Lymphocytotoxic antibodies in cancer

Summary To study the relationship of cold-reactive lymphocytotoxic antibodies (LCA) to immune defects in cancer, LCA were measured in sera from 71 patients with different types of malignancies and 31 healthy controls. A significantly increased incidence (10 of 24) of LCA was noted in a group of patients with lymphoreticular malignancies excluding chronic lymphocytic leukemia (CLL). No significantly increased incidence of LCA was found in patients with CLL (0 of 9), melanoma (5 of 22), or carcinoma (1 of 16) as opposed to normal controls (1 of 31). In the lymphoreticular malignancy group, no correlation was noted between LCA levels and delayed hypersensitivity skin tests, skin croton oil response, total lymphocytes, T cells, or B cells. We conclude that LCA is increased in patients with certain lymphoreticular malignancies in contrast to other types of cancer. Its presence, however, is unrelated to major defects in the cell-mediated immune system seen in some of these patients.     

Polyclonal antibodies against human cell-surface antigen CD34

An efficient and inexpensive laboratory approach for the generation and the purification of polyclonal antibodies to human antigen CD34 was developed. It was shown that cloned refolded and purified from Escherichia coli recombinant extracellular fragment of CD34 antigen retained immunogenic determinants of cell-surface expressed CD34. Immunization of mice with unglycosylated truncated recombinant protein elicit polyclonal antibodies specific for the native human antigen CD34. The antibodies generated are applicable for phenotyping of CD34+ cells using immunocytochemistry and flow cytometry assays.     

Lymphocytotoxic and microbial antibodies in Crohn's disease and matched controls

Patients with Crohn's disease and age, sex and seasonally matched healthy controls were studied for cold lymphocytotoxic and a variety of micriobial antibodies. Lymphocytotoxic antibody titers were increased significantly in the Crohn's patients compared to controls. but did not correlate with any of the microbial titers. Antibodies to a Pseudomonas-like bacterium and Bacteroides vulgatus were also increased in the patients, but titers to Chlamydia trachomatis, rotavirus and Norwalk virus were not elevated above control values. Analysis of microbial antibody within the patient and control groups revealed a significant (P=.001) correlation between antibodies to the Pseudomonas-like bacterium and Bacteroides vulgatus. The elevated titers to the Pseudomonas-like organisms and B. vulgatus are of potential importance, but their significance is known at this time. There is no evidence of an increased incidence of infection with C. trachomatis, rotavirus or Norwalk virus in Crohn's disease.     

Monoclonal antibodies ICO-20 against human thymocyte antigen T10

Monoclonal antibodies (Mab) Ig G2a isotypes reacting in indirect immunofluorescence assay with 68.7 +/- 4.1% of thymocytes, 7% of T-cells and not determining the antigen on other blood cells were obtained. Mab ICO-20 reacted in complement-dependent cytotoxic test. The antigen was expressed on colony-forming cells of granulocyte-macrophage row. Mab ICO-20 reaction with 100% of thymocytes was defined by flow cytometry. Antigen molecular mass is 45000 Dalton. The antigen was expressed on blast cells of patients with ALL and AML. Mab ICO-20 reaction was more more often with T-cell ALL.     

Monoclonal anti-idiotopic antibodies against myasthenia-inducing anti-acetylcholine receptor monoclonal antibodies. Preponderance of nonparatope-directed antibodies affecting antigen binding

To analyze components of the idiotypic network in experimental autoimmune disease, we produced 17 isogeneic anti-idiotopic monoclonal antibodies (anti-Id) against two experimental autoimmune myasthenia gravis-producing anti-acetylcholine receptor (anti-AChR) monoclonal antibodies. We studied the binding of five of the anti-Id to the anti-AChR monoclonal antibodies bearing the complementary idiotopes (Id-mAb). They bound with Kd values ranging from 0.06 to 0.86 nM, values comparable to those of Id-mAb:AChR complexes (0.26 and 0.34 nM). All of the anti-Id tested moderately inhibited the binding of AChR to Id-mAb, whereas for each anti-Id, AChR either strongly inhibited anti-Id binding or had no effect on anti-Id binding. Hence, the inhibition of Id-mAb:AChR binding by anti-Id was not reciprocal with the inhibition of anti-Id:Id-mAb binding by AChR. For each anti-Id, the relative affinities of anti-Id and AChR for Id-mAb together with the lack of symmetry of inhibition by anti-Id compared to inhibition by AChR indicate that these two "ligands" are not competitive inhibitors. Consequently, anti-Id and AChR do not bind to overlapping sites on the Id-mAb, suggesting that the observed inhibition is mediated allosterically. This may be a common mechanism of anti-Id:Id binding, which would have important implications for the mechanism of anti-Id-induced suppression.     

