High Specific Affinity Research Articles

Immunodetection of human atherosclerotic plaque with 125I-labeled monoclonal antifibrin antibodies

To test the affinity of a new F(ab′) 2 monoclonal antibody (TRF1) against human fragment D dimer of cross-linked fibrin for atherosclerotic plaques free of detectable thrombi, 6 atherosclerotic segments of carotid and femoral artery, and as a control 5 segments of atherosclerosis-free internal mammary artery, were drawn from 11 male patients undergoing bypass surgery. All segments were carefully washed in order to remove possible endoluminal thrombi, and cut to obtain pairs of intimal fragments of similar weight, containing either plaques ( n = 16), or fatty streaks ( n = 12), or normal endothelium ( n = 20). Each fragment underwent a direct binding test to TRFI, or to a non-specific antibody, both labeled with 125I. The activity in each fragment was measured after 3 h of incubation at 37°C, and after washing the fragments every hour for 3 h. TRF1 binding (as percentage of initial activity) was significantly higher ( P < 0.001) in atherosclerotic than in normal fragments (26% ± 11.5%, vs. 9.2% ± 3.9% in fatty streaks, and 1.9% ± 0.6% in normal endothelium), and indirect immunofluorescence confirmed TRF1 uptake within the plaque wall. By contrast, the non-specific antibody did not show any significant binding. These preliminary results demonstrate the high specific affinity of TRF1 for atherosclerotic plaques, probably due to the hemorheologic phenomena that activate platelets and provoke the formation of fragment D dimers of cross-linked fibrin on the plaque surface.

Ectoenzyme activity and bacterial secondary production in nutrient-impoverished and nutrient-enriched freshwater mesocosms

This report presents results on relationships between the kinetics (Vmax and Km) of β-glucosidase (GLCase) and aminopeptidase (AMPase) activity, and dissolved organic carbon (DOC) and bacterial secondary production in freshwater mesocosms of differing degrees of eutrophication. These relationships varied in different mesocosms and depended on the trophic status of water and the exudation rates of organic carbon (EOC) by phytoplankton. Close coupling of bacterial production to Vmax of GLCase activity was observed only in nutrient-enriched mesocosms. The relationship between GLCase and DOC content was also significant in enriched water. There was no correlation between the Vmax, of GLCase and DOC and bacterial production in nutrient-impoverished and control (mesotrophic) enclosures. However, the Vmax of AMPase correlated well to DOC and bacterial production in these mesocosms. AMPase activity did not correlate with DOC and bacterial production in nutrient-impoverished mesocosms. There was no relationship between bacterial biomass and enzyme activity in all studied mesocosms. Comparison of the rates of phytoplankton production of EOC and rates of the bacterial organic carbon demand (BOCD) in nutrient-impoverished mesocosms showed that EOC flux constituted, on average, 90% of BOCD. However, in nutrient-enriched mesocosms EOC contributed only, on average, 27% to the BOCD; thus, in these mesocosms, bacteria were probably organic-carbon limited. It is hypothesized that to bypass substrate limitation, bacteria produced GLCase and AMPase. These enzymes had a high specific activity and high affinity to their substrates and efficiently hydrolyzed polysaccharides and proteins, thereby supplying microorganisms with readily utilizable products of enzyme catalysis.

Pharmacological characterization and autoradiographic localization of histamine H2 receptors in human brain identified with [125I]iodoaminopotentidine.

