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IgA, Goat anti_Mouse; ELISA

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[#YSRTSTAR137] IgA, Goat anti_Mouse; ELISA


YSRTSTAR137 | IgA, Goat anti_Mouse; ELISA, 0.5 mg.
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(1) Evaluation of a microsphere-based immunofluorescence assay for the determination of Immunoglobulin A concentrations in cerebrospinal fluid of dogs.[TOP]

Pubmed ID :22858001
Publication Date : //
The simultaneous increase of immunoglobulin A (IgA) in serum and cerebrospinal fluid (CSF) is a characteristic finding in dogs suffering from canine steroid-responsive meningitis-arteritis (SRMA). The study aimed at developing and evaluating a microsphere-based immunofluorescence assay (MIA) for the measurement of IgA, trying to fulfill the need of a quicker method using only small volumes of CSF. Microsphere beads were coated with goat-anti-dog IgA antibodies and bound IgA was detected by a mouse-anti-dog IgA antibody in combination with a PE-labeled goat-anti-mouse IgG. CSF from 44 dogs were tested for IgA and compared with an in-house utilized ELISA. Using clinical relevant reference ranges, the new method showed a good agreement (84.17%) with the ELISA. A method comparison revealed a moderate agreement only. These findings indicate that the MIA will not replace the ELISA, but it opens the possibility for further research with microsphere-based assays.

Authors : Roerig A, Carlson R, Tipold A,

(2) Frequent false positive beta human chorionic gonadotropin tests in immunoglobulin A deficiency.[TOP]

Pubmed ID :15996198
Publication Date : //
A patient with IgA deficiency had a series of positive serum pregnancy tests which led to medical and surgical procedures for suspected molar pregnancy. These tests were found to be falsely positive due to heterophile antibody. The aim of this study was to determine the frequency of false positive betahCG assays in sera of IgA deficient patients. Sera from a panel of IgA deficient (IgA < 7 mg/dl) patients were tested for the presence of betaHCG using three different assays, and also for IgG anti-goat and anti-mouse antibodies. Patients were seen at Mount Sinai Medical Center and included 54 patients (ages 1-80 years, 32 females, 22 males) with IgA deficiency. Thirty percent of 54 IgA deficient patient sera yielded positive pregnancy tests by one or more of the three betahCG assays, however, none of the patients were pregnant. In comparison to sera of normal controls, 39% of the patient sera contained significant amounts of anti-goat antibody and 18% contained significant amounts of anti-mouse antibody. While heterophile antibodies are common in IgA deficient serum, false positive assays for betahCG in IgA deficient serum have not been previously reported. The possibility of false positive test results should be considered prior to invasive procedures in IgA deficient patients.

Authors : Knight A K, Bingemann T, Cole L, Cunningham-Rundles C,

(3) Development of a time-resolved immunofluorometric assay for quantitation of mucosal and systemic antibody responses.[TOP]

Pubmed ID :11687241
Publication Date : //
We developed a solid phase immunoassay that measured mucosal and systemic antibody responses from mice inoculated with either a staphylococcal enterotoxin B vaccine (SEBv) or noninfectious virus-like particles (VLP) of lentiviral origin. The assay used time-resolved fluorescence (TRF) with affinity-purified goat anti-mouse IgA and IgG conjugated to samarium and europium chelates, respectively. By employing these fluorogenic conjugates with different spectral emissions, IgA and IgG specific for SEB or VLP were readily detected in serum and saliva from mice inoculated intranasally. The TRF assay detected antigen-specific IgA in saliva 10 min after the addition of enhancement solution, while a conventional alkaline phosphatase-based assay for salivary IgA required 18 h after substrate addition. The TRF assay also provided a significantly higher signal-to-noise ratio and exhibited greater sensitivity. TRF assays detected both IgA and IgG in the same well, thereby reducing sample and reagent requirements.

Authors : Hale M L, Campbell T A, Campbell Y G, Fong S E, Stiles B G,

(4) Human anti-animal antibody interferences in immunological assays.[TOP]

Pubmed ID :10388468
Publication Date : //
The scope and significance of human anti-animal antibody interference in immunological assays is reviewed with an emphasis on human anti-animal immunoglobulins, particularly human anti-mouse antibodies (HAMAs).

