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1.
Nasu T Fukuda Y Hashino J Nagahira K Kawashima H Noguchi C Oikawa S Nakanishi T 《Immunology letters》1999,67(1):57-62
Monoclonal antibodies (MoAbs) against N-domain of carcinoembryonic antigen (CEA), C249, K348, K1338, and K1444, that inhibit CEA-mediated cell adhesion, did not crossreact with nonspecific cross-reacting antigen (NCA). To determine amino acid sequences involved in the adhesion, epitopes of the MoAbs were mapped with recombinant NCAs carrying CEA-NCA chimeric N-domain. The data showed that the epitopes of C249, K1338, K1444 are located within the regions 1-32, 1-32, and 33-59 of CEA, respectively, and that two discrete regions 1-32 and 60-93 may be related to the epitope of K348. Comparison of amino acid sequences between CEA and NCA suggested that four residues (21, 27-29), eight residues (21, 27-29, 66, 78, 79, 89), and three residues (43, 44, 46) are important for recognition by C249 (or K1338), K348, and K1444, respectively. These residues seem to participate in the cell adhesion mediated by CEA. 相似文献
2.
Specificities and binding properties of eight monoclonal antibodies against carcinoembryonic antigen
Eight different monoclonal antibodies against CEA were derived from fusions with spleen cells of mice immunized with highly purified CEA. All eight antibodies were IgG l,
and had isoelectric points between pH 6.5 and 7.5. They reacted strongly with native CEA, Smith degraded CEA (SI-stage) and cealowonly marginally with reduced and alkylated CEA and not at all with orosomucoid, indicating that they were directed against conformation dependent protein determinants. Two antibodies cross-reacted strongly with nonspecific cross-reacting antigen (NCA) while none of the antibodies cross-reacted with biliary glycoprotein I (BGP I). At least six different epitopes in the peptide moiety of CEA were recognized by this series of monoclonal antibodies. At least two of these appeared to occur twice in the molecule, possibly indicating that CEA contains two or more homology regions. The affinity constants of three of the CEA-‘specific’ antibodies were found to be very high, e.g. 1.2, 3.3 and 7.4 × 108M−1, respectively. 相似文献
3.
High affinity human monoclonal antibodies directed against hepatitis B surface antigen 总被引:2,自引:0,他引:2
C Desgranges J Paire C Pichoud S Souche D Frommel C Trepo 《Journal of virological methods》1987,16(4):281-292
Peripheral blood mononuclear cells from donors immunized with hepatitis B vaccine (Pasteur Hevac B) were transformed with Epstein-Barr virus. Two polyclonal cell lines, producing antibodies to hepatitis B surface antigen were established and cloned. Seven clones were isolated; they secreted between 10 and 20 micrograms/ml of HBs specific IgG1 kappa or lambda antibody with anti-HBs titer of 300-800 IU/ml. These human antibodies expressed the anti 'a' specificities and had high affinity and avidity; their potential use as reagents for hepatitis B virus detection and for passive immunotherapy is under study. 相似文献
4.
Mazhar Rishi Albert Kovatich Hormoz Ehya Rosa M. Davila 《Diagnostic cytopathology》1994,11(4):358-362
To assess the usefulness of polyclonal and monoclonal antibodies against carcinoembryonic antigen (CEA) in the differential diagnosis of hepatocellular carcinoma (HCC) vs. metastatic adenocarcinoma (MA), we studied 25 cases of fine-needle aspirates (FNA) of hepatic lesions. The material consisted of 9 primary HCCs, 8 MAs, and 8 benign hepatic aspirates. For immunostaining, the avidin-biotin complex technique was performed on paraffin sections of cell blocks, using a standardized automatic immunostainer. Specific bile canalicular immunostaining with polyclonal CEA (pCEA) antibody was present in five of eight (5/8) benign hepatic aspirates and eight of nine (8/9) HCCs. Diffuse cytoplasmic immunostaining with pCEA antibody was present in four of eight (4/8) MAs. None of the aspirates showed any positive immunostaining with monoclonal CEA (mCEA) antibody. We conclude that: (1) pCEA antibody is useful in the evaluation of hepatic FNAs. Diffuse cytoplasmic staining is seen in MAs, whereas canalicular immunostaining pattern is an indication of benign or malignant hepatocytes. (2) Paraffin-embedded cell blocks made from hepatic aspirate material are suitable for immunostaining with polyclonal CEA antibody. (3) mCEA antibody has no value in the diagnosis of HCC. Diagn Cytopathol 1994; 11:358–362. © 1994 Wiley-Liss, Inc. 相似文献
5.
