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At least 73 records · Page 4Linked to original sources
PubMed2001

Detection of 'antiphospholipid' antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding beta(2)-glycoprotein I and prothrombin.

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and beta(2)GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta(2)GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0.05) and at lower antibody concentrations (12.5 microg/ml versus 100 microg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to beta(2)GPI (r = 0.90; P < 0.001) but not to CL (r = 0.06; P = 0.76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-beta(2)GPI or antiprothrombin antibodies. All MoAbs binding beta(2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta(2)GPI had more LA effect.

Animals↗
PubMed2004

Antibodies to guanosine triphosphate misidentified as anti-double-stranded DNA antibodies in a patient with antinuclear antibody-negative lupus, due to buckling of insolubilized assay DNA.

OBJECTIVE: To investigate why the serum of a pediatric patient with systemic lupus erythematosus was persistently (>30 months) and strongly positive for antibodies to double-stranded DNA (dsDNA) as revealed by enzyme-linked immunosorbent assay (ELISA), but yielded negative results on the antinuclear antibody test (HEp-2 immunofluorescence [IF]). METHODS: The patient's antibodies were isolated on dsDNA and single-stranded DNA (ssDNA) supports, which were then examined by dsDNA ELISA and HEp-2 IF. Tests included the use of various inhibitors to determine the fine specificity of the antibodies. Other tests performed included immunoblotting, immunoprecipitation, Crithidia luciliae IF, and neutrophil IF. RESULTS: The antibodies isolated from the dsDNA and ssDNA supports were similar, in that they were of the IgG type, bound well in the dsDNA ELISA, and recognized a normally hidden nucleolar RNA antigen in HEp-2 cells. With both the dsDNA ELISA and nucleolar antigens, inhibition studies revealed that the epitope recognized was guanosine 5'-triphosphate (GTP). Binding of the antibodies was better to GTP than to guanosine 5'-monophosphate or cytidylyl (3'-5') guanosine, and, in turn, was better than to guanosine, while N7-methylated GTP was unreactive. The antibodies did not bind to dsDNA present in solution or in HEp-2 or Crithidia cells, but bound transfer RNA well and recognized a cytoplasmic RNA antigen in neutrophils. CONCLUSION: A new problem in dsDNA ELISA is revealed in the occurrence of a hitherto-unknown and unusual buckling of the insolubilized DNA molecule, which, absent in dsDNA found in solution or in whole cells, presumably creates gaps of single-strandedness in the molecule. A new antibody specific for GTP is described in this patient, which may be clinically important.

Antibodies, Antinuclear↗
PubMed1994

Isolation of tumor cell-specific single-chain Fv from immunized mice using phage-antibody libraries and the re-construction of whole antibodies from these antibody fragments.

Enhanced expression of epidermal growth factor receptor (EGFR) occurs on a variety of malignant tissues thus making anti-EGFR antibodies possible agents for the diagnosis and therapy of human tumors. Standard hybridoma technology has been used successfully to isolate anti-EGFR antibodies from immunized mice and rats. This report demonstrates that phage-antibody libraries are an alternative, and more versatile, method for isolating antibodies from immunized mice. Anti-EGFR antibodies were isolated from phage-antibody libraries constructed not only from the spleen of an immunized mouse but also from the draining lymph node of an immunized mouse and from in vitro immunized mouse cells. Two of the single-chain Fv isolated from the phage-antibody libraries were engineered to create partially humanized whole antibody molecules.

Amino Acid Sequence↗
PubMed1996

Antiphospholipid antibody panels and recurrent pregnancy loss: prevalence of anticardiolipin antibodies compared with other antiphospholipid antibodies.

OBJECTIVE: To describe the prevalence of antiphospholipid antibodies in addition to cardiolipin in women with recurrent pregnancy loss. DESIGN: Retrospective data analysis of test results from an antiphospholipid antibody panel. SETTING: A university-based private patient referral center. PATIENTS: Included 866 women with a history of recurrent pregnancy loss and 288 parous women without a history of reproductive problems. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Enzyme-linked immunosorbent assay, with referenced standards and known positive and negative sera on each plate, was used to measure anticardiolipin, antiphosphatidyl inositol, antiphosphatidylglycerol, antiphosphatidylserine, and antiphosphatidylethanolamine. Statistical analyses used the two-tailed Fisher's exact test. RESULTS: Positive anticardiolipin antibodies were detected in 17.3% of patients with recurrent pregnancy loss compared with only 4% in the control population. Eighty-seven of the 866 women (10.1%) were negative for anticardiolipin antibodies but had positive levels of another antiphospholipid antibody. Isolated positive antibody levels occurred most frequently in the immunoglobulin (Ig) G class of phosphatidylinositol, cardiolipin, and phosphatidylethanolamine. Isolated IgA was only found in phosphatidylethanolamine. CONCLUSION: In women with recurrent pregnancy loss, 150 of 866 (17.3%) had positive anticardiolipin antibodies. Additionally, 87 of 866 (10.1%) women were positive for another antiphospholipid antibody. Patient demographics were similar in both groups. We emphasize the importance of careful standardization, quality control, and interpretation of positive results.

