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M Haruno

Publications and source records attributed to M Haruno.

At least 19 recordsLinked to original sources

Mosaic model for sensorimotor learning and control.

Humans demonstrate a remarkable ability to generate accurate and appropriate motor behavior under many different and often uncertain environmental conditions. We previously proposed a new modular architecture, the modular selection and identification for control (MOSAIC) model, for motor learning and control based on multiple pairs of forward (predictor) and inverse (controller) models. The architecture simultaneously learns the multiple inverse models necessary for control as well as how to select the set of inverse models appropriate for a given environment. It combines both feedforward and feedback sensorimotor information so that the controllers can be selected both prior to movement and subsequently during movement. This article extends and evaluates the MOSAIC architecture in the following respects. The learning in the architecture was implemented by both the original gradient-descent method and the expectation-maximization (EM) algorithm. Unlike gradient descent, the newly derived EM algorithm is robust to the initial starting conditions and learning parameters. Second, simulations of an object manipulation task prove that the architecture can learn to manipulate multiple objects and switch between them appropriately. Moreover, after learning, the model shows generalization to novel objects whose dynamics lie within the polyhedra of already learned dynamics. Finally, when each of the dynamics is associated with a particular object shape, the model is able to select the appropriate controller before movement execution. When presented with a novel shape-dynamic pairing, inappropriate activation of modules is observed followed by on-line correction.

Brain↗

Highly enhanced hepatic masses seen on CT during arterial portography: early hepatocellular carcinoma and adenomatous hyperplasia.

BACKGROUND: To describe computed tomographic (CT) features of highly enhanced hepatic masses as seen on CT during arterial portography (CTAP) and to survey the varieties of hepatic lesions associated with such findings. METHODS: CTAP files for 400 patients were reviewed, on the basis of which six patients with highly enhanced hepatic masses were selected. These six patients also subsequently underwent CT during hepatic arteriography (CTHA) on the same day. All the patients had chronic liver damage, which was cirrhotic in five cases. Five had a current diagnosis and one had a history of hepatocellular carcinoma (HCC). RESULTS: Solitary highly enhanced masses were observed on CTAP in three patients, three masses were seen in one patient and multiple (10-12) masses in the other two patients. All the CTAP-enhanced masses except one were round in shape and homogeneous in attenuation. The size of the mass ranged from 6 to 25 mm in diameter. In all except two nodules in one patient, the masses were hypoattenuated on CTHA. On histopathological examination of five nodules in three patients, the nodular lesions were consistent with so-called early HCC (well-differentiated HCC of Edmondson I) in four nodules and adenomatous hyperplasia in the other nodule. CONCLUSIONS: Highly enhanced hepatic masses relative to the surrounding liver parenchyma have been sporadically noted on CTAP, especially in patients with liver cirrhosis. When present, such nodules are typically hypoattenuated on CTHA and histological features are consistent with early HCC and adenomatous hyperplasia.

Adenoma↗

Optimal injection protocol for CT evaluation during hepatic arterial infusion chemotherapy.

OBJECTIVE: As an imaging modality for follow-up during continuous or repeated hepatic arterial infusion chemotherapy using a hepatic intra-arterial indwelling catheter, the usefulness of CT while infusing contrast through the indwelling catheter (reservoir port) was examined. METHODS: Using reservoir ports implanted in eight patients with hepatic metastasis from colon cancer, radioisotope perfusion scintigraphy (RI), CT (three rates of infusion of contrast were used), and digital subtraction angiography (AG) were performed to compare the modalities' ability to visualize the intrahepatic and abnormal extrahepatic distributions. RESULTS: CT (infusion rate 0.1 mL/sec) was superior to AG and RI in terms of the ability to visualize intrahepatic distribution, particularly in small areas, and facilitated 3D delineation of the distribution. In evaluating extrahepatic distribution, CT also outperformed the other modalities. CONCLUSIONS: For imaging study follow-up during hepatic arterial infusion chemotherapy, CT proved to be more useful than conventional RI and AG.

Aged↗

Antihyperglycemic effects of N-containing sugars from Xanthocercis zambesiaca, Morus bombycis, Aglaonema treubii, and Castanospermum australe in streptozotocin-diabetic mice.