125I-Aminopotentidine (125I-APT), a reversible probe of high specific radioactivity and high affinity and selectivity for the H2 receptor, was used to characterize and localize this histamine receptor subtype in human brain samples obtained at autopsy. On membranes of human caudate nucleus, specific 125I-APT binding at equilibrium revealed a single component, with a dissociation constant of 0.3 nM and maximal capacity of about 100 fmol/mg of protein. At 0.2 nM, 125I-APT specific binding, as defined with tiotidine, an H2-receptor antagonist chemically unrelated to iodoaminopotentidine, represented 40-50% of the total. Specific 125I-APT binding was inhibited by a series of typical H2-receptor antagonists that displayed apparent dissociation constants closely similar to corresponding values at the reference biological system, i.e., guinea pig atrium. This indicates that the pharmacology of the H2 receptor is the same in the human brain as on this reference system. However, histamine was about 10-fold more potent in inhibiting 125I-APT binding to membranes of human brain than of guinea pig brain. 125I-APT binding was also inhibited by amitriptyline and mianserin, two antidepressant drugs, in micromolar concentrations corresponding to effective plasma concentrations of treated patients. The distribution of H2 receptors was established autoradiographically with 125I-APT on a series of coronal sections of human brain after assessing the pharmacological specificity of the labeling. The highest density of 125I-APT sites was found in the basal ganglia, various parts of the limbic system, e.g., hippocampus or amygdaloid complex, and the cerebral cortex. H2 receptors displayed a laminar distribution in cerebral cortex and hippocampal formation. A low density of sites was found in cerebellum as well as in hypothalamus, the brain area where all the perikarya and the largest number of axons of histaminergic neurons are found. The widespread distribution of H2 receptors in the human brain is consistent with the alleged modulatory role of histamine mediated by this subtype of receptor.

Molecular recognition processes at functionalized lipid surfaces: a neutron reflectivity study

The specific binding of proteins to functionalized monolayers on aqueous subphases has been characterized by neutron reflectivity measurements. As a model for the investigation of a recognition process on a molecular length scale, streptavidin and biotin were used because of the high specific affinity between them. Reflectivities from the aqueous surfaces covered with biotinylated monolayers before and after the adsorption of proteins were collected with a novel fixed wavelength liquid surface neutron reflectometer. In quantitative terms, binding was found to occur at a biotin surface concentration as low as 1 unit/1250 Å 2. The data are consistent with the formation of a monomolecular protein layer at the interface, directly underneath the lipid monolayer. The thickness of the protein film is 44 ± 2 Å, comparable in size to the smallest axis of the unit cell of three-dimensional streptavidin crystals (48 Å). It is found that the composition of the functionalized surface monolayer critically influences the two-dimensional aggregration behavior of the protein underneath.

Analysis of cytotoxicity of 131I-labelled OC125 F(ab′) 2 on human epithelial ovarian cancer cell lines

Monoclonal antibody (mAb) OC125 detects the cell surface-antigen CA 125, which is expressed in more than 80% of epithelial ovarian cancers but not in normal adult ovaries. Its high specificity and binding affinity makes OC125 a potential candidate for use in radioimmunotherapy (RIT) in patients with recurrent ovarian cancer. Initial biodistribution studies using radiolabelled specific mAbs have demonstrated significant increase in tumor uptake of dose as compared to radiolabelled irrelevant antibody. We report here an isodose comparison of the cytotoxicity of 131I-labelled OC125 F(ab′) 2, 131I-labelled nonspecific protein and external beam irradiation using a cesium-137 gamma source. Enhancement of cytotoxity due to the specific binding of the mAb could only be observed when a critical activity of 131I localized at the cell membrane. At a specific activity labelling of less than 4.1 mCi/mg, the antigen specificity of OC125 does not contribute to cell kill. Using a specific activity of 10.2 mCi/mg, the relative biological effectiveness of 131I-labelled OC125 (F(ab′) 2 was increased by a factor of 5 compared with external-beam X-ray therapy, and the specificity of mAb OC125 was found to enhance the cytotoxicity of the radioimmunoconjugate (RIC) by a factor of 2.7. This low value is in accordance with previously reported theoretical calculations for long range, low-LET isotopes and may be one of the reasons why RIT using 131I has severe limitations. In conclusion, it is necessary to maximize the specific activity of RICs with low-LET isotopes such as iodine-131 in order to maximize the ratio of the dose delivered specifically by membrane-bound mAb versus free-floating nonspecific protein.