Abortion, Habitual↗
PubMed2000

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 1. Development and characterization of monoclonal antibodies and molecular modeling studies of antibody recognition.

Sulfonamide antibiotics are used to treat a variety of bacterial and protozoan infections in cattle, swine, and poultry. Current residue methods for the analysis of sulfonamides in animal-based food products include bioassays, chromatographic methods (HPLC, GLC), and immunoassays. Most immunoassays have employed highly specific polyclonal antibodies. In this paper, we describe the isolation of monoclonal antibodies against sulfadimethoxine (SDM) that vary in their sensitivities and cross-reactivities against a large number of sulfonamides. The most sensitive monoclonal antibody, designated SDM-18, exhibits an IC(50) value for SDM of 1.53 ppb. Another monoclonal antibody, designated SDM-44, exhibits IC(50) values for six sulfonamides well below the established threshold level of 100 ppb for animal tissues. Molecular modeling studies of the cross-reactive drugs suggest that, depending on the monoclonal antibody, both steric and electronic features govern antibody binding. Due to the diversity of these monoclonal antibodies, it should be possible to design both compound- and class-specific monoclonal antibody-based immunoassays.

Animals↗
PubMed1969

Regulation of antibody formation by serum antibody. I. Removal of specific antibody by means of immunoadsorption.

Rabbits were injected intravenously with bovine serum albumin (BSA) and bacteriophage T(2) (T(2)). 2-3 wk later, anti-BSA was removed from such animals by a procedure which involved exposure of removed plasma to an immunoadsorbent ((125)I-BSA bound to bromoacetyl cellulose) and return of the adsorbed plasma to the animal. This resulted in removal of the majority of antibody activity to BSA without affecting antibody levels to T(2). 1-2 days later, anti-BSA levels began to rise, and reached peak levels usually 5 days after the removal of antibody. Antibody levels to T(2) did not change. No evidence was obtained that BSA was released from the immunoadsorbent into the circulation of the rabbits. Thus, only trace amounts of radioactivity were released into the plasma; most of the radioactivity was equally coprecipitable with BSA or human gamma globulin and their specific antibodies; the released material was not demonstrated to be immunogenic in primed rabbits; and the released material did not elute with BSA on gel filtration. The results are interpreted as evidence that serum antibody acts as a regulatory mechanism for antibody formation during the conventional antibody response to a metabolizable antigen.

Animals↗
PubMed1989

Murine monoclonal IgG3 antibodies to human colorectal tumor-associated antigens: production and characterization of antibodies active in both antibody-dependent cellular cytotoxicity and complement-mediated cytolysis.

Murine monoclonal antibodies have been developed against human colorectal tumors using immunogens consisting of extracts from xenografted human colon carcinoma bound to lectin-conjugated agarose beads. Antibodies of the IgG3 isotype were selected from the resulting fusions, since we and other investigators have reported that this class of antibody can mediate antibody-dependent cellular cytotoxicity (ADCC). They were characterized with respect to specificity, ADCC activity, and complement-mediated cytolytic activity. One antibody, NR-Co-01, was produced using wheat germ agglutinin-agarose, whereas NR-Co-03, NR-Co-04, and NR-Co-05 were independently derived after immunization with a Dolichos biflorus lectin-agarose carrier. All four antibodies reacted intensely with the cytoplasm and cell surface of colonic adenocarcinomas and showed a restricted normal tissue reactivity. Recognition of epithelial cells in some normal adult tissues was revealed by immunoperoxidase staining of frozen tissues. No binding was found to normal lymphocytes, monocytes, granulocytes, and erythrocytes by immunofluorescence flow cytometry. All four of the antibodies were reactive with neutral glycolipids partitioned by Folch extraction of human colon carcinoma cell lines and also with a glycoprotein extracted from ovarian cyst mucin, suggesting that they may recognize one or more carbohydrate structures. The NR-Co-01, NR-Co-03, NR-Co-04, and NR-Co-05 are potent inducers of ADCC with human peripheral blood mononuclear effector cells and of complement-mediated cytolysis with human complement. However, they exhibited no ADCC activity with nude or normal mouse peripheral blood mononuclear cells at target:effector ratios ranging from 40:1 to 160:1. Their tumor reactivity, restricted normal tissue distribution, and ability to direct ADCC and complement-mediated cytolysis make these antibodies promising candidates for cancer therapy.

