Detection of a soluble serum antigen by a new monoclonal antibody ED8 in patients with breast cancer

A murine hybridoma, secreting monoclonal antibody ED8, was generated by immunization with the breast cancer cell line H466B. ED8 reacts with a 300 kDa antigen present on H466B cell surface. The antigen can be detected in serum from breast cancer patients, using ELISA and immunoblotting.     

Detection of common flagella antigen in Bacillus cereus by monoclonal antibody

Bacillus cereus has been classified into 23 types by immunochemical analyses of flagella antigen, but a common antigenic determinant of flagella had not been determined. When the immunochemical method of classification had changed from "agglutination method" to "enzyme-linked immunosorbent assay (ELISA)," the cross-reactivity between each flagella type was increased. These results indicated that the application of ELISA method would enable detection of the common antigenic determinant of B. cereus. Therefore, we attempted to make monoclonal antibody against common flagella antigen that could be detected by ELISA. Monoclonal antibody provided was specific for B. cereus (H-1 to H-23) and did not show the cross-reactivity with Escherichia coli NIH and Bacillus subtilis.     

Immunohistochemical studies of colorectal carcinoma with a monoclonal antibody against carcinoembryonic antigen

Immunoperoxidase localization of carcinoembryonic antigen (CEA) was performed on tissue sections of colorectal carcinoma using a monoclonal antibody (MAb) against CEA. CEA has been demonstrated in 20 out of 22 rectum carcinomas (90.9%), in all of 23 colonic carcinomas, in none of 4 hyperplastic polyps and in 2 out of 6 adenomatous polyps (33.3%). CEA was found more often, and the intensity of the staining was stronger in well-differentiated carcinomas than in moderately and poorly differentiated carcinomas. No correlation was found between the presence of CEA in colorectal carcinoma and the stages of the disease. The mean values of serum CEA in patients with colorectal carcinoma and polyps with negative, weakly and strongly positive staining were 5.4 +/- 3.9 ng/ml, 28.3 +/- 23.8 ng/ml and 99.8 +/- 145.3 ng/ml respectively. Elevation of serum CEA occurred in 30 out of 39 (78.9%) cases with strongly positive CEA staining, in 4 out of 6 (66.7%) with weakly positive and in 1 out 9 (11.1%) with negative staining. A significant difference was found in serum CEA activity between the group with negative CEA staining and positive CEA staining (P less than 0.01). Our results suggest that the monoclonal antibody (MAb C27) can be used for the localization of CEA in conventionally prepared tissues of colorectal carcinomas by immunoperoxidase techniques for routine immunopathological diagnosis.     

Preoperative carbohydrate antigen 195 (CA195) and CEA serum levels as prognostic factors in patients with colorectal cancer

We evaluated in 214 patients with primary colorectal cancer the prognostic value of the preoperative serum levels of CEA and CA195. For CEA these levels were above the cutoff of 6 ng/ml in 31.3% of patients, whereas for CA195 they were higher than 12 U/ml in 35.9% of patients. The simultaneous use of both antigens increased the sensitivity to 49%, which was significantly higher than that of CEA (p < 0.001) and CA195 (p < 0.01) taken singly. The mean preoperative CEA levels were significantly (p < 0.001) correlated with Dukes' stage only, while there was a significant correlation between preoperative serum levels of CA195 and Dukes' stage (p < 0.001), grade of differentiation (p < 0.01) and tumor location (p < 0.05). The results indicated that high preoperative serum levels of CEA and CA195 were associated with a shorter overall survival (p < 0.0001). In addition, separate Cox multivariate analysis showed that preoperative CA195 was, after Dukes' stage, the strongest factor to predict overall survival (p < 0.0001).     