Authors : Kricka L J,

(5) Early induction and augmentation of parasitic antigen-specific antibody-producing B lymphocytes in the non-Peyer's patch region of the small intestine.[TOP]

Pubmed ID :9792905
Publication Date : //
In this study, B lymphocytes from the small intestine of immunized rats were examined for their expression of specific antibodies against Trichinella spiralis (TS) antigen. The isotypes of the antigen-specific antibodies on B cells were examined via immunofluorescence microscopy. Monoclonal mouse anti-rat IgE, IgG1, IgG2a, IgG2b, IgG2c, IgA and IgM primary antibodies in conjunction with FITC-conjugated goat anti-mouse Ig secondary antibody and XRITC-conjugated 9D4 T. spiralis antigen were used to study the dynamics of the appearance of activated B lymphocytes in the small intestine, Peyer's patch, both the germinal center (PP-GC) and the non-germinal center (PP-NGC), the mesenteric lymph node (MLN), and the spleen. The results demonstrate that activated B cells are elicited by TS in the non-Peyer's patch region of the small intestine to express all isotypes of antibodies against TS antigen. IgG- and IgE-producing cells (Ab-PC) began proliferation only 1 and 2 days after infection, respectively. The strongest response was mounted by the IgE-PC in the lamina propria of the intestine. The response by IgA-PC generated was not only significantly delayed and also much weaker than that of the IgE- and IgG-PC. Peyer's patches failed to be a significant contributor in this immune response. Although this antigen-specific immune response was produced in the MLN and the spleen, it was weaker than that of the small intestine. The study indicates the potential ability of an immunized host to generate an early, yet effective, humoral immunity against T. spiralis in the non-Peyer's patch region of the small intestine."

Authors : Wang C H, Richards E M, Block R D, Lezcano E M, Gutierrez R,

(6) Efficient induction of apoptosis in cultured rat glomerular mesangial cells by dimeric monoclonal IgA anti-Thy-1 antibodies.[TOP]

Pubmed ID :8995731
Publication Date : //
Apoptosis of glomerular cells (GMC) has been observed in the early phase as well as the resolution phase of Thy-1 nephritis. Recently, we and others reported that IgG2a (ER4G) and IgG1 (OX7) monoclonal mouse anti-Thy-1 antibodies (anti-Thy-1 MoAb) are able to induce apoptosis of rat GMC in vivo. The purpose of this study was to investigate whether cross-linking of Thy-1 would influence the degree of apoptosis in cultured rat GMC using monomeric and dimeric IgA anti-Thy-1 MoAb. IgA anti-Thy-1 MoAb (ER4A) was generated by class switching of the IgG producing ER4 (ER4G) hybridoma. The ER4A clone spontaneously produces monomeric (m-ER4A) and dimeric IgA anti-Thy-1 MoAb *di-ER4A). Unaltered epitope specificity of ER4A was confirmed by blocking experiments of the binding of fluorescence labeled ER4G to cultured rat GMC with unlabeled ER4A on FACS. For the experiments of apoptosis, quiescent rat GMC were incubated for eight hours with medium alone or with medium in the presence of 10 micrograms/ml of m-ER4A, di-ER4A or control IgA MoAb of corresponding sizes. Apoptosis was assessed by morphological studies, agarose gel electrophoresis and quantitative FACS analyses using terminal deoxynucleotidyl transferase (TDT) method and the annexin V method. The TDT method detects specific-DNA nicking in apoptosis. The annexin V method detects early membrane changes during apoptosis. In morphological studies, cells incubated with m-ER4A and di-ER4A showed typical apoptotic features such as nuclear condensation and fragmentation. DNA isolated from the cells incubated with di-ER4A was cleaved into a distinctive ladder pattern compatible with apoptosis. In contrast, both medium alone and control IgA MoAb did not reveal detectable changes in morphological studies and agarose gel electrophoresis. In quantitative analyses by FACS using the TDT method and the annexin method, both m-ER4A and di-ER4A induced significantly higher percentages of apoptosis in rat GMC as compared to the controls. Furthermore, di-ER4A was considerably more efficient than m-ER4A in inducing apoptosis possibly through additional cross-linking of Thy-1 on the cell surface. This notion was confirmed by experiments, in which the addition of goat anti-mouse kappa antibodies enhanced apoptosis of rat GMC pre-sensitized with m-ER4A. Taken together, our results indicate that apoptosis of rat GMC by anti-Thy-1 antibodies is enhanced by cross-linking of Thy-1 on the cell surface. These studies are of importance for our understanding of mechanisms that may play a role in glomerular diseases.