The aqueous MeOH extract of the leaves and root of Xanthocercis zambesiaca (Leguminosae) and eight structurally related nitrogen-containing sugars, fagomine (1), 4-O-beta-D-glucopyranosylfagomine (2), 3-O-beta-D-glucopyranosylfagomine (3), 3-epifagomine (4), 2,5-dideoxy-2,5-imino-D-mannitol (5), castanospermine (6), alpha-homonojirimycin (7), and 1-deoxynojirimycin (8) were evaluated for antihyperglycemic effects in streptozotocin (STZ)-diabetic mice. The insulin-releasing effects of 1 were also investigated. The blood glucose level fell after i.p. injection of the extract (50 mg/kg). Compounds 1, 2, 5, and 6 reduced the blood glucose level after i.p. injection of 150 mumol/kg. Compound 1 increased plasma insulin level in STZ-diabetic mice and potentiated the 8.3-mM glucose-induced insulin release from the rat isolated-perfused pancreas. The 1-induced potentiation of insulin release may partly contribute to antihyperglycemic action.

Animals↗

Radioimmunoscintigraphy using technetium-99m-labeled parental mouse and mouse-human chimeric antibodies to carcinoembryonic antigen in athymic nude mice bearing tumor.

Biodistribution and imaging characteristics of Tc-99m-labeled parental mouse and mouse-human chimeric antibodies to carcinoembryonic antigen (CEA), designated F11-39 and ChF11-39, respectively, were evaluated in athymic nude mice bearing the human CEA-producing gastric carcinoma (MKN-45) xenografts. Group F monoclonal antibodies such as F11-39 and ChF11-39 have been found to recognize the protein epitopes present on the domain B3 of the CEA molecule and to discriminate CEA in tumor tissues from the CEA-related antigens. The Tc-99m labeling was performed by immediately mixing a reduced antibody by 2-mercaptoethanol with Tc-99m pertechnetate in the presence of stannous chloride. The labeling yields of the two antibodies were greater than 95% when estimated using gel chromatography. Although these Tc-99m-labeled antibodies were stable in neutral saline solution, Tc-99m from both labeled antibodies was associated with cysteine solution. Technetium-99m ChF11-39 was more susceptible to transchelation than was Tc-99m F11-39. The immunoreactivity of each Tc-99m-labeled antibody was confirmed using MKN-45 cell-binding assay. Biodistribution studies in tumor-bearing mice were performed at 1 h, 5 h, and 20 h after being given IV injections of 3.7 MBq of either Tc-99m F11-39 or Tc-99m ChF11-39. All tumor-to-organ uptake ratios increased with time for both Tc-99m-labeled antibodies. Imaging results also showed selective and progressive accumulation of both Tc-99m antibodies at the tumor site. Both these Tc-99m-labeled antibodies have proved to be good radiotracers giving satisfactory scintigrams of the CEA-producing tumor.

Animals↗

Tumor-specific accumulation of 125I-labeled mouse-human chimeric anti-CEA antibody in a xenografted human cancer model demonstrated by whole-body autoradiography and immunostaining.

Whole-body autoradiography (WBAR) was used to study the biodistribution of 125I-labeled mouse-human chimeric antibody (Ch F11-39) to carcinoembryonic antigen (CEA) in athymic nude mice bearing the CEA-producing MKN-45 human gastric carcinoma xenografts. Significantly high uptake of 125I-Ch F11-39 in the tumors obtained by tissue-counting technique was confirmed by WBAR of mice of 12, 24, 48, and 96 h postinjection of 125I-Ch F11-39. When compared with histochemical or immunohistochemical staining results of the tumor tissue sections, imaging profiles of 125I-Ch F11-39 obtained by WBARs were topographically correlated with histopathological findings of tissues and immunohistochemical localization of CEA in the tumor tissues, indicating that the accumulation of 125I-Ch F11-39 at the tumor site is based on its specificity for CEA. These results demonstrate that this chimeric antibody may serve as a potential useful diagnostic and/or therapeutic reagent for human CEA-producing cancers.

Animals↗

Reducing interference from heterophilic antibodies in a two-site immunoassay for carcinoembryonic antigen (CEA) by using a human/mouse chimeric antibody to CEA as the tracer.