Detection of a glycosphingolipid antigen in bladder cancer cells with monoclonal antibody MRG-1

Summary The monoclonal antibody MRG-1 has been evaluated for the immunohistochemical detection of the type 3 chain of blood group A in human normal bladder epithelium and bladder tumours. Light microscope examination of paraffin sections demonstrated that this antigen was present in normal epithelium and superficial bladder tumour in patients with blood group A or AB, but was absent in the invasive type of bladder tumour. In normal epithelium, the plasma membrane was positive for this antigen, and the cytoplasm was diffusely stained. In superficial transitional cell carcinoma, the plasma membrane was negative, whereas the cytoplasm was intensely stained in the perinuclear region. This pattern was different from that observed for type 1 and 2 group A antigen, which was recognized mainly at the plasma membrane. However, in superficial transitional cell carcinoma, the staining was also seen on the plasma membrane. The pattern of the localization of this antigen in this carcinoma was influenced by the treatment of organic solvents. Electron microscopical observations confirmed that this antigen was localized on the plasma membrane and also in the Golgi apparatus of the superficial tumour.These results proved that the type 3 chain of blood group A is present in human bladder epithelium and low grade tumours in correspondence with the blood type, but disappears in tumours with high malignant potential. However, its expression is independent of the expressions of the other subtypes which have been studied. Furthermore, the changes in the staining pattern caused by pretreatment with organic solvents suggested possible differences in the microenvironment of the glycolipids containing this type of sugar chain.     

Detection of leishmania antigen in kala azar patients using monoclonal antibodies.

Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovani antigenic determinants ranging from 42-116 kDa were selected as 'capture antibodies' and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb.17 could effectively detect circulating leishmania antigen in 85.4%. The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.     

Conformational structure of a monoclonal anti-idiotypic antibody to the monoclonal anti-adenocarcinoma-associated carbohydrate antibody YH206.

The mAb AI206 (IgG1) is an anti-Id antibody of mAb YH206 (IgM) to adenocarcinoma-associated carbohydrate Ag and inhibits the reaction of mAb YH206 to YH206 Ag at low concentrations. By Western blot analysis, mAb AI206 only reacted with unreduced mAb YH206, whereas it did not react with reduced mAb YH206. Furthermore, mAb AI206 reacted with IgM subunit (180 kDa), F(ab')2 (110 kDa), and F(ab) (50 kDa) of pepsin-treated unreduced mAb YH206. Thus, mAb AI206 recognized the structure of F(ab) of mAb YH206. The mAb YH206 reacted with unreduced mAb AI206, F(ab')2 (110 kDa), and F(ab) (50 kDa) of pepsin-treated unreduced mAb AI206. It is presumed that mAb YH206 and mAb AI206 recognize each other in an unreduced condition but not a reduced condition. The recognition of such a conformational Id on F(ab) is important. Because mAb YH206 recognized the carbohydrate on YH206 Ag as well as the peptide on mAb AI206, the conformation on F(ab) of mAb AI206 may mimic the carbohydrate structure on YH206 Ag. In fact, YH206 antibody activity was induced in syngeneic mouse serum immunized with mAb AI206. These observations suggest that the internal image of YH206 carbohydrate Ag is preserved within the conformational Id on F(ab) of mAb AI206.     

Induction of anti-idiotypic humoral and cellular immune responses by a murine monoclonal antibody recognizing the ovarian carcinoma antigen CA125 encapsulated in biodegradable microspheres

The use of biodegradable poly(dl-lactic-co-glycolic acid) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses was investigated using a murine monoclonal antibody B43.13 that recognizes the human ovarian cancer antigen CA125. Immunization of mice with mAb B43.13 encapsulated in poly(dl-lactic-co-glycolic acid) microspheres resulted in enhanced humoral and cellular immune responses compared with mAb B43.13 alone or mAb B43.13 mixed with microspheres. The antibody responses could be further enhanced by the co-encapsulation of mAb B43.13 with monophosphoryl lipid A, a non-toxic adjuvant, in microspheres. Anti-idiotypic humoral responses were shown to result in Ab2 antibodies mimicking the nominal antigen CA125 and Ab3 antibodies recognizing CA125. Further, microsphere delivery of mAb B43.13 also resulted in induction of T cell responses involving T2 cells reactive with mAb B43.13 epitopes and T3 cells recognizing CA125. These results indicate that microsphere delivery of Ab1 can induce both humoral and cellular anti-idiotypic responses relevant to cancer antigens. This raises the possibility of the use of such formulations for anti-idiotypic induction immunotherapy for cancer. Received: 27 August 1997 / Accepted: 24 April 1998     