Effect of Cross-Reactivity of Alpha-Fetoprotein Monoclonal Antibody on Quantitation of Serum AFP and Radioimmunodetection of Hepatocellular Carcinoma

Murine hybridomas were generated by immunizing mice with purified alpha-fetoprotein (AFP) to determine the cross-reactivity of AFP antibodies with human serum albumin (HSA). Eighteen out of 23 hybridoma products were cross-reactive with HSA. All 23 monoclonal antibodies (MAbs) could be subgrouped into 3 groups by their binding affinity to AFP and HSA, and one MAb from each group, IIB3, IIIB1 and IIIC6, were chosen for this study. The affinity to AFP was highest for IIB3 followed by IIIC6 and IIIB1, and that to HSA was in the order IIIB1 greater than IIB3. Immunohistochemical staining revealed no cross-reactions of these antibodies with HSA at the tissue level. IIB3 was the most efficient in the quantitative assays, indicating high affinity and specificity (non-cross-reactivity) of the antibodies, both of which are prerequisites for in vitro assays. Scans with good tumor localization were obtained from mice which received 131I-labeled IIB3 or IIIC6 after xenografting of the human hepatocellular carcinoma. The images obtained with IIB3 were inhibited in the presence of either AFP or HSA in the circulation. These findings indicated that the use of cross-reactive and/or high-affinity antibodies against AFP provides an inhibitory factor for tumor localization. MAb IIIC6, because of its high specificity and moderate affinity to AFP, seems to be the best candidate for immunoscintigraphy of the tumors in future studies.

Neurohypophyseal Hormone Receptors in the Rat Thymus, Spleen, and Lymphocytes

Receptor sites for the neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) have been identified and characterized in some tissues involved in immune function in the rat. Novel radioiodinated ligands for the detection of neurohypophyseal hormone receptors, with a high specific radioactivity and affinity, enabled the selective detection of OT receptors in the thymus and vasopressin (VP) receptors in the spleen. OT receptors were detected in thymic membrane preparations and on thymocytes, which had a ligand selectivity similar to that of uterine OT receptors. AVP receptors of the V1 pressor type were present in a splenic membrane preparation. Specific AVP-binding sites, probably of the V1 type, were also present on splenic lymphocytes. Binding sites for AVP and OT could not be detected on mononuclear cells in peripheral blood of the rat. This study demonstrates that the use of the newly developed radioiodinated AVP and OT receptor ligands, with high specific radioactivity and affinity, enables the selective characterization of receptor sites for the neurohypophyseal hormones, even in the thymus, where previously no binding sites could be detected.

Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity.

alpha-Particles such as those emitted by 211At may be advantageous for radioimmunotherapy since they are radiation of high linear energy transfer, depositing high energy over a short distance. Here we describe a strategy for labeling monoclonal antibodies and F(ab')2 fragments with 211At by means of the bifunctional reagent N-succinimidyl 3-(trimethylstannyl)benzoate. An intact antibody, 81C6, and the F(ab')2 fragment of Me1-14 (both reactive with human gliomas) were labeled with 211At in high yield and with a specific activity of up to 4 mCi/mg in a time frame compatible with the 7.2-hr half-life of 211At. Quantitative in vivo binding assays demonstrated that radioastatination was accomplished with maintenance of high specific binding and affinity. Comparison of the biodistribution of 211At-labeled Me1-14 F(ab')2 to that of a nonspecific antibody fragment labeled with 211At and 131I in athymic mice bearing D-54 MG human glioma xenografts demonstrated selective and specific targeting of 211At-labeled antibody in this human tumor model.

Localisation of fluorescence-labelled recombinant tissue-type plasminogen activator (rt-PA) on thrombi in vitro

Recombinant tissue-type plasminogen activator exhibits a high specific affinity for fibrin and is therefore of great value as a thrombolytic agent. However, its distribution after binding to a thrombus has not yet been demonstrated. In this study the binding of fluorescence-labelled rt-PA to human whole blood thrombi in vitro was investigated. After labelling with dichlorotriazinylaminofluorescein (DTAF) rt-PA retained fibrin affinity as well as enzymatic activity. Aged thrombi were incubated with increased concentrations of DTAF-rt-PA. Fluorescence in cryosections of the clots, as visualised by incidence microscopy, was observed only at the thrombus surface. The following control experiments confirmed that the fluorescence was specifically derived from the labelled protein: 1. after incubation of thrombi with DTAF alone no fluorescence could be observed in cryosections prepared from these thrombi; 2. incubation of a thrombus, exhibiting DTAF-rt-PA derived surface fluorescence, with unlabelled rt-PA resulted in the complete loss of surface fluorescence; 3. the fluorescence was randomly distributed throughout the whole cryosection if DTAF-rt-PA was present during thrombus formation. The findings suggest that during clot lysis, rt-PA binds initially to the thrombus surface but does not penetrate into the body of the clot. Thrombolysis is, therefore, likely to proceed via successive fibrin removal from the clot surface.