Eight monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) were characterized. Five clones are IgG(1), two clones are IgM and one clone is IgG(2b); all have kappa light chain. The affinities are in the range of 1.1 x 10(-7) approximately 2.4 x 10(-9) M; the affinities of two IgM clones could not be estimated because of their low enzyme-linked immunoadsorbent assay (ELISA) signal. Each clone was constructed as single-chain Fv (scFv) and expression was performed in E. coli. Four clones out of 8 could express scFv soluble to culture media and the expression was confirmed further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The nucleotide and amino acid sequences of V(H) and V(L) of four scFvs were deduced and their family and subgroup were analyzed. We found that the clones that do not express the scFv have aberrant kappa chain (incorrect V/J recombination or stop codon); in contrast, their heavy chain sequences proved correct. The E. coli-expressed scFvs showed 1.5 x 3.4-fold lower affinities (2.8 x 10(-8) approximately 3.6 x 10(-9) M) than those of hybridoma-derived parental antibodies except the one clone (C5), which exhibited approximately 10(-6) M of affinity. 相似文献
6.
Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA) 总被引:1,自引:0,他引:1
Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma. 相似文献
7.
Characterization of monoclonal antibodies to carcinoembryonic antigen with increased tumor specificity 总被引:1,自引:0,他引:1
S Jothy S A Brazinsky M Chin-A-Loy A Haggarty M J Krantz M Cheung A Fuks 《Laboratory investigation; a journal of technical methods and pathology》1986,54(1):108-117
Nine monoclonal antibodies reacting with carcinoembryonic antigen (CEA) were produced after immunization of mice with either purified CEA or a CEA-producing human cell line. Their specificities were assessed by immunohistochemistry on tissue sections of neoplastic and nonneoplastic lesions. These monoclonal antibodies have different patterns of tissue reactivity. Two of them, D14 and B18, were found to have a high degree of specificity for colonic carcinoma and did not react with formalin-fixed paraffin-embedded sections of normal colon with standardized staining conditions. Most cases of noncolonic adenocarcinomas and normal epithelial structures were not stained by these two monoclonal antibodies. The specificity of the monoclonal antibodies was further investigated immunochemically using intact, reduced, and alkylated or chemically fragmented CEA. Liquid phase radioimmunoassays and antibody competition immunoenzymatic assays confirmed that the antibodies recognize different epitopes of CEA. These data support the concept of CEA heterogeneity and the reactivity of the D14 and B18 monoclonal antibodies with colonic adenocarcinomas indicates that they are useful immunohistochemical probes. 相似文献
8.
The reaction between monoclonal antibodies and surface-immobilised hapten was studied by ellipsometry, a method allowing absolute measurement of the surface concentration of proteins. Monoclonal antibodies against 2-phenyloxazolone were used and their affinity for the antigen in solution was determined by calculations of the equilibrium constant from data obtained by measuring fluorescence quenching of the hapten due to antibody binding. The binding rate of antibody to surface-immobilised hapten and the dissociation rate of the complex were measured by ellipsometry. The equilibrium constant of the heterogeneous antigen-antibody reaction was determined by a Scatchard plot. The affinity of the antibodies for the antigen was found to be higher in the heterogeneous than in the homogeneous reaction by a factor which varied between different monoclonal antibodies. 相似文献
9.
Interference of complement with the binding of carcinoembryonic antigen to solid-phase monoclonal antibodies 总被引:1,自引:0,他引:1
O P B?rmer 《Journal of immunological methods》1989,121(1):85-93
Six mouse monoclonal antibodies against carcinoembryonic antigen were evaluated for use in a solid-phase immunometric assay. Three IgG2 antibodies, when attached to polymer particles or microtiter wells, were found to be severely inhibited by fresh serum. The remaining three antibodies, which were of the IgG1 subclass, were inhibited only slightly or not at all when used in the same way. With the aid of labelled antibodies against C1q and C3, it was shown that antibody inhibition was accompanied by the binding of large amounts of these complement factors. Experiments involving heat inactivation or dilutions of serum, or the addition of EDTA, consistently revealed the same correlation between binding of complement factors and inhibition of CEA binding to antibody. It is suggested that the significant inhibition of the solid-phase IgG2 antibodies was caused by classical pathway activation of complement by the solid-phase antibody even in the absence of antigen. The slight inhibition of solid-phase IgG1 antibodies observed at high serum concentrations was probably due to complement binding by the alternative pathway. Preliminary evidence suggests that complement interference is not restricted to CEA assays, but is a potential problem in all types of serum immunoassays using solid-phase antibodies. 相似文献
10.