Novel tumor-associated antigen of hepatocellular carcinoma defined by monoclonal antibody E4-65

A monoclonal antibody, E4-65, produced by immunizing mice with SMMC-7721 cells, a human hepatocellular carcinoma (HCC) cell line, was used to identify and characterize an unreported HCC-associated antigen. Indirect immunofluorescence studies showed that E4-65 antibody reacted with five out of eight HCC cell lines, but not with 10 non-HCC tumor cell lines or a normal liver cell line. Using immunohistochemical examination, E4-65 antigen was detected on the cell membranes and in the cytoplasm of human liver tumor tissues, but was not found in most other tumors, or normal adult or fetal tissues, except for a weakly positive reaction in tissues of the digestive system. Western blot analysis showed that E4-65 antibody bound to a 45 kDa protein in the human HCC cell line and tissue lysates. Enzyme treatment and lectin blotting did not detect the carbohydrate chain in E4-65 antigen. This HCC-associated protein represents a potentially useful target for diagnoses and immunotherapy of human HCC.     

Altered cell surface antigen expression in bladder carcinoma detected by a new hemagglutinating monoclonal antibody

A hemagglutinating monoclonal IgM antibody (MoAb145) was produced against a high incidence red blood cell membrane antigen. By the specific red cell adherence test, the antibody also reacted with human bladder epithelium; in addition, expression of the MoAb145 antigen was lost in some cases of transitional cell carcinoma of the bladder, in a manner similar to the ABH blood group. Hemagglutination studies with a panel of erythrocytes lacking specific high incidence red blood cell membrane antigens indicated that MoAb145 did not recognize ABH specificity but rather a determinant absent from rare MN variant erythrocytes, including En(a-) erythrocytes, which lack glycophorin-alpha. Failure of MoAb145 to stain, by indirect immunofluorescence, the erythroleukemia cell line K562, which expresses glycophorin-alpha and the MN blood group, and failure to inhibit MoAb145 hemagglutination with an erythrocyte sialoglycoprotein fraction that contained MN blood group activity suggests that MoAb145 does not recognize either glycophorin-alpha or the MN blood group, but rather another membrane determinant, which is altered in En(a-) erythrocytes. This study demonstrates a new epitope detected by MoAb145 that is shared between human erythrocyte membranes and bladder epithelia, and is affected by neoplastic transformation in transitional cell carcinoma of the bladder.     

A novel carbohydrate, differentiation antigen on fucogangliosides of human myeloid cells recognized by monoclonal antibody VIM-2

A mouse monoclonal antibody, VIM-2, specific for human blood cells of myelomonocytic lineage, was found to bind to a series of minor gangliosides isolated from the cells of patients with chronic myelogenous leukemia (Uemura, K., Macher, B.A., DeGregorio, M., Scudder, P., Buehler, J., Knapp, W., and Feizi, T. (1985) Biochim. Biophys. Acta 846, 26-36). TLC immunostaining studies with the VIM-2 antibody of gangliosides from normal human neutrophils, acute myeloid leukemia, and chronic myelogenous leukemia cells showed that the total amount and the ratio of the VIM-2 gangliosides varies among these different myeloid cells and appears to be related to the level of cellular differentiation. Purification of these gangliosides from chronic myelogenous leukemia cells was aided by a sensitive enzyme-linked immunosorbent assay procedure used in conjunction with high performance liquid chromatography. Structures for two of the immunoreactive gangliosides (a ceramide decasaccharide, VIII3NeuAcV3-Fuc-nLc8Cer and a ceramide dodecasaccharide X3-NeuAcVII3Fuc-nLc10Cer) are proposed from negative ion fast atom bombardment mass spectrometry of the native gangliosides, methylation analysis, and the combined use of glycosidase treatment and TLC immunostaining with carbohydrate sequence specific antibodies. The VIM-2 antigen was thus characterized as involving the sialofucooligosaccharide sequence.     

Hepatitis B core antigen. Detection of antibody by radioimmunoprecipitation.