Interaction of phosphatidylinositol-4-monophosphate with a low activity form of dna polymerase alpha: a potential mechanism for enzyme activation

1. 1. DNA polymerase alpha isolated from Norman murine myxosarcoma exhibited two isozyme forms, one with low specific activity and low DNA binding affinity (a 1), and one with high specific activity and high DNA binding affinity (A 2). 2. 2. DNA polymerase alpha a 1, but not A 2, showed a significant increase in specific activity after treatment with phosphatidylinositol, ATP and phosphatidylinositol kinase, or with phosphatidylinositol-4-monophosphate. 3. 3. Treatment of DNA polymerase alpha a 1 with the phospholipase C hydrolysis product of phosphatidylinositol-4-monophosphate, inositol-1,4-bisphosphate, was sufficient to effect the transient increase in activity of polymerase a 1 to a form not chromatographically distinguishable from isozyme form A 2

Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes.

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.

Site-specific mutagenesis of human apolipoprotein E. Receptor binding activity of variants with single amino acid substitutions.

Apolipoprotein (apo) E, an important protein involved in cholesterol transport in the plasma, binds with high specificity and high affinity to the apoB, E (low density lipoprotein) receptor. Several lines of evidence have indicated that key basic residues in the vicinity of residues 140-160 of apoE are important in mediating binding to the receptor. Furthermore, apoE variants exhibiting defective receptor binding are associated with the genetic lipid disorder type III hyperlipoproteinemia. To determine whether other basic amino acids in this region of apoE also affect receptor binding activity, site-specific mutagenesis of apoE in a bacterial expression system was undertaken. This system had been used successfully to produce apoE3 that was structurally and functionally equivalent to human plasma apoE3. Variants of apoE in which neutral amino acids were substituted for basic residues at positions 136, 140, 143, and 150 were produced. The variants all displayed defective binding; their activity ranged from 9 to 52% of normal (a range similar to that seen with naturally occurring variants of human apoE). In addition, to determine whether the conformation of this region is important for receptor binding, we designed variants in which proline was substituted for leucine 144 or alanine 152. Both variants were defective, exhibiting 13 and 27% of normal binding, respectively. In contrast, a double mutant in which arginine was substituted for serine 139 and alanine for leucine 149 displayed slightly enhanced receptor binding activity. These studies confirm that the middle of the apoE molecule is important in receptor binding and indicate that only certain amino acid substitutions in this region interfere with receptor binding activity.

1 Localisation of fluorescence labelled recombinant tissue-type plasminogen activator (rt-PA) on thrombi [formula omitted

Abstract Recombinant tissue-type plasminogen activator exhibits a high specific affinity for fibrin and is therefore of great value as a thrombolytic agent. However, its distribution after binding to a thrombus has not yet been demonstrated. In this study the binding of fluorescence-labelled rt-PA to human whole blood thrombi in vitro was investigated. After labelling with dichlorotriazinylaminofluorescein (DTAF) rt-PA retained fibrin affinity as well as enzymatic activity. Aged thrombi were incubated with increased concentrations of DTAF-rt-PA. Fluorescence in cryosections of the clots, as visualised by incidence microscopy, was observed only at the thrombus surface. The following control experiments confirmed that the fluorescence was specifically derived from the labelled protein: 1. after incubation of thrombi with DTAF alone no fluorescence could be observed in cryosections prepared from these thrombi; 2. incubation of a thrombus, exhibiting DTAF-rt-PA derived surface fluorescence, with unlabelled rt-PA resulted in the complete loss of surface fluorescence; 3. the fluorescence was randomly distributed throughout the whole cryosection if DTAF-rt-PA was present during thrombus formation. The findings suggest that during clot lysis, rt-PA binds initially to the thrombus surface but does not penetrate into the body of the clot. Thrombolysis is, therefore, likely to proceed via successive fibrin removal from the clot surface.

