A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both ¹²⁵I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20 % of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of ³H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton chain and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen     

The unique antigen receptor signaling phenotype of B-1 cells is influenced by locale but induced by antigen

Normal animals contain an autoreactive B lymphocyte subset, the B-1 subset, which is controlled by undefined mechanisms to prevent autoimmunity. Using a V(H)11V(kappa)9 Ig transgenic mouse, with a specificity prototypic of the subset, we have explored conditions responsible for the previously reported Ag hyporesponsiveness of these cells. We report that peritoneal V(H)11V(kappa)9 B cells exhibit typical B-1 behavior with high basal intracellular free Ca(2+) and negligible receptor-mediated calcium mobilization. However, splenic B cells from this mouse, while phenotypically similar to their peritoneal counterparts, including expression of CD5, mount robust B-2-like responses to Ag as measured by calcium influx and altered tyrosine phosphorylation responses. When these splenic cells are adoptively transferred to the peritoneal cavity and encounter their cognate self-Ag, they acquire a B-1 signaling phenotype. The ensuing hyporesponsiveness is characterized by increases in both basal intracellular calcium and resting tyrosyl phosphorylation levels and is highlighted by a marked abrogation of B cell receptor-mediated calcium mobilization. Thus, we show that self-Ag recognition in specific microenvironments such as the peritoneum, and we would propose other privileged sites, confers a unique form of anergy on activated B cells. This may explain how autoreactive B-1 cells can exist while autoimmunity is avoided.     

Chemical degradation of liposomes by serum components detected by NMR

Interaction between serum components and liposomes is an oxygen-dependent exothermic process. We studied the interaction of 100 nm extruded liposomes (bearing positive, negative or no charge) with foetal calf serum by 1H NMR and 13C NMR, in order to further our understanding of these reactions. Studies of aqueous or organic extracts obtained after 2 h, 1 day or 1 week, showed hydrolysis to be a degradation process concomitant with the interaction with serum. Oxidation was identified as additional to hydrolysis in the process of degradation. Oxidation produced aldehydes, acids and alcohols, although aldehydes and alcohols were prone to further decomposition and only appeared transiently. Alkenes and other oxidized compounds predominated in those products derived from oxidation. In stearylamine-containing liposomes some aldehydes and a nitroderivative were found as degradation products. Such metabolites are apolar and their presence might explain the intrinsic toxicity of this kind of liposome in cell cultures. The work described in the present study revealed the chemical degradation of liposomes in the serum used. In all cases the results obtained were compared with liposomes not incubated with serum.     

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both 125I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20% of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of 3H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen.     

Serologically detected lymphocyte antigens in Holstein cattle

Lymphocytes from 103 Holstein cattle were tested with 11 antilymphocytotoxic sera. Four of these sera were produced by whole blood immunization; these generally yielded lymphocytotoxicity against a large number of animals in each test panel. The majority of the sera were collected from parous cows which had never been immunized. Observations about this latter group of sera are (1) lymphocyte antibodies, may occur by alloimmunization in foetal-maternal interaction, (2) normal sera from non-immunized parous cows generally had a narrower specificity of antibodies than from parous cows immunized with blood from unrelated animals and in some cases these antisera may be monospecific, and (3) these sera from parous cows are easy to obtain without the need for laborious immunizations. Analysis of these lymphocytotoxic sera indicate several reagents were obtained which possessed different antibodies; these were useful in detecting polymorphism of cattle lymphocyte antigens. Using these 11 sera, an analysis of the reactions patterns among 103 Holsteins, including parent-offspring data resulted in the postulation of several alleles. These data suggest that cattle lymphocyte antigens are very polymorphic and inherited.     

Tumor-associated neural differentiation antigens detected on C 1300 neuroblastoma cells by hybridoma monoclonal autoantibodies

Immune isoantisera and hybridoma monoclonal autoantibodies against syngeneic C1300 neuroblastoma (NB) cells were produced from BALB/c mice. Isoantisera were obtained (i) from mice immunized with membrane preparations from cloned NB cells and (ii) from mice bearing NB tumors. After repetitive absorptions on several different syngeneic or allogeneic tumor cell lines and syngeneic normal kidney, liver, spleen, bone marrow, and brain mouse tissue powders, these sera still retained antibodies reacting with tissue-differentiation antigens present on both NB cells and normal nerve sympathetic cells on cryostat whole body sections of neonatal mice. Monoclonal autoantibodies against NB cells were the products of the fusion between plasmacytoma cells and spleen cells from mice bearing syngeneic NB tumors. These anti-NB monoclonal antibodies revealed a restricted spectrum of distinct alloantigenic specificities against syngeneic bone marrow, fetal and adult brain cells, and nerve sympathetic cells present on neonatal rather than adult mice. A mixture of four monoclonal antibodies, recognizing, respectively, an epitope of the Ia complex and three distinctive neuronal-restricted antigens, proved to be a powerful and specific probe for histological immunodiagnosis of neuroblastoma, on cryostat sections of NB tumors, metastases, and tumor-draining lymph nodes.     

Salivary gland amylase polymorphism in pigs and cattle detected by affinity electrophoresis

New allelic variants of salivary gland alpha-amylase in pigs (AMY-1C, AMY-1D) have been detected using affinity electrophoresis. In yak, zebu and in hybrids (yak x cattle, zebu x cattle) a new AMY-1 allelic variant (AMY-1D) has been found using the same method.     