Values of the equilibrium dissociation constant and the number of attachment sites for the binding of radiolabeled F(AB) fragments prepared from four monoclonal antibodies to southern bean mosaic virus have been determined. Monoclonal antibodies B6 and B10 produced against the bean type strain of southern bean mosaic virus (SBMV-B) and 1C4 and 1D6 produced against the cowpea type strain (SBMV-C) were more reactive with the untreated virus than with EDTA-swollen particles. B6 and B10 reacted with 180 and 90 sites, respectively, on the SBMV-B particle and were not reactive with SBMV-C. Antibody 1D6 reacted with 180 sites on both SBMV-B and SBMV-C, whereas 1C4 bound to 180 sites on SBMV-C and 60 sites on SBMV-B. The equilibrium dissociation constants for B10, 1C4, and 1D6 were similar but antibody B6 bound with an 8- to 10-fold lower affinity. Solid phase competitive antibody inhibition analysis showed that 1C4 and 1D6 were mutually inhibitory and that these antibodies also inhibited the binding of B6 and B10. Antibody B6 partially inhibited the binding of 1D6 but did not affect 1C4 or B10 binding. No inhibition of B6, 1C4, or 1D6 binding was observed when B10 was the competing antibody. Possible sites of reaction of these monoclonal antibodies on the SBMV particle are discussed. 相似文献
11.
12.
Selection of monoclonal antibodies for use in an immunometric assay for carcinoembryonic antigen. 总被引:1,自引:0,他引:1
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. 相似文献
13.
Four hybridoma clones (BKL1, BKL2, BKL5 and BKL6), secreting monoclonal antibodies against the decapeptide luteinizing hormone releasing hormone (LHRH), were obtained by the fusion of Sp 2/0-Ag14 myeloma cells with spleen cells of a Balb/k mouse immunized with a conjugate of thyroglobulin-LHRH. All four monoclonal antibodies belong to the IgG1 subclass. The antibodies cross-reacted in RIA from 9.3 to 21% with 4-10 LHRH and less than 1% with 7-10 LHRH, 1-3 LHRH and LHRH-OH; 4-6 LHRH showed no cross-reaction. A RIA based on the BKL2 antibody and able to detect 4 pg LHRH per tube was developed. An extract of rat hypothalamus was submitted to Sephadex G50 separation and a single peak of LHRH immunoreactivity corresponding to synthetic LHRH, was detected with the antibody BKL2. When this material was further analyzed by HPLC, we found that the major peak of immunoreactivity co-migrated with synthetic LHRH. The data shows that the antibodies should be useful tools for biochemical and physiological studies on LHRH. 相似文献
14.
《Immunotechnology》1999,4(1):49-57
Background: Carcinoembryonic antigen (CEA) is a human tumor antigen with the domain structure N-A1-B1-A2-B2-A3-B3, in which each domain is predicted to have an Ig-like fold and is known to bind epitope specific anti-CEA antibodies. Objective: To determine the affinity constants of several domain specific anti-CEA antibodies using purified recombinant or synthetic domains. Results and Conclusion: We have determined the kinetic and affinity constants of several anti-CEA antibodies for CEA, CEA domains (A3-B3) expressed in HeLa cells, and a synthetic peptide corresponding to the A3 domain using a BIAcore biosensor. There was no difference in affinity for CEA among a murine (mT84.66), a mouse/human chimeric form (cT84.66) or a disulfide deleted version (ΔSScT84.66) of this antibody. There was less than a five-fold drop in affinity of murine T84.66 for the A3-B3 domain expressed in HeLa cells compared to CEA. The synthetic A3 domain had an affinity constant for mT84.66 which was ten-fold less than for CEA. The affinity constants for CEA with several other anti-CEA monoclonal antibodies, including three antibodies which have almost identical CDR sequences (CEA.281, CEA.11 and CEM231) were also determined. CEM231 which had a two-fold higher affinity constant for CEA than either CEA.281 or CEA.11 had a two-fold faster on-rate which accounts for its higher affinity constant. This difference may be due to one or more of the amino acid differences present in H1 (N vs. S or D) and H3 (A vs. V). 相似文献
15.
A new graphical method for determining the affinity constants of monoclonal antibodies to enzymes 总被引:1,自引:0,他引:1
A new graphical method is presented for determining the affinity constants of antibodies to enzymes. The method does not require purification of reactants or separation steps at equilibrium. The plot 1/v versus [AT]/V - v) ([AT] = total concentration of antibody binding sites, V = enzyme activity measured in the absence, and v = activity measured in the presence, of antibodies) yields straight lines in the case of simple antibody-enzyme interactions. More complex interaction models show different curve shapes, which can be used for model discrimination as shown by computer simulation studies. The applicability of the method was demonstrated by the determination of the affinity constant of the monoclonal antibody IB 10B8 to alkaline phosphatase of calf intestine. This antibody inhibits enzymic activity completely. 相似文献
16.