Design of potent, orally effective, nonpeptidal antagonists of the peptide hormone cholecystokinin.

We describe the design and synthesis of nonpeptidal antagonists of the peptide hormone cholecystokinin. Several of these compounds have high specificity and nanomolar binding affinity and are active after oral administration. To our knowledge, the design of such agents has not previously been accomplished for any peptide hormone. The structural similarities between these synthetic compounds and the anxiolytic 1,4-benzodiazepines are noted, and the potential of this structural feature for future design of ligands for other peptide hormone receptors is discussed.

Thymidine kinase. A novel affinity chromatography of the enzyme and its regulation by phosphorylation in Physarum polycephalum.

dThd bound to epoxy-activated Sepharose results in a high specific affinity adsorbent, which was used to purify Physarum dThd kinase 8000-fold. With this purified enzyme, it was proved that dThd kinase exists in five enzyme variants in Physarum polycephalum, the acidic forms being phosphorylated. This protein modification is discussed under the aspect of the regulation of this enzyme.

Ontogenesis of beta-adrenergic receptors in the ovine placenta

Radioligands with high specific activity and high affinity have recently become available to study the β-adrenergic receptor (BAR). BAR may be important in the placenta for mediating a variety of metabolic and hemodynamic effects of catecholamines including placental hormone synthesis and secretion, placental glycogenolysis, and placental blood flow. Little is known regarding the development of the BAR. We have used the tritiated radioligand dihydroalprenolol to study the ontogenesis of BAR in the fetal and maternal portions of the ovine placenta. The receptor number decreased from 415 fmoles · mg−1 of protein at 120 days' gestation to 226 fmoles · mg−1 at 145 days; this trend was highly significant (r = −0.822, p < 0.01). Similar changes were noted in the dissociation constant. Downward regulation as a result of fetal neurosympathetic maturation is proposed as an explanation for this observation.

Tissue distribution of radioiodinated Ricinus lectins

Two Ricinus lectins, ricin D and castor bean hemagglutinin (CBH) were iodinated with different numbers of diiodotyrosine (DIT) per molecule and trace labeled with iodine-125. The distribution in rat tissues after intraperitoneal and intravenous injection was investigated as a function of the degree of iodination of the lectins. A high specific affinity to the pancreas was found after intraperitoneal injection. About 6% of the administered dose/gram was localized in the pancreas at 30 min after injection of 3 DIT, 125I-CBH and the pancreas to liver ratio was 16:1 (% dose/g). In the case of ricin D, the uptake in the pancreas was about 3% dose/g, at 30 min after injection of 3.6 DIT, 125I-ricin D and the pancreas to liver ratio was 12:1 (% dose/g). No uptake, except in the liver, was observed after intravenous injection.

Oestrogen binding function of alpha 1-fetoprotein

The discovery of a protein with high specific affinity for oestrone and oestradiol in the sera of rat and mouse at a period of intensive cellular development and sexual maturation has directed our researches primarily towards the possible role of this protein in the functions of the oestrogen hormones. One of our approaches has been a detailed study of the hormonal milieu during the perinatal growth in the rat. We have thus been able to demonstrate, along with “true” oestrogens, the presence of alternative serum ligands for alpha 1-fetoprotein, either reactive or unreactive towards specific antioestrogen antibodies. We shall mainly concern ourselves with the preliminary characterization of the latter class of alpha 1-fetoprotein ligands and show their ability to compete for oestrogen binding sites on oestrophilic alpha 1-fetoproteins. Their possible role in the mechanism of action of oestrogens will be discussed.