Opsonizing and bactericidal activity of antibodies against common antigen of Enterobacteriaceae

Domingue, Gerald J. (State University of New York at Buffalo, Buffalo, N.Y.), and Erwin Neter. Opsonizing and bactericidal activity of antibodies against common antigen of Enterobacteriaceae. J. Bacteriol. 91:129-133. 1966.-In addition to the well-known O, H, K, and Vi antigens, Enterobacteriaceae produce a common antigen, which was first identified by hemagglutination tests with Escherichia coli O14 antiserum. Studies on the biological significance of this antigen by in vitro phagocytic experiments have revealed that opsonization is markedly enhanced in the presence of the corresponding antibody, rabbit polymorphonuclear leukocytes, and normal rabbit serum. This effect is specific, because Pseudomonas aeruginosa, which is devoid of this antigen, is not opsonized under these conditions, and removal of the antibody by absorption markedly reduces the uptake of enteric bacteria containing the antigen. Opsonization thus represents another method for the study of this antigen-antibody system. Bactericidal tests have revealed that antibodies against this antigen, engendered in rabbits by different strains of enteric bacteria and various procedures, are bactericidal for E. coli O14 but not for other enteric bacteria, possibly due to previously demonstrated differences between the antigen moieties obtained from these microorganisms.     

Preparation of monoclonal antibodies against duck hepatitis B core antigen and analysis of the monoclonal antibodies

Mice were immunized against duck hepatitis B virus core (DHBc) particles isolated from the liver of asymptomatic carrier ducks of duck hepatitis B virus (DHBV) by ultracentrifugation. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 12 clones of hybridoma cells secreting antibodies against DHBc (anti-DHBc) were isolated. According to the reactivity to core particles and core peptide obtained from DHBc particles treated with SDS-2ME, the 12 antibodies were classified into two groups. Two monoclonal antibodies reacted against both core particles and core peptide (B-type), the other ten monoclonal antibodies reacted against core particles but did not react against core peptide obtained from DHBc particles treated with SDS-2 ME. (A-type). Solid phase enzyme immuno assay (EIA) using these two types of antibodies could detect core antigenisity not only in the liver homogenate but also in the DHBV infected serum. Sucrose gradient analysis and gel filtration analysis revealed this DHBc antigenisity in the serum is not carried by core particles but carried by core peptide, equivalent to HBe antigen in the serum of Hepatitis B virus (HBV) carrier. This EIA may provide sensitive test monitoring both serum DHBe antigen levels and DHBc antigen levels in the liver during DHBV infection.     

Monoclonal antibodies against hepatitis B virus surface antigen: preparation and characterization

Hybridomas secreting HBsAg antibodies were obtained by fusing murine myeloma cell line P3-X63-Ag8 to spleen cells of BALB/c mice sensitized with HBsAg. The surface antigen used for immunization of mice was prepared by purification from pooled human plasma specimens. Resulting monoclonal antibodies were detected by the SPRIA method. Clones producing highest anti-HBs titres were used to prepare mouse ascitic fluids. Monoclonal antibodies in ascitic fluid reached a titre of 10(6) to 10(7) at a protein concentration of 1 mg per ml. Two of the prepared monoclonal antibodies, HBS-01 and HBS-02, both belonging to IgG1 subclass of immunoglobulins, were selected for further study in order to assess their potential useability in the commercial ELISA kit. The pI values for HBS-01 ranged from 6.60 to 6.85, for HBS-02 from 5.6 to 6.1. In solid phase ELISA test the use of HBS-01 antibody improved accuracy of the assay by increasing its detection sensitivity for HBsAg subtypes adw and ayw in the reference serum; this sensitivity was evidently much better than that seen with the commercially available rabbit polyclonal anti-HBsAg antibody. The monoclonal antibody HBS-01 is specific to the determinant "a", which makes it suitable for use in ELISA test aimed at HBsAg detection. The antibody HBS-02 showed a markedly better reaction with HBsAg subtype adw than subtype ayw and can thus be used with advantage for their discrimination.     

Serotype-specific monoclonal antibodies against the H12 flagellar antigen of Escherichia coli

The flagellar filaments of morphotype E isolates of Escherichia coli characteristically possess an apparent helically arranged sheath structure, surrounding the central core of the filament. Re-examination of the type strains of H-serotypes belonging to morphotype E showed that all but serotype H34 possessed the expected morphology. Heterogeneity was observed in both the diameter of filaments from individual morphotype E strains and in the Mr of individual flagellins. There was no apparent correlation between these two features. Monoclonal antibodies (MAbs) of the IgM class were raised against serotype H12 flagella. In Western immunoblotting and agglutination tests, the MAbs recognized the H12 antigen of six isolates with different O:K antigen combinations. The MAbs were H-serotype-specific, with no significant reaction with the H-antigens of other morphotype E strains. The location of the serotype-specific H12 epitope(s) was studied by immunolabelling with colloidal gold markers. The epitope was surface-exposed and appeared to be helically arranged on the flagellar filament. The pattern of colloidal gold labelling was consistent with the possibility that the H12 serotype-specific epitope resides in the apparent sheath structure.     

