Tarumi Senba

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.

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.

Enhanced antitumor activity of a combination treatment with a mouse/human chimeric anti-MK-1 antibody and lymphokine-activated killer cells in vitro and in a severe combined immunodeficient mouse xenograft model

Abstract Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarcinoma, recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas and seems to be valuable in immunodiagnosis and immunotherapy of various cancers. In a recent study, we constructed a mouse/human chimeric antibody, designated Ch FU-MK-1, by fusing the FU-MK-1 VH and Vκ genes to the human Cγ1 and Cκ genes, respectively. In the present study, we tested combination immunotherapy of Ch FU-MK-1 with human lymphokine-activated killer (LAK) cells in vitro and in mice with severe combined immunodeficiency (SCID) bearing human MK-1-expressing tumors. In in vitro experiments, Ch FU-MK-1 effectively mediated antibody-dependent cell-mediated cytotoxicity (ADCC) against MK-1-expressing MKN-74 cells, which was completely blocked by an anti-FcR antibody. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by LAK cells and Ch FU-MK-1 against MKN-74 cells. The implication of the apoptosis during ADCC was demonstrated by means of both a terminal-deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay and a propidium iodide staining method. In vivo antitumor activity of combination treatment with LAK cells and Ch FU-MK-1 was estimated using SCID mice inoculated s.c. with MKN-74 cells. The i.v. administration of LAK cells and i.p. administration of Ch FU-MK-1 and interleukin-2 (IL-2) produced a marked growth inhibition of MKN-74 tumors in SCID mice. When the actual tumor weights were measured 16 days after initiation of treatment, more than 70% reduction was observed in the group receiving LAK cells plus Ch FU-MK-1 plus IL-2 as compared to the control untreated group. Together these results suggest that Ch FU-MK-1 may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

Establishment and Evaluation of a New Chemiluminescent Enzyme Immunoassay for Carcinoembryonic Antigen Adapted to the Fully Automated ACCESS ® System

We have established a new chemiluminescent enzyme immunoassay for carcinoembryonic antigen (CEA), designated ACCESS CEA, which is adapted to the fully automated ACCESS immunoassay analyzer. The assay is based on a one step sandwich-type method using two monoclonal antibodies, one of which is immobilized on micrometer-size paramagnetic particles and the other is conjugated to alkaline phosphatase. Ten microliters of calibrators or sera are incubated for 5 minutes at 37 degrees C with the particles and with the alkaline phosphatase conjugate. The particles are then magnetically separated and washed to remove unbound components. Time needed to obtain the first result is less than 15 minutes. The assay range was 0.04-1000 micrograms/l of CEA, and the possible high-dose hook effect was prevented at CEA concentrations up to 100000 micrograms/l in this working range. The coefficient of variation (CV) for intra-assay precision was 3.0 to 4.7%, and inter-assay CV was 3.4 to 5.6%. The sample carryover was less than 0.001%. The analytical recovery ranged from 98 to 104% and a dilution linearity was demonstrated. No interference was detected in any sample with levels up to 300 mg/l for bilirubin, 12000 mg/l for haemoglobin, 50000 mg/l for human serum albumin, 8500 mg/l for triacylglycerol, and 500000 IU/l for rheumatoid factor. The ACCESS CEA assay also showed very homogeneous reactivity with purified CEA preparations from different tumours and could discriminate CEA from four CEA-related normal antigens tested. Serum samples (n = 362) from patients with malignant or non-malignant disease, as well as from healthy individuals, were analyzed by the ACCESS CEA assay and by the established IMx CEA assay. The CEA values determined by the ACCESS CEA assay were in good agreement with those determined by the IMx CEA assay, and the ACCESS CEA assay significantly increased the sensitivity and specificity of tumour diagnosis as compared with the IMx CEA assay.

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.