3 x 10(9) for the antibodies on the 7 and 10 micron particles, respectively. A dilution series of CEA samples was incubated with aliquots of the particle mixture and secondary biotin-streptavidin-phycoerythrin-conjugated antibody directed against a different epitope on the CEA molecule. The fluorescence intensity of the two particle types was measured flow cytometrically, and a double standard curve plotted from the mean logarithmic fluorescence values. The precision profile derived from the standard curve demonstrated that an increase in the dynamic range of about 50% (from 2 to 3 log) was obtained by using a mixture of high and low affinity particles, compared to using the high affinity particles alone.

Antibodies, Monoclonal↗