Immunological characteristics of monoclonal antibodies against human carcinoembryonic antigen (CEA)

Four hybridomas secreting monoclonal antibodies (MAbs) of the IgG1 subclass against human carcinoembryonic antigen (CEA) were obtained from fusion of P3-NS1/1-Ag4 myeloma cells with splenic cells from mice immunized with purified CEA. None of the MAbs showed cross-reactivity to perchloric acid extractable antigens from the normal human colon by an inhibition radioimmunoassay. However, MAb C27 showed the highest affinity to CEA. The intensity of immunofluorescence staining of human colorectal cancer cells with MAb C27 correlates well to the cellular CEA content of cancer cells. LS174T showed the highest intensity of fluorescence (95%) while COLO320DM and COLO320HRS were the lowest (0.5%). None of the normal human organs - colon, lungs, liver, spleen or kidneys-showed positive staining by immunoperoxidase anti-peroxidase (PA) techniques, while tissues from colorectal carcinoma (CRC), gastric carcinoma, hepatoma and lung cancer gave a positive rate of 100% (30/30), 96.6% (28/29), 32.1% (9/28) and 82.1% (69/84) respectively. Results suggest that MAb C27 can be used in immunodetection and radiolocalization of colorectal carcinoma.     

An antigen chimera of poliovirus induces antibodies against human papillomavirus type 16.

It has been established that the surface of poliovirus type 1 can be extensively modified to incorporate antigenic domains from other poliovirus serotypes and from unrelated viruses. The fact that the modified (chimeric) viruses exhibit dual antigenicity and immunogenicity led us to explore the possibility of using the Sabin vaccine strain of poliovirus type 1 as a vector for the presentation of antigenic domains from human papillomavirus type 16 (HPV-16), a virus associated with the development of cervical carcinoma. We report here the construction and characterization of a chimeric poliovirus containing a 16-residue sequence derived from the major capsid protein (L1) of HPV-16. This virus chimera stimulated the production in rabbits of antibodies which recognized the HPV-16-derived peptide and an L1 fusion protein synthesized in Escherichia coli and detected HPV-16 in human biopsy material by immunoperoxidase staining. The possibility that poliovirus-HPV chimeras could be used as vaccines against HPV-16 is discussed.     

Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen.

Five murine hybridoma lines that produce monoclonal antibodies against Epstein-Barr virus membrane antigen (MA) were established. Immunoprecipitation experiments demonstrated that three of the antibodies precipitated both the 236,000 (236K) MA and the 212K MA. The other two antibodies precipitated the 86K MA. Antibodies against the 236K-212K MA and the 86K MA mediated complement-dependent cytolysis of Epstein-Barr-virus-infected cells. The antibodies against the 86K MA neutralized both the B95-8 and P3HR-1 viruses.     

Monoclonal antibodies against SSEA-1 antigen: binding properties and inhibition of human natural killer cell activity against target cells bearing SSEA-1 antigen

We describe the properties of three monoclonal antibodies (Mab) against stage-specific embryonic antigen-1 (SSEA-1) in terms of their binding activity to HL60, K562, OTF9, and SOTF9 tumor target cells and their functional activity in modulating human natural killer (NK) cytotoxicity assays in vitro against these target cells. Indirect binding, competition, and Western blot analyses indicate that the Mab AEC3A1-9 (3A1), ASSEA-1, and AECAB1-32 (AB1) recognize cell-defined SSEA-1 antigen with activity characteristic of the cell source (HL60 greater than OTF9 greater than K562 much greater than SOTF9). The addition of anti-SSEA-1 Mab to the NK cytotoxicity assay resulted in an inhibition of LU per 1 X 10(6) PBL that correlated closely with the expression of SSEA-1 antigen on the target cell. No significant inhibition was seen for seven other Mab. Inhibition of NK activity (greater than 30%) was observed in the presence of anti-SSEA-1 Mab for 18 of 21 and 6 of 7 human donors examined for HL60 and OTF9 target cells, respectively. The pretreatment of fixed competing cells with anti-SSEA-1 Mab reduced the efficacy of those cells to act as cold competitors in a standard NK cytotoxic assay. Taken together these data suggest that SSEA-1 determinants are important at some stage in the cytolysis produced by NK cells.     

Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8

Human herpesvirus 8 (HHV-8; also designated Kaposi's sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA-glutathione S-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.