To reduce heterophilic antibody interference in a two-site immunoassay for carcinoembryonic antigen (CEA), we utilized a human/mouse chimeric antibody to CEA as the tracer. One mouse monoclonal antibody (MAb), F82-61, which reacts with an epitope present on the domain N of CEA, was immobilized on 96-well polystyrene microtiter plates. A human/mouse chimeric antibody (Ch F11-39), which recognizes an epitope present on the domain B3 of CEA, was biotinylated for the tracer (Ch F11-39 system). Another MAb F11-39, the parental MAb of Ch F11-39, was also biotinylated and used as the control tracer (F11-39 system). For a fair comparison, the same 503 serum samples from healthy individuals were simultaneously assayed in the present study. When a tentative common reference limit of 5 ng/ml was used, the false positive rate with the Ch F11-39 system was only 2.8% (14/503) and that with the F11-39 system was 29.0% (146/503). Adding normal mouse serum (NMS; 1%) or a mixture of purified mouse IgG subclasses (heterophilic blocking reagent (HBR, 15 micrograms/test)) to the F11-39 system reduced the false positive rate from 29.0% to 6.2% (31/503) or 4.8% (24/503), respectively, suggesting that heterophilic antibodies reactive with mouse IgG gave rise to the high positive rate in normal populations with the F11-39 system. On the other hand, the false positive rate with the Ch F11-39 system was only slightly reduced from 2.8% to 2.6% (13/503) or to 2.0% (10/503) by adding NMS or HBR to the Ch F11-39 system. The false positive rates with two commercially available assay systems, CEA Roche EIA.DM or Abbott IMx CEA, were 5.4% (27/503) and 5.8% (29/503), respectively, which both corresponded roughly to that with the F11-39 system including NMS or HBR. These results indicate that the application of human/mouse chimeric antibodies in two-site immunoassays is more effective for reducing interference from heterophilic antibodies than the adding of NMS or purified mouse IgG in the assay using conventional MAbs.

Animals↗

[Construction and clinical application of chimeric antibodies].

To reduce the high immunogenicity of mouse monoclonal antibodies (MAbs) in man, mouse/human chimeric antibodies have been generated in two ways. The more simple method is to construct chimeric antibodies in which the variable regions from the mouse MAbs are linked to human constant regions. The second is to graft the complementarity determining regions from the variable regions of mouse MAbs into human variable regions. So far, more than a hundred of chimeric antibodies have been reported and about twenty-five have been tried for immunotherapy of various human diseases. In the near future, many mouse/human chimeric antibodies will be commercially available.

Animals↗

Measuring oxygen uptake and carbon dioxide production in critically ill patients using a standard blood gas analyzer.

OBJECTIVE: The measurement of oxygen uptake and CO2 production in critically ill patients requires invasive monitoring or complex analysis equipment. This study investigates the hypothesis that oxygen uptake and CO2 production can be accurately determined by measuring oxygen and CO2 concentrations in samples from inspiratory and expiratory ventilator circuitry, using a standard blood gas analyzer. DESIGN: Prospective comparison of CO2 production and oxygen uptake measurements determined by use of a blood gas analyzer vs. a mass spectrometer. SETTING: University teaching hospital medical and surgical intensive care units (ICUs). PATIENTS: Critically ill patients (n = 46) receiving mechanical ventilation in the ICUs. INTERVENTIONS: PO2 and PCO2 were obtained with two new techniques and compared simultaneously with measurements on a mass spectrometer in critically ill, mechanically ventilated patients. Two methods were evaluated: a) arterial blood gas analyzer measurements of PO2 and PCO2 from fluid collected in traps on the inspiratory and expiratory limbs of the ventilator circuitry; b) PO2 and PCO2 measurements of inspiratory and expiratory gas samples collected in bags and injected directly into an arterial blood gas analyzer. Oxygen consumption and CO2 production were compared, using both methods of gas measurements. MEASUREMENTS AND MAIN RESULTS: Direct injection of gas samples collected in a bag from inspiratory and expiratory limbs of a breathing circuit into the arterial blood gas analyzer correlated very closely with mass spectrometer measurements for all variables (n = 32 sample measurements in 25 patients): fractional oxygen (r2 = .99, slope = 1.02, bias = 0.37%, precision = 0.54), fractional expired CO2 (r2 = .90, slope = 0.86, bias = -0.10%, precision = 0.15), oxygen uptake (r2 = .87, slope = 0.99, bias = 21.6 mL/min, precision = 38.0), and CO2 production (r2 = .98, slope = 0.95, bias = 7.90 mL/min, precision = 15.3). In contrast, although fractional oxygen and CO2 concentrations were approximated by analysis of fluid collected from inspiratory and expiratory traps, the values did not correlate well enough with mass spectrometer values to yield reasonable oxygen uptake or CO2 production results. CONCLUSION: We have demonstrated that direct Fick oxygen uptake and CO2 production can be accurately determined in mechanically ventilated patients, using direct injection of collected gas samples into standard blood gas analyzers. This simple, inexpensive technique can be performed using equipment readily available in any hospital.