Radioactive cores were prepared from concentrated Dane particles by DNA polymerase reaction followed by CsCl density gradient centrifugation. Two radioactive peaks were obtained: one peak with an average density of 1.36 g/cm-3 in CsCl contained cores that possessed full serologic reactivity; the other peak, with a density of 1.28 to 1.32 g/cm-3, contained cores associated with globulin. A double antibody immunoprecipitation test was developed, using the radioactive heavy cores as a source of antigen. The test was at least 300 times as sensitive as complement fixation for detecting antibody to core.     

Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64

The immunohistochemical characteristics of the monoclonal antibody IND.64 are very similar to those of the monoclonal antibody Ki-67. The aim of this study was to further characterize this new antibody and to compare it with Ki-67 using immunobiochemical methods. Our results demonstrate that the similarity between the antibodies holds true even at the molecular level. Immunoblot analysis of IM-9-cell lysates with both antibodies showed a double band with apparent molecular weights of 395 kD and 345 kD, respectively. Competition ELISAs using a synthetic peptide derived from the thus far determined Ki-67 cDNA sequence as competitor, indicate that IND.64 may recognize the same epitope as Ki-67. The IND.64 epitope resides at least within a 20 amino acid sequence which also contains the Ki-67 epitope. Since IND.64 is of the IgG2b subclass, while Ki-67 is of the IgG1 subclass, the two antibodies may be useful for double immunostaining. In addition, IND.64 may help in determining the still unknown function of the anigen it recognizes.     

Detection of bluetongue virus group-specific antigen using monoclonal antibody based sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.     

Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and a monoclonal antibody.

The role of renal expression of the glycosphingolipid verotoxin receptor, globotriaosylceramide, in susceptibility to verotoxin-induced hemolytic uremic syndrome is unclear. We show that a single glycosphingolipid can discriminate multiple specific ligands. Antibody detection of globotriaosylceramide in renal sections does not necessarily predict verotoxin binding. The deoxyglobotriaosylceramide binding profile for verotoxin 1, verotoxin 2 and monoclonal anti-globotriaosylceramide are distinct. Anti-globotriaosylceramide had greater dependency on the intact alpha-galactose and reducing glucose of globotriaosylceramide than verotoxin 1, while verotoxin 2 was intermediate. These ligands differentially stained human kidney sections. Glomerulopathy is the primary verotoxin-associated pathology in hemolytic uremic syndrome. For most samples, verotoxin 1 immunostaining within adult glomeruli was observed (type A). Some samples, however, lacked glomerular binding (type B). Anti-globotriaosylceramide (and less effectively, verotoxin 2) stained all glomeruli. Verotoxin 1/anti-globotriaosylceramide tubular staining was comparable. Type B glomerular/tubular globotriaosylceramide showed minor, but significant, fatty acid compositional differences. Verotoxin 1 type B glomerular binding became evident following pretreatment with cold acetone, or methyl-beta-cyclodextrin, used to deplete cholesterol. Direct visualization, using fluorescein isothiocyanate-verotoxin 1B, showed paediatric, but no adult glomerular staining; this was confirmed by anti-fluorescein isothiocyanate immunostaining. Acetone induced fluorescein isothiocyanate-verotoxin 1B glomerular staining in type A, but poorly in type B samples. Comparison of fluorescein isothiocyanate-verotoxin 1B and native verotoxin 1B deoxyglobotriaosylceramide analogue binding showed an alteration in subspecificity. These studies indicate a marked heterogeneity of globotriaosylceramide expression within renal glomeruli and differential binding of verotoxin 1/verotoxin 2/anti-globotriaosylceramide to the same glycosphingolipid. Verotoxin 1 derivatization can induce subtle changes in globotriaosylceramide binding to significantly affect tissue binding. Heterogeneity in glomerular globotriaosylceramide expression may play a significant (cholesterol-dependent?) role in determining renal pathology following verotoxemia.     

Detection of Ki-ras proto-oncogene protein by a specific monoclonal antibody.

We have used a commercially available mouse monoclonal antibody and shown it to bind specifically to cellular Ki-ras p21 proteins and not to cellular N- and Ha-ras p21 proteins. In conjunction with electrophoresis and Western blotting, this antibody can be used, with further detailing, to assess levels of the cellular Ki-ras p21 against a background of total p21s.     

Detection of antibody to hepatitis delta virus in human serum by double antigen sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein's purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.