Authors : Sato T, van Dixhoorn M G, Schroeijers W E, van Es L A, Daha M R,

(7) Characterization of the procoagulant-inducing factor derived from the plasma of BXSB mice.[TOP]

Pubmed ID :7734036
Publication Date : //
Plasma procoagulant activity inducing factor (PIF) is a spontaneously occurring, potent inducer of macrophage procoagulant activity (PCA) in the male BXSB murine model of systemic lupus erythematosus. The physical characteristics of PCA induction by PIF, aggregated mouse IgG, and lipopolysaccharide (LPS) were compared. Both aggregated IgG and PIF-induced PCA were heat, acid and alkali sensitive. In contrast, LPS-induced PCA was heat resistant and only partially acid and alkali sensitive. Plasma containing PIF was fractionated on Sephacryl S-300. The PIF activity localized to the first protein peak, molecular weight 400,000 to 900,000 daltons. Analysis of peak 1 by an enzyme-linked immunosorbent assay showed the presence of IgM, IgA and IgG. This was confirmed by Western blot analysis using 125I-labelled goat anti-mouse IgM, IgA and IgG probes. The concentration of PIF increased with Sephacryl S-300 chromatography and was reduced by removal of IgG, but not IgA or IgM by affinity chromatography. Peak 1 did not contain DNA as revealed by ethidium bromide staining. Thus, IgG from the plasma of BXSB mice, a strain which develops lupus nephritis, stimulates macrophages to express PCA, accounting for PCA induction in the BXSB model of murine lupus.

Authors : Cole E H, Neil G, Myers-Mason N, Fung L S, Levy G A,

(8) Characterization of a marker of differentiation for tracheal ciliated cells independent of ciliation.[TOP]

Pubmed ID :8338674
Publication Date : //
Although morphologic features have been used to follow cell lineage and differentiation, an objective assessment of differentiation can be best established by characterizing the expression of specific proteins that form the phenotypic profile of differentiated cells. Thus, specific markers or probes are required to unequivocally identify the various types of cells resulting from differentiation in a cell lineage. We report characterization of an IgM monoclonal antibody (5B4/H3), which recognized a surface antigen of approximately 130 kD unique to ciliated cells. The antibody reacted with the lumenal surface of the ciliated cells in transmission electron micrographs, in immunohistochemical staining of tracheal sections, and in cultured monolayers of tracheal epithelial cells. Flow cytometry, performed on enzymatically dispersed tracheal epithelial cells tagged with 5B4/H3 and fluorescent-labeled goat anti-mouse IgA/IgG/IgM, produced a population of fluorescent ciliated cells and a mixed nonfluorescent, nonciliated cell population. Ciliated cells were followed in vitro by time-lapse video microscopy for 48 to 72 h. Some of the ciliated cells lost their cilia under these culture conditions, but these cells were still found to react with the 5B4/H3 antibody. The antigen detected by this antibody remained on the surface of the cells after they lost their cilia. These results indicate that 5B4/H3 recognized a cell surface antigen that is specific to the ciliated cells and is independent of cell morphology. This marker will be useful in tissue culture studies of airway epithelial lineage, or differentiation, in which cell morphology is variable and cannot be used as a reliable marker of differentiation.

Authors : Aitken M L, Villalon M, Pier M, Verdugo P, Nameroff M,

(9) Immunoblot analysis of IgG subclass antibody response against Coxiella burnetii in Balb/c mice.[TOP]

Pubmed ID :1350177
Publication Date : //
Balb/c mice were inoculated with live or inactivated organisms of Coxiella burnetii, strain Nine Mile, phase I. Sera collected after different time intervals were subjected to immunoblot analysis and results compared with ELISA values. Immunoblots were performed with different horseradish peroxidase labelled conjugates (goat anti-mouse IgG1, IgG2a, IgG2b, IgG3) and