Bias↗

A rapid colorimetric assay for carcinoembryonic antigen (CEA)-mediated cell adhesion and analysis of CEA domains involved in the adhesion.

A colorimetric microadhesion assay that allows the quantitative determination of carcinoembryonic antigen (CEA)-mediated homophilic cell adhesion to CEA immobilized on 96-well polyvinyl chloride plates is described. Chinese hamster ovary (CHO) cells transfected with a full-length CEA cDNA were used as indicator cells. After dislodging nonadherent cells, specifically bound cells were quantified by a colorimetric analysis based on the ability of live cells to reduce the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan product. The domains of CEA responsible for the homophilic cell adhesion were analyzed by inhibition assays using anti-CEA monoclonal antibodies whose reactive domains were already known. The involvement of domain N and possibly subdomain A3 of CEA in the homophilic cell adhesion has been suggested.

Animals↗

Measurement of cardiac output by automated single-breath technique, and comparison with thermodilution and Fick methods in patients with cardiac disease.

Accurate noninvasive methods are needed for determination of cardiac output. Current methods are generally complex or may be unreliable. A previously described method, based on absorption of acetylene gas during a constant exhalation that enables calculation of cardiac output by estimating pulmonary capillary circulation, is incorporated in a new, automated commercial product (SensorMedics 2200). In this study, cardiac output by single-breath acetylene blood flow measured with this device was compared with the standard thermodilution and direct Fick methods in 20 patients undergoing cardiac or pulmonary artery catheterization. Patients inhaled test gas mixture to total lung capacity and exhaled at a constant rate through an adjustable resistor. Lung volumes and noninvasive acetylene blood flow value were calculated automatically. Correlation between the automated single-breath technique and both thermodilution and Fick cardiac output determinations was very high (correlation coefficients were 0.90 and 0.92, respectively), regression slopes were close to identity (0.98 and 0.90), and bias (-0.39 and -0.79 liter/min) and precision (0.94 and 1.02) were good; when shunt correction was applied, bias was reduced to 0.06 and 0.35 liter/min, respectively. Rapid, accurate, noninvasive measurement of cardiac output was easily obtained using the automated device. This technique may have a wide applicability for noninvasive evaluation of patients with cardiac disease and for monitoring effects of therapeutic interventions.

Absorption↗

Construction and expression of two mouse-human chimeric antibodies with high specificity and affinity for carcinoembryonic antigen.

We have previously reported that a group of monoclonal antibodies (MAbs) to carcinoembryonic antigen (CEA), designated Group F MAbs, are able to discriminate CEA in tumor tissues from the CEA-related normal antigens and that CEA assay systems utilizing at least one Group F MAb show the improved cancer diagnosis. In this study, we cloned the genes coding for two Group F MAbs (F11-35 and F11-39) and deduced the amino acid sequences of the variable regions for their heavy and light chains. The variable region for the heavy chain of F11-35 contained a possible N-glycosylation site (Asn/Asp/Thr) at amino acid positions 89-91. Then, we constructed two mouse-human chimeric antibodies by using the F11-35 and F11-39 variable region genes of heavy and light chains (VH and V kappa) and human heavy and light chain constant region genes (gamma 1 and kappa) derived from a human plasma cell leukemia line (ARH77). The chimeric gene constructs were sequentially co-transfected into murine non-Ig-producing myeloma (P3-U1) or hybridoma (Sp2/0) cells by electroporation. The resulting chimeric heavy chain of F11-35 showed a slightly but significantly higher molecular weight than that of F11-39, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were similar, indicating the glycosylation at the possible N-glycosylation site in the variable region of the Ch F11-35 heavy chain. Both chimeric antibodies exhibited the same specificity and affinity for CEA as those of the parental murine hybridoma antibodies, respectively. Ascites production of Sp2/0 transfectomas is sufficiently high (600-900 micrograms/ml) for initial clinical studies with the chimeric antibodies.

Amino Acid Sequence↗

Reaction profiles of seven enzyme immunoassay kits for carcinoembryonic antigen (CEA) analyzed with purified preparations of CEA and related normal antigens.