Circulating melanoma-associated antigen detected by monoclonal antibody NKI/C-3

Summary NKI/C-3 and NKI/black-13 are monoclonal antibodies recognizing different epitopes on a melanoma-associated antigen that is preserved after fixation in formalin and embedding in paraffin in virtually all melanoma tissues. The antigen, a predominantly cytoplasmic vesicle membrane-bound heterogeneous glycoprotein of 25–110×10³ daltons, was shown to be a single 25×10³ dalton polypeptide when incorporation of N-linked carbohydrates was inhibited by tunicamycin. The antigen was measured in a double determinant enzyme immunoassay (DDEIA) using NKI/C-3 as catcher antibody. Results from in vitro experiments indicated that the antigen is actively shed from living cells. In sera from melanoma patients with a small tumor burden, the antigen concentrations were in the range of those of controls (0–22 U/ml). Significantly increased values (33–600 U/ml) were found in sera from patients with a moderate or large tumor burden. The antigen concentrations in sera from patients with multiple metastases of other tumors were within the range of controls. Several sera from patients with multiple metastases of colon, pancreatic, and stomach carcinoma, however, contained increased antigen concentrations (45–80 U/ml). These results correspond with the reactions of NKI/C-3 in tissue sections of some malignancies other than melanoma.During the follow-up of melanoma patients the concentrations of circulating antigen correlated with tumor progression. The predictive value of the NKI/C-3 assay was no better than determination of serum lactate dehydrogenase, alkaline phosphatase or gamma glutamyl transferase activity.     

Serologically detected lymphocyte antigens in Holstein cattle1

Lymphocytes from 103 Holstein cattle were tested with 11 antilymphocytotoxic sera. Four of these sera were produced by whole blood immunization; these generally yielded lymphocytotoxicity against a large number of animals in each test panel. The majority of the sera were collected from parous cows which had never been immunized. Observations about this latter group of sera are (1) lymphocyte antibodies may occur by alloimmunization in foetal-maternal interaction, (2) normal sera from non-immunized parous cows generally had a narrower specificity of antibodies than from parous cows immunized with blood from unrelated animals and in some cases these antisera may be monospecific, and (3) these sera from parous cows are easy to obtain without the need for laborious immunizations. Analysis of these lymphocytotoxic sera indicate several reagents were obtained which possessed different antibodies; these were useful in detecting polymorphism of cattle lymphocyte antigens. Using these 11 sera, an analysis of the reactions patterns among 103 Holsteins, including parent-offspring data resulted in the postulation of several alleles. These data suggest that cattle lymphocyte antigens are very polymorphic and inherited.     

Lymphocytotoxic antibodies against cattle J antigen

The J substance is unique compared to all other known red cell antigenic characters of cattle blood because it is detected by naturally occurring antibodies and occurs dissolved in body fluids. Also it could be demonstrated by absorption techniques on the lymphocytes and platelets of the J-positive animals (Eyquem et al., 1956). The presence of the J antigen on these tissues raises the question whether cytotoxic antibodies exist against it. This question has been studied and the first results are presented.     

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.     

Deficiency of neutrophil membrane antigen detected by monoclonal antibody in rheumatoid arthritis

Monoclonal antibodies (mAbs) against cell surface antigens and receptors are instrumental in defining specific membrane markers. mAbs GF 26.7.3 and MF 25.1 against human neutrophils modulated the activation mechanism of superoxide anion production induced by formyl-peptide and PMA in all subject. However, treatment with mAb MF 25.1 of neutrophils from patients with rheumatoid arthritis did not have any effect. This may suggest that the antigen which MF 25.1 binds is absent in rheumatoid conditions. This confirms our previous data showing that defective expression of membrane components is associated with neutrophil dysfunction.     

Antigenic determinants of blood serum proteins detected in the structure of hepatitis B surface antigen by the method of affinity chromatography]

Tropism of the antigen of hepatitis B to the antibodies against normal serum proteins was revealed by the method of affine chromatography; this pointed to the possibility of the presence in the HBs-antigen structure of some antigenic determinants of the serum proteins or to the association of serum proteins with the HBs-antigen. A change of the isoelectric spectrum of the HBs-antigen and its tropism in affine chromatography to the antibodies against serum proteins possibly depended both on the nonhomogeneity of the antigenic determinants included into the composition of the antigen, and on the presence of the HBs-antigen--HBs-antibody or HBs-antigen--serum proteins complexes.     

Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.     

The volatilization of ammonia from cattle urine applied to soils as influenced by soil properties

The amounts of ammonia volatilized, following the application of cattle urine to 22 soils, were measured in the laboratory during an incubation period of 10 days. The urine contained 12.0 g N dm^-3 and was applied to small columns of soil at a rate equivalent to 26.5 g N m^-2. The soils were from fields of both grassland and arable cultivation and varied widely in properties. Ammonia volatilization ranged from 6.8 to 41.3% of the total urinary N, with a mean value of 26.4%. The soil property most closely related to the extent of volatilization was cation exchange capacity (CEC), and this was so whether all 22 soils were considered together or whether the 14 grassland and 8 arable soils were considered separately. In general, the higher the CEC the less the amount of ammonia volatilized. However, for a given value of CEC, volatilization tended to be greater from a grassland than from an arable soil. The pH of a soil/urine mixture measured after 24 hours was also quite closely correlated with the amount of ammonia volatilized, but the initial pH and titratable acidity of the soil were poorly correlated with ammonia volatilization. ei]H Marschner ei]H Lambers