Properties and kinetics of a population of B-lymphocytes during rat ontogenesis]

Cells bearing surface immunoglobulins (Ig+-cells) detected by the indirect immunofluorescent method and cells forming rosettes (RFC) with sheep erythrocytes coated with antibody and complement (EAC rosettes) were found in the liver and the spleen on the 15th and the 20th days of prenatal life of rats. The percentage of Ig+-cells and RFC in the liver was high and remained unchanged and at about the same level during the whole postnatal life. The spleen and the bone marrow displayed an increase of the Ig+-cells and RFC increased throughout the 1st month of postnatal life with the maximum at the 30th day after birth; a sharp decrease occurred in old animals. In the thymus the Ig+-cells and the RFC were either absent or present in very small amounts only at some periods of study. Ig+-cells with "capping" were discovered in the spleen and bone marrow on the 5th--10th days of postnatal life; their count increased considerably in 30-day and adult rats. Such cells were absent in the lymphoid tissues of old 40-month rats.     

Changes in the properties of human erythrocyte membranes in the presence of isoimmune antibodies

Light diffusion was used to study the properties of the red cell membrane of human heparinized blood under the action of different concentrations of isoimmune anti-erythrocyte anti-Rh antibodies. An increase was found in the temperature of phase transitions and density of the red cell membrane, which depended on the antibody activity. These changes were also observed in the presence of low antibody concentrations, provoking no conspicuous agglutination.     

Cytotoxic serum against B-lymphocytes obtained by immunization with bone marrow cells]

Immunization of rabbits with the bone marrow cells of intact mice permitted to obtain antisera selectively reacting with the cells of the bone marrow origin. A method of obtaining the anti-B-sera by immunization of animals directly with the bone marrow cells permitted to exclude irradiation and thymectomy of mice, this serving as necessary steps of the methods described formerly.     

Detection of antibodies to adipose tissue cell membranes in human blood]

In serum of some healthy women and patients with fibroadenomatosis of the mammary gland antibodies to the cell membranes of adipocytes were detected. Interconnections between these antibodies and corresponding antigens in blood, on the one hand, and hormonal-metabolic status of probands, on the other hand, were observed. Possible autoimmune origin of phenomenon detected and its relation to the normal and pathological processes in adipose tissue are discussed.     

Monoclonal antibodies against human beta-glucocerebrosidase

Monoclonal antibodies were obtained against the membrane-bound lysosomal enzyme beta-glucocerebrosidase (acid beta-glucosidase), which is deficient in Gaucher's disease. BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against beta-glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme. Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine-T procedure whereas a second one is not.     

Monoclonal antibodies against human chondrocytes

Summary Cell-specific antigens are mainly found in cells or membrane surfaces rather than in the surrounding matrix. However, until now it was not possible to produce antibodies specific for cellular structures of chondrocytes. In 1989, Lance (Immunol. Lett. 21:63–73; 1989) first established specific monoclonal antibodies for human articular chondrocytes tested only by immunofluorescence. Studies describing the specificity of these five antibodies (HUMC 1–5) and their relevance for immunohistological analysis of cartilage tissue were not available until now. Therefore, the aim of the following study was to investigate the distribution of HUMC 1, 2, 3, 4, and 5 in mesenchymal cellsin vivo andin vitro immunohistochemically. Further investigations concentrate on the localization of chondrocyte specific antigens using immunoelectron microscopy. Immunohistological studies showed positive immunostainings with all five antibodies in human chondrocytesin vivo andin vitro. A cross-reaction with human fibroblasts and osteoblasts for the antibodies HUMC 2 and HUMC 5 was observed. furthermore, a parallel loss of immunoreactivity for HUMC 1, HUMC 3, and HUMC 4 was observed in cultured chondrocytes indicating that the specific antigens vanish during differentiation observedin vitro. Subsequent immunoblot analysis employing collagens as antigens did not show any reactivity. Using immunoelectron microscopy, gold particle labeling was observed in intracytoplasmatic vesicles of isolated chondrocytes. Our results indicate that HUMC 1, HUMC 3, and HUMC 4 are specific for cartilage cells and might be suitable for immunohistological analysis of different cartilage tissues and pathologically altered chondrocytes.     

Monoclonal antibodies against human fibroblast interferon

A stable hybridoma clone derived by infusion of mouse myeloma cells (cell line Fo) and spleen cells of immunized mice has been isolated which secretes monoclonal antibodies against human fibroblast interferon (interferon-beta). The antibody inhibits the antiviral activity of human fibroblast interferon in an antiviral assay using human FS4 fibroblast, reacts immunologically with interferon-beta separated by sodium dodecylsulfate/polyacrylamide gel electrophoresis and subsequent transfer to nitrocellulose and absorbs interferon-beta immunologically when bound to CNBr-activated Sepharose. It also inhibits the antiviral activity of human fibroblast interferon-beta from which the sugar moiety has been cleaved off by enzymatic treatment. The antibody is therefore probably directed against the protein moiety of the interferon molecules.     