Antigenic reactivities of 4 purified CEA preparations and 4 different CEA-related normal antigens (NCA from lungs, NCA-2 from meconium, and NFA-1 and NFA-2 from adult feces) were comparatively analyzed with seven commercially available EIA kits [Abbott CEA-EIA Monoclonal, D-ZYME CEA, Imzyne CEA, CEA MITSUI II, CEA Roche EIA, Immunoball-CEA (N), and Glaozyme CEA]. All kits employ a sandwich-type solid-phase method using polystyrene beads and monoclonal anti-CEA antibodies as either capture antibody and/or tracer antibody. In general, all CEAs reacted well with these assay kits. Reactivity differences in weight among the CEA preparations were, however, observed in all but one assay kit (D-ZYME CEA) in which all CEAs showed fairly homogeneous reactivity. The degree of reaction intensity among the preparations used varied depending on the assay kits used. NCA, which is well known to be partially cross-reactive with CEA, revealed a negligible reactivity in all kits. NFA-1 which is also partially cross-reactive with CEA but antigenically unrelated to NCA, showed very strong reactivity in 2 kits [CEA Roche EIA and Immunoball-CEA (N)] and weak reactivity in another kit (CEA MITSUI II). NCA-2 in meconium and NFA-2 in normal adult feces, which both have immunochemical and physicochemical properties very similar to those of CEA, reacted to a greater or lesser extent with all kits. However, one assay kit (D-ZYME CEA), whose reactivity to different CEAs was fairly homogeneous, could discriminate CEAs from NCA-2 and NFA-2. Although the reactivity of NFA-2 was less than that of any CEA in all kits, that of NCA-2 was higher than that of some CEAs in 3 kits (Abbott CEA-EIA Monoclonal, CEA MITSUI II, and CEA Roche EIA). These differences in reactivity and specificity of currently available EIA kits for CEA should be borne in mind when selecting an assay kit.

Antibodies, Monoclonal↗

Epitope mapping of the carcinoembryonic antigen with various related recombinant proteins expressed in Chinese hamster ovary cells and 25 distinct monoclonal antibodies.

Antigenic epitopes of carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA) were analysed in relation to their domain structures [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M for CEA and domains N, I (A1-B1), and M for NCA]. We reconstructed cDNAs for CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M (CEA-whole), NCA-N, NCA-N-I and NCA-N-I-M (NCA-whole), which were expressed in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. Their mol. wts judged from Western blotting were 17,000-26,000 for CEA-N, 70,000 for CEA-N-I, 150,000 for CEA-N-I-II, 165,000 for s-CEA-whole which was spontaneously released from cells into culture medium, 180,000 for p-CEA-whole which was solubilized with phosphatidylinositol specific phospholipase C (PI-PLC) from cells, 18,000-25,000 for NCA-N, 63,000 for NCA-N-I, and 96,000 for p-NCA-whole which was solubilized with PI-PLC from cells. The divergence of the observed mol. wts from those calculated from cDNA sequences seems to indicate that these recombinant proteins are highly N-glycosylated. By enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 25 distinct anti-CEA monoclonal antibodies (MAbs), each representative of 25 different subgroups within five groups (Groups 1-5) previously classified by us in terms of the reactivity with CEA and CEA-related antigens. Twenty-one MAbs previously shown to react with different protein epitopes of the CEA molecule allow to define six groups (A-F) of epitopes according to their expression by different domains of the CEA and NCA molecules. Among four epitopes common to CEA and NCA, two were found to be present on domain N (Group A) and two on domain I (Group B). Among 15 epitopes absent from NCA but expressed by CEA and normal fecal antigens (NFAs), four were on domain N (Group C), five on domain I (Group D) and six on domain II (Group E). Two epitopes were previously described as "CEA distinctive", because they were recognized by MAbs reacting with CEA but not with the NFAs. These two epitopes (Group F) were found to be expressed by p-CEA-whole but not by s-CEA-whole. The latter results suggest that the Group F epitopes are located on a part of the domain III close to the anchoring device of the CEA molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Biochemical characterization of 25 distinct carcinoembryonic antigen (CEA) epitopes recognized by 57 monoclonal antibodies and categorized into seven groups in terms of domain structure of the CEA molecule.