Epitope specificity and cross-reactivity pattern of a large series of monoclonal antibodies to carcinoembryonic antigen 总被引:1,自引:0,他引:1
Monoclonal antibodies (MAbs) were produced against purified carcinoembryonic antigen (CEA) from liver metastases of colo-rectal and lung adenocarcinoma. Three and eight anti-CEA MAbs from the two groups were analyzed in detail. All antibodies were IgG1. With one exception they recognized epitopes present on all eight individual CEA preparations investigated irrespective of whether they were from colo-rectal or lung carcinoma. The exceptional MAb reacted with an epitope present on most but not all CEA preparations. With two, or possibly three, exceptions the MAbs recognized conformation dependent epitopes in the peptide moiety of CEA. One MAb reacted strongly with reduced and carboxymethylated CEA but only weakly with native CEA. Four MAbs appeared to be CEA-specific in that they did not react with any of the known CEA-cross-reactive substances including nonspecific cross-reactive antigen of 160,000 mol. wt (NCA-160). A total of nine different epitopes were detected in native CEA using this and our previous series [Hedin A., Hammarstr?m S. and Larsson A. (1982) Molec. Immun. 19, 1641] of anti-CEA MAbs. With one exception practically all molecules (70-90%) in purified CEA preparations contained these epitopes. 相似文献
17.
The affinity constants (Ka) of monoclonal antibodies (MAb) for binding to their corresponding antigens (Ag), unlabelled and in buffered solution were determined by the following procedure: 1. Incubation of MAb (fixed concentration) with Ag (concentration dilution series). 2. Rapid bound/free separation by adding immobilized second antibody, followed by centrifugation. 3. Determination of free Ag in the supernatant using a gold sol particle agglutination immunoassay (SPIA) in a microtitration plate format. 4. Calculations and interpretation were based on Scatchard and Sips plots. Ka values found by this procedure were found to be similar to those obtained by a radio-immunoassay (RIA) procedure. The present method avoids possible artefacts in Ka values introduced by the procedure or chemical modification due to labelling of MAb or Ag. It enables rapid, simultaneous screening of a considerable number of different MAbs under non-specialized (i.e. RIA) laboratory conditions. 相似文献
18.
A generally applicable method for the selective precipitation of antigens from solution phase is presented. The method is based on the rapid and quantitative binding of avidin to biotin-labeled antigen in the presence of biotinylated carrier protein. Using carcinoembryonic antigen (CEA) as a model antigen biotin-labeled CEA was precipitated to greater than 97% at amounts of up to 5000 ng/tube. The method was applied to the determination of epitope specificities and affinities of monoclonal anti-CEA antibodies in solution phase. Non-specific precipitation did not exceed 2% for radiolabeled Fab fragments and 6% for radiolabeled IgG. Because antigen-antibody binding takes place in solution, artifacts introduced by the immobilization of reactants to solid supports are omitted. 相似文献
19.
Monoclonal antibodies (McAbs) specific to methamphetamine (MA) were produced using p-amino MA coupled to bovine serum albumin (BSA) with glutaraldehyde (GA) as an immunogen and with conventional hybridoma techniques. Hybridoma clones secreting the McAbs were selected by an enzyme-linked immunosorbent assay (ELISA) system using both the above conjugate and BSA modified with GA as screening antigens. In the ELISA system were used avidin and biotinyl-alkaline phosphatase which converts nicotinamide adenine dinucleotide phosphate (NADP) into NAD. The final enzyme activity was determined using diformazan of nitroblue tetrazolium formed together with the NAD produced, alcohol dehydrogenase and phenazine methosulfate. The McAbs from 9 clones were characterized by a crossreactivity test using the ELISA. The McAbs recognized MA (100%), methoxyphenamine (8.0%), ephedrine (2.3%), but did not react with metylephedrine, amphetamine, OH-amphetamine, dimethylamphetamine, beta-phenylethylamine, norephedrine, phentermine and ranitidine. An inhibition curve for MA was obtained in the range of 0.75 to 50 ng. 相似文献
20.
Summary Four monoclonal antibodies generated against the recombinant core antigen of hepatitis B virus are investigated for antigen binding. All exhibit a similar affinity to polystyrene-sorbed antigen but only one of them interacts with native form of HBcAg (an assembled particle) in solution. The presence of 0.1% sodium dodecylsulphate is required for the binding of other three antibodies. The phenomenon can be interpreted as inaccessibility of the corresponding epitopes unless the multimeric antigen structure is disrupted. The core antigen coated on polystyrene is considered as a similar exposed structure. 相似文献