Masatora Haruno

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.

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.

BINDING REACTIVITY OF MONOCLONAL ANTI-CARCINOEMBRYONIC ANTIGEN (CEA) ANTIBODIES WITH CELL MEMBRANE-BOUND CEA AND WITH FREE CEA IN SOLUTION

Binding reactivities of 62 anti-CEA MAbs from 10 different research groups with cell membrane-bound CEA and with free CEA in solution were compared by inhibition of MAb binding to CEA-expressing tumor cells by free CEA. Bindings of 30 MAbs to the cell membrane-bound CEA (280 ng CEA/2 x 10(5) cells) were inhibited by approximately equal amounts of free CEA, indicating that binding affinities of about half the MAbs for cell membrane-bound CEA are similar to those for free CEA, respectively. Bindings of 15 MAbs to the cell membrane-bound CEA were easily inhibited by free CEA of less than half the amount of the cell membrane-bound CEA, while inhibition of bindings of the remaining 17 MAbs required twice more free CEA than the amount of cell membrane-bound CEA, showing that about one-fourth of the MAbs have higher affinities for free CEA and the remaining about one-fourth of the MAbs possess higher affinities for cell membrane-bound CEA. These results help form the basis for selecting the anti-CEA MAbs for use in clinical applications, such as serum CEA assay, tumor imaging and immunotherapy.

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.

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.

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.

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.

Immunoserological and histological differences between autoimmune hepatitis with acute presentation and chronic autoimmune hepatitis

The histological features of clinically chronic autoimmune hepatitis (AIH) have been well established, with interface hepatitis and plasma cell infiltration as hallmark lesions, however, the immunoserological and histological features of recent‐onset and acute AIH remain undefined. The goal of this study was to define the immunoserological and histological differences between AIH with acute presentation and chronic AIH.

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.

[A case of drug-induced liver injury caused by Keishi-karyukotsu-boreito and Shin-i-seihaito].

A 43-year-old woman was admitted to our hospital due to liver dysfunction. She had a history of liver injury induced by the herbal medicine Keishi-karyukotsu-boreito, which occurred at the age of 35 years. On the present occasion, she had taken the herbal medicine Shin-i-seihaito to treat her sinusitis for one month. We diagnosed liver injury caused by Shin-i-seihaito, and her liver dysfunction normalized after discontinuation of Shin-i-seihaito. This is the first reported case of drug-induced liver injury caused by the herbal medicines Keishi-karyukotsu-boreito and Shin-i-seihaito.