The chemical nature of 25 distinct carcinoembryonic antigen (CEA) epitopes, which were recognized by 57 different monoclonal antibodies and categorized into 7 groups (Groups A to G) in terms of domain structure of the CEA molecule, was analyzed and the findings obtained were compared with the results of our previous studies using recombinant CEA proteins. All 21 epitopes of Groups A to F defined by 48 MAbs were resistant to periodate oxidation and were to a greater or lesser extent retained after deglycosylation of CEA, indicating that they are all protein in nature. The 21 epitopes were detected in recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Seven of the 21 epitopes of protein nature were partially or completely sensitive to reduction and alkylation of CEA and not detected or only slightly revealed in the recombinant CEA proteins expressed in E. coli, indicating that those epitopes are dependent on the tertiary structure of the peptide chain, which is formed by disulfide bonds. All 4 epitopes of Group G defined by 9 MAbs were sensitive to mild periodate oxidation and deglycosylation, but resistant to reduction and alkylation and to digestion with pepsin or pronase, indicating that those 4 epitopes are carbohydrate in nature. Although none of the 4 epitopes of Group G were detected in the recombinant CEA proteins expressed in E. coli, two were detected in those expressed in CHO cells. The biochemical studies reported here thus provide information as to the nature of the epitopes on the CEA molecule and help form the basis for selecting the anti-CEA MAbs for use in biological study and potential clinical applications of CEA.

Animals↗

Epitope mapping of the nonspecific cross-reacting antigen using various related recombinant proteins expressed in Chinese hamster ovary cells and eight distinct monoclonal antibodies.

Antigenic epitopes of nonspecific cross-reacting antigen (NCA) recognized by 8 different monoclonal antibodies (MAbs) were analyzed in relation to the domain structures of NCA [domains N, I (A1-B1) and M] and CEA [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M]. We reconstructed cDNAs for NCA-N, NCA-N-I-M, CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M in a eukaryotic expression vector, pdKCR-dhfr, and expressed them in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested against eight different MAbs reactive with NCA. All 8 MAbs had been shown to recognize the protein epitopes of the NCA molecule and classified into two groups in terms of the reactivity with NCA and CEA; Group X, 5 clones reactive with both NCA and CEA; and Group Y, 3 clones reactive only with NCA. The epitopes recognized by two of five Group X MAbs were found to be present on the domain N of the NCA molecule as well as of the CEA molecule, and those of the three others were on the domain I (A1-B1) of both molecules, respectively. All three epitopes of Group Y MAbs, which were unique to NCA, were present on the domain I (A1-B1) but not on the domain N of the NCA molecule. The epitope mapping reported here helps form the basis for understanding the relation between the chemical structure and antigenic activities of the NCA molecule and may be useful to study the functions of the NCA molecule, especially those of the respective domains.

Animals↗

Determination of epitope specificities of a large number of monoclonal antibodies by solid-phase mutual inhibition assays using biotinylated antigen.

A generally applicable method for the determination of the epitope specificities of a large number of monoclonal antibodies (MAbs) is presented. The method is based on the solid-phase mutual inhibition assay using 96-well plates coated with the respective MAbs, competitor MAbs, biotinylated antigen and avidin-peroxidase conjugate. Using carcinoembryonic antigen (CEA) as a model antigen the method was applied to the determination of epitope specificities of anti-CEA MAbs. A constant amount of biotinylated CEA was incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated CEA bound to the immobilized antibody were then reacted with avidin-peroxidase conjugate and the activity of the bound peroxidase was determined by the use of o-phenylenediamine and hydrogen peroxidase. The method used here alleviates the laborious procedures of labeling all antibodies to be tested and the confusion caused by differential labeling among different MAbs, and is convenient for mapping analysis of many MAbs if the corresponding purified antigen is available.

Animals↗

Immunological and biochemical characterization of the nonspecific cross-reacting antigen epitopes using twenty-three monoclonal antibodies.

Twenty-three monoclonal antibodies (MAbs) reactive with nonspecific cross-reacting antigen (NCA) were prepared and used for constructing a serological map of the NCA molecule. The MAbs were generated using purified NCA or carcinoembryonic antigen (CEA) as immunogen. The MAbs could be divided into two groups: Group X, 10 clones reactive with NCA and CEA; and Group Y, 13 clones specific for NCA. Cross-competition enzyme immunoassays between MAbs of the individual groups revealed that at least 8 different subgroups can be defined i.e., 5 and 3 subgroups in Groups X and Y, respectively. The chemical nature of the epitopes recognized by those MAbs was tested using chemically or enzymatically treated antigens; all MAbs reacted with periodate-treated NCA and deglycosylated NCA, indicating that all the epitopes identified appeared to be protein in nature. Reduction and alkylation, pepsin digestion or pronase treatment of NCA, however, gave some differential results with respect to MAb binding. The serologic mapping and biochemical studies reported here thus provide information as to the range and nature of the epitopes on the NCA molecule and help form the basis for selecting the anti-NCA MAbs for use in biological and immunological study of NCA.

Animals↗