Animals↗
PubMed1997

Influence of cardiolipin antibodies on the binding of treponemal specific antibodies in the fluorescence treponemal antibody absorption test and the Treponema pallidum immobilisation test.

The aim of the present study was to investigate the biological role of cardiolipin antibodies during Treponema pallidum infection. Inhibition of the binding of treponemal specific antibodies at the early and late stages of infection by cardiolipin antibodies was shown in the fluorescence treponemal antibody absorption (FTA-ABS) test and T. pallidum immobilisation (TPI) test. Incubation of treponemes with cardiolipin antibodies followed by a second incubation with treponemal specific antibodies resulted in a reduction of the titres of the FTA-ABS test and the TPI test. The findings suggest that cardiolipin antibody production should be considered as a virulence mechanism of pathogenic treponemes with the purpose of evading the host defence mechanisms.

Animals↗
PubMed1983

Method for detection of antibody against low molecular weight antigen (hapten)--production and detection of antibody against histamine H2 receptor of antibody against histamine H2 receptor antagonist "Famotidine".

Antibodies against histamine H2-receptor antagonist "Famotidine (FAMO)", molecular weight 337, chemical name; N-sulfamoyl-3-(2-guanidinothiazol-4-ylmethylthio) propionamide, were produced by subcutaneously injecting rabbits with an albumin and FAMO conjugate covalently bound with 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide (ECDI). Two new detection systems for antibody titration were developed and employed. In one method, the antigen FAMO was tagged to sheep red blood cells (SRBC) and analysed qualitatively by a fluorescence activated cell sorter (FACS) using a second fluorescence isothiocyanate (FITC) labeled antibody. In the other method, CH-Sepharose beads were employed in place of SRBC and Horse Radish Peroxidase (HRP) was labeled to the second antibody instead of FITC used in the former method. HRP of the immune complex was colorimetrically measured with DAB-H2O2 to analyse the fine antibody titer. These high sensitive detection methods revealed the existence of IgG type of FAMO antibody. The detection sensitive detection methods revealed the existence of IgG type of FAMO antibody. The detection sensitivity was approximately 50 to 100 times higher in the later method with HRP than in the former. Furthermore these two methods could be deemed to be a good model system for a receptor assay.

Animals↗
PubMed1984

Monoclonal antibody-toxin conjugates reactive against human T lymphocytes. A comparison of antibody linked to intact ricin toxin with antibody linked to ricin A chain.

A non-complement-binding monoclonal antibody, TA-1, recognizing determinants on human T lymphocytes, was linked to the plant seed toxin ricin, either the intact molecule or purified ricin A chain. Peripheral blood lymphocytes were pretreated with conjugate for 2 hr, washed, and then measured in vitro for T cell proliferation. Studies showed that antibody-intact ricin conjugates were up to 39-fold more inhibitory than antibody-A-chain conjugates. Killing was selective because an unreactive control antibody linked to toxin had minimal inhibitory effect. Dose response curves obtained in human studies were nearly identical to curves obtained in an animal model n which a monoclonal anti-murine T cell antibody (anti-Thy 1.1) was linked to ricin and ricin A chain. In the human system, longer exposures of peripheral blood cells to conjugates did not alter our findings. TA-1-ricin conjugates were tested against human ALL cell lines. KOPN-1, a cell line bearing the determinant reactive with TA-1 was selectively eliminated within 2 days after pretreatment with 500 ng/ml. Even 10-fold greater concentrations of TA-1-A chain were not adequate for leukemic cell destruction. These findings (1) show for the first time in a human model that monoclonal antibodies, directed against certain differentiation antigens when linked to ricin A chain are not as effective in normal or malignant cell killing as when linked to intact ricin; (2) contribute to the growing body of evidence showing that monoclonal antibody A chain conjugates do not permit the acquisition of levels of toxin sufficient to destroy target cells; and (3) are important relative to increasing interest in use of antibody-toxin conjugates for graft-versus-host disease prophylaxis in allogeneic bone marrow transplantation.

Antibodies, Monoclonal↗
PubMedDate not supplied

Antisperm antibodies (ASAs) in infertile males: subclass distribution of IgA antibodies and the effect of an IgA1 protease on sperm-bound antibodies.