Generation of human anti-MUC3 IgG antibodies after in vitro immunization of naive peripheral blood B-lymphocytes

It has been demonstrated that IgG antibodies can be generated to self-antigen peptides as well as against viral antigens by an antigen-specific in vitro immunization system of resting human peripheral B-lymphocytes. Using a synthetic peptide from the consensus variable tandem-repeat region of the MUC3 mucin (TSSITTTGTTSHSTPSP) as the B cell epitope, we immunized blood donor B-lymphocytes in vitro and tested for MUC3-specific antibodies by ELISA. After the primary activation step all antibodies were IgM. At the end of the secondary immunization step we obtained 1.8% (21/1138) of the cultures with IgG-switched antibodies. In a competitive inhibition ELISA using the MUC1, MUC2, MUC3, MUC4 and PIP2 peptides, only one culture (F8.1) gave satisfactory specific inhibition. Using this antibody in fluorometric studies, it stained cells from two colon carcinoma cell lines predominantly in the cytoplasm, whereas those from a breast cancer cell line stained predominantly the cell surface. In a preliminary immunohistological evaluation with formalin-fixed sections, the antibody appeared to moderately stain colon sections, but not breast sections or lymph node. This method of in vitro immunization may be a useful tool in generating IgG antibodies specific to self-antigens and could find applications in tumour targeting and immunotherapy. Received: 12 October 2000 / Accepted: 11 January 2001     

Monoclonal antibodies against human placental sphingomyelinase

Monoclonal antibodies against human placental acid sphingomyelinase have been raised. The antibodies are all of the IgG1 type, and are quite specific for the enzyme. One of the antibodies has been used to demonstrate a common antigenic identity of three polypeptides (mol.wts. 90,000, 72,000, and 48,000) of a purified sphingomyelinase preparation.     

Monoclonal antibodies against a human juxtaglomerular epithelioid granular cell tumour

With the exception of the renin-angiotensin system, the molecular composition of juxtaglomerular epithelioid granular cells (JEG cells) is unknown. We demonstrate the molecular peculiarities of these modified smooth muscle cells using monoclonal antibodies produced against a benign human JEG-cell tumour. Six out of 29 different clones produced antibodies that label JEG cells nearly exclusively. Antibodies from the other clones recognize JEG cells but also granularly or homogeneously distributed antigens of proximal tubule cells. This suggests antigenic similarity between granules of JEG cells and lysosomes of proximal tubule cells. Other clones produce antibodies which tag different cytoplasmic membranes or even mast cells. The antibodies directed exclusively against JEG cells promise to be useful tools to study their physiology and pathology.     

A selective bi-site immunoenzymatic procedure for human Lp[a] lipoprotein quantification using monoclonal antibodies against apo[a] and apoB

A selective bi-site ELISA assay procedure for quantification of Lp[a] lipoprotein in human plasma based on linkage of apo[a] to apoB is described. The lipoproteins referred to as apo[a]:B were captured by a mixture of two anti-apo[a] monoclonal antibodies (K07, K09) and were revealed by a mixture of six anti-apoB monoclonal antibodies coupled to peroxidase. Since apo[a] and plasminogen have striking similarities in protein structure, the selective binding of Lp[a]:B in our assay depended upon the marked difference in affinity of the K07 and K09 mixture for Lp[a]:B (Kd = 0.32 x 10(-10) M) versus plasminogen (Kd = 0.47 x 10(-7)M). The high sensitivity (the Lp[a]:B working range 0.06-0.40 micrograms/ml) and the use of anti-apoB as antibody tracer added to the selectivity of the assay. The expression of K07 and K09 epitopes determined by competitive inhibition method and the reactivity of Lp[a]:B particles measured by bi-site ELISA were similar on individual lipoproteins, independent to their plasma levels. The assay is precise, and intra- and interassay coefficients of variation were 4.7% and 9.6%, respectively. It yields quantitative Lp[a]:B values that correlate highly with Lp[a] levels obtained by electroimmunoassay with polyclonal antibody (r = 0.73) or with Lp[a] levels measured by the other bi-site ELISA using only K07 and K09 antibodies (r = 0.96). However, upon analyzing each individual plasma with an arbitrary Lp[a]-cut off of 15 mg/dl, evidence of the qualitative aspect of the lipoprotein was obtained. The group with Lp[a] less than 15 mg/dl had higher frequency of subjects (65%) with the ratio Lp[a]/Lp[a]:B above 1.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Abstract Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae . It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Surface immunoglobulins (SmIg) in human B-lymphocytes: effect of enzymatic digestion]

In order to determine the number of circulating B lymphocytes which can synthetize their surface membrane immunoglobulins, we have evaluated by immunofluorescence their presence before and after enzymatic exposure to pronase. After pronase treatment the majority of lymphocytes carrying SmIg has been disappeared (from 7.8% to 1.9%; p < .0.0005). After 18 hours incubaton only a small proportion (4.6%) of B lymphocytes risynthetized their SmIg. This observation support the hypothesis that a subset of lymphocytes present in the peripheral blood carries on its membrane only cytophilic SmIg and therefore may interfere in the determination of the number of B lymphocytes.     

Studies on human sera for antibodies against Clostridium butyricum strain Jean H 8]

In 235 human sera, antibodies against Clostridium butyricum strain Jena H8 could be demonstrated in 33 patients (15%); only 189 sera (80%) reacted negatively, and in 5% (13 sera) a questionable reaction was obtained. The results are discussed, pointing out the necessity of preliminary titer determination if the clostridia-tumor phenomenon is to be utilized for early diagnosis of cancer.