The Interaction of Glyceraldehyde 3-Phosphate Dehydrogenase with Human Erythrocyte Membranes

Abstract Human erythrocyte ghosts retain most of the enzyme glyceraldehyde 3-phosphate dehydrogenase bound to the membrane. The bound dehydrogenase, 3 x 105 molecules per ghost, can be eluted with increasing concentrations of salt, with 50 % elution occurring at 0.16 m NaCl, pH 7.5. Exogenous glyceraldehyde phosphate dehydrogenase can bind to the membrane. Two types of binding sites have been detected. The high affinity sites bind a maximum of 3 x 105 molecules of dehydrogenase per ghost with a Kd less than 10-8 m for the enzyme obtained from either erythrocytes or rabbit muscle. The low affinity sites bind up to 63 x 105 molecules per ghost with Kd greater than 10-7 m. Enzyme bound to the membrane in this way can be re-eluted with NaCl, indicating freely reversible interaction between the enzyme and membrane. Kant and Steck (J. Biol. Chem. (1973) 248, 8457–8464) have reported reversible binding of erythrocyte glyceraldehyde phosphate dehydrogenase with ghost membranes; however, their conditions did not permit the demonstration of two types of binding sites. There is specificity in the enzyme-membrane interactions. Both the erythrocyte and rabbit muscle glyceraldehyde phosphate dehydrogenase bind to the high and low affinity sites, but yeast enzyme interacts only weakly with the membrane. A large amount of yeast enzyme (62 x 105 molecules per ghost) binds with a Kd of 10-4 m, binding which resembles the interaction of cytochrome c with this membrane. Ghosts prepared and sealed in the presence of MgSO4 bind very little glyceraldehyde phosphate dehydrogenase and no binding occurs with high affinity. This result agrees with the conclusion of Kant and Steck that the dehydrogenase specific sites are located on the cytoplasmic surface of the membrane. Ghosts incubated at 37° in NaCl become partially sealed. Such ghosts also bind the dehydrogenase both at the high and low affinity sites. However, at each type of site part of the bound enzyme is cryptic, i.e. its activity is masked unless a detergent, Triton X-100, is present. It appears that under these conditions there is an allotopic effect on some of the dehydrogenase when it interacts with the membrane. The number of high affinity glyceraldehyde phosphate dehydrogenase binding sites on the ghost membrane is similar to that of other known membrane proteins of erythrocytes and about equals the average number of the dehydrogenase molecules in the intact erythrocyte. These facts, together with the high specificity and high affinity, suggest that the interaction between this enzyme and the membrane may have a physiological function. Because both the enzyme and membrane are well characterized they form a valuable model system for examining enzyme-membrane interactions in vitro.

Comparative affinity of embryonal and maternal serums for corticosterone and progesterone in the rat

The binding parameters of corticosterone and progesterone have been measured comparatively in the blood sera from rat, embryos and pregnant rats, at the 19th day of pregnancy. The level of corticosterone binding is significantly higher in the sera of foetal rats than in in the sera of pregnant rats. In contrast, the fixation of progesterone appears quantitatively similar in the embryo and in the pregnant rat. Scatchard analysis shows that a single protein species, with high specific affinity for corticosterone is present in the foetal as well as in the pregnant serum. The association constants have similar values, i.e.Kass= 3 to 5 × 108 M‐1 at 25 ° C; these values are characteristic for the corticosterone‐binding globulin of the rat. However, the binding capacity [concentration of binding sites (n) × number of moles of binding protein (M)] is 2 to 3 times higher in the foetal serum. The results are interpreted as indicating that the higher levels of fixation of this steroid in the embryo, compared to the mother, are due to a quantitative rather than qualitative difference between their binding proteins. The systems involved in the binding of progesterone are more complex in the embryo as well as in the pregnant rat. The values of the association constants and the results of competition and heat‐inactivation experiments suggest a probable contribution of the corticosterone binding globulin to these systems. However, the progesterone seems to bind mainly to one or several proteins with low affinity (Kass= 6 × 106 M‐1 at 25 °C) and great binding capacity (n×M= 215 × 10‐8/g of serum proteins). The foetal and pregnant rat sera appear to behave similarly in the process of progesterone binding.