PROBLEM: (1) To determine the IgA subclass distribution of antibodies in the serum and on the sperm of infertile male patients. (2) To determine the effect of an IgA1 protease on the binding of IgA antisperm antibodies (ASA). METHOD: Fifteen infertile males with ASA in serum (10) or on sperm (5) were recruited for this study. Duration of infertility was at least one year. Monoclonal antibodies to human IgA1 and IgA2 were conjugated to immunobeads. The distribution of IgA1 and IgA2 subclass ASA was determined for positive serum and sperm samples. The effects of an IgA1 protease (isolated from Neisseria meningitidis strain HF13) on sperm-bound antibodies was evaluated by immunobead binding. RESULTS: In serum, the IgA1 subclass ASA was predominate (91%) when compared with IgA2 (9%) subclass. Direct sperm-bound antibodies displayed a distribution more characteristic of the secretory immune system with IgA1 accounting for 63% and IgA2 accounting for 37% of the total IgA ASA. Enzyme treatment dramatically reduced the amount of serum IgA antibodies bound to sperm (P < 0.05). Similarly, a significant reduction in direct sperm-bound antibodies was observed after enzymatic treatment with no loss in sperm motility. CONCLUSIONS: (1) Although IgA1 direct sperm-bound antibodies were dominant, when compared with serum there was a higher proportion of IgA2 subclass, which suggests a local production of IgA. (2) Specific IgA1 protease treatment is capable of reducing the amount of immunobead-detectable IgA on sperm. Hamster oocyte sperm-penetration assays are ongoing to determine if this treatment might improve sperm penetration rates.

Antibodies, Monoclonal↗
PubMedDate not supplied

Binding of antibodies onto the thylakoid membrane. I. Maximal antibody binding and adsorption of antibodies to lipids.

The binding of antibodies onto the lamellar system of Antirrhinum majus was determined in dependence on the serum addition. The unspecific adsorption of serum proteins was taken into account or eliminated. The binding of antibodies as a function of the amount of serum added is seem from a saturation curve. From an antiserum obtained by hyperimmunization with stroma-freed chloroplasts, the chloroplasts bind maximally 1 gram antibodies per gram stroma-freed chloroplasts. From an antiserum to the proteins of the thylakoid membrane prepared in the same way an equal amount of antibodies is adsorbed. It is assumed that with this amount the surfaces of the lamellar system accessible to antibodies is completely covered by antibodies. For an antiserum to monogalactosyl diglyceride a maximal antibody binding of 0.16 g, for sulphoquinovosyl diglyceride 0.12 g and for phosphatidyl glycerol 0.13 of antibodies per gram stroma-freed chloroplasts are obtained. The significance of these results with respect to the molecular surface structure of the thylakoid membrane is discussed.

Agglutination↗
PubMedDate not supplied

Relationship of common avian pathogen antibody titers in so-called chicken anemia agent (CAA)-antibody-positive chicks to titers in CAA-antibody-negative chicks.

Antibody titers for infectious bursal disease virus (IBDV), infectious bronchitis virus, Newcastle disease virus, and reovirus from chicks with chicken anemia agent (CAA) antibodies were compared with antibody titers from their CAA-antibody-negative counterparts. These comparisons were made in 396 chickens that were 1 day, 2 weeks, 8-9 weeks, 10 weeks, 17 weeks, or 29-32 weeks old. Only one serum sample was collected from any given chick or chicken. There were no significant differences between the antibody titers at any age for any antigen, with one exception: at 29-32 weeks, the IBDV titers were higher (t = 2.62, df = 142, P less than 0.01) in chickens with CAA antibody. Although not at all likely, we believe that the observation of high IBDV antibody titers in CAA-antibody-positive chicks could have been a spurious one.

Anemia↗
PubMed1995

Granulocyte/macrophage-colony-stimulating factor augments the induction of antibodies, especially anti-idiotypic antibodies, to therapeutic monoclonal antibodies.

A group of 86 patients with advanced colorectal carcinoma were treated with the mouse (m) (IgG2A) or chimeric (c) monoclonal antibody (mAb) 17-1A. Prior to therapy, no patient had detectable levels of antibodies to mAb17-1A. All mmAb17-1A-treated patients (n = 76) developed antibodies against both idiotypic and isotypic determinants. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of anti-idiotypic (ab2) as well as anti-isotypic antibodies. Of the mmAb17-1A-treated patients, 16 developed type I allergic reactions. These patients had significantly higher concentrations of anti-(mouse Ig) antibodies than patients without type I reactions. Of these 16 patients, 5 had received mmAb17-1A alone; they constituted 9% of this group (5/56). The remaining 11 patients had been given mmAb17-1A together with GM-CSF, and represented 55% of this treatment group (11/20). The difference was statistically significant (P < 0.001). Of 10 patients, 9 (90%) treated with cmAb17-1A and GM-CSF developed ab2. The ab2 concentration in this patient group was significantly lower compared to those treated with mmAb-17A. Anti-(mouse Ig) antibodies caused clinical symptoms requiring therapeutic intervention in fewer than 10% of the patients treated with mmAb17-1A alone. With the addition of GM-CSF, the antibody concentration as well as the frequency of allergic side-effects calling for medical action increased significantly. Significantly more patients with a high ab2 concentration (at least 15 micrograms/ml) 1 month after completion of mAb therapy responded to mAb treatment as compared to those with a low ab2 concentration (P < 0.05). Moreover, patients with a high ab2 concentration (at least 15 micrograms/ml) had a median survival time of 15 months while those with a lower concentration survived for a median time of 9 months (P = 0.01).

Adolescent↗
PubMed1985

A reevaluation of rabbit anti-allotype antibody for the presence of cross-reactive idiotypes. I. A species-specific idiotype on rabbit anti-a1 antibody is recognized by guinea pig anti-IdX antibody.

Rabbit reagents previously thought to display specificity for a cross-reactive idiotype on anti-VHa allotype antibody from all tested rabbits have recently been shown to be contaminated with an induced (latent) molecule similar or identical to the original antigen (rabbit a1 or a2 allotype). In an attempt to circumvent this problem, we have immunized guinea pigs with rabbit anti-a1 allotype antibody to produce heterologous anti-idiotype antibody. The resulting guinea pig antibody (GP anti-R IdX) recognizes anti-a1 antibody from each of 17 immunized rabbits, and in four tested samples reacts with 22 to 100% of the molecules. Neither goat nor guinea pig anti-a1 reacts with the guinea pig anti-R IdX antibody, even though the goat, guinea pig, and rabbit anti-a1 Ab all recognize a similar set of a1 determinants. The reaction between IdX-bearing rabbit anti-a1 and guinea pig anti-R IdX is inhibited by the original antigen (a1 IgG), demonstrating that the IdX is at or near the antigen combining site of anti-a1 antibody. Immunoelectron microscopy of immune complexes supports this conclusion and demonstrates that the reactive site on the GP anti-R IdX is at or near its antigen combining site.

Animals↗
PubMed1988

Studies on murine IgE with monoclonal antibodies. I. Characterization of rat monoclonal anti-IgE antibodies and the use of these antibodies for determinations of serum IgE levels and for anaphylactic reactions.

Rat monoclonal antibodies were constructed by fusion of immunized rat spleen cells with a nonsecreting mouse myeloma cell. Two monoclonal antibodies (6HD5 and HMK-12) were selected for further study. Both reacted with various IgE molecules of different specificities and different allotypes, but did not react with immunoglobulins of other isotypes and with light chains. These antibodies were therefore anti-isotypic (IgE) and not anti-allotypic or anti-idiotypic. It was shown by competition studies that these antibodies recognize different epitopes on the FcR epsilon fragment. A sensitive ELISA for the quantitation of murine IgE was developed with these monoclonal antibodies; the sensitivity was between 2 and 250 ng/ml for detection of serum IgE levels. Good correlation was obtained with protein amounts as determined by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA) activities. Both monoclonal antibodies were used to study anaphylactic reactions elicited by IgE antibodies. Both could inhibit PCA reactions and both could elicit reverse PCA reactions.

Animals↗
PubMed1975

Allotypic suppression in rabbits: competition for target cell receptors between isologous and heterologous antibody and between native antibody and antibody fragments.

Neonatal injection with various foreign proteins (normal goat serum, human Cohn fraction II, human albumin) caused a stimulation of immunoglobulin synthesis. This effect was not antigen-specific and did not constitute a conventional antibody response directed against the injected substance. When this stimulatory effect of foreign protein was minimized, the heterologous (goat) anti-rabbit allotype antibody and rabbit antibody F(ab') 2 fragments not only failed to induce suppression but also competed with the suppression-inducing native rabbit antibody. Allotypic suppression in a rabbit can thus only be induced by an antibody molecule possessing an intact Fc portion of isologous, rabbit origin. Antibody to Ae14, an allotypic specificity located on the Fc portion, failed to induce suppression or stimulation of immunoglobulin synthesis. This was attributed to the position of Ae14 in the cell membrane which reduced its accessibility to antibody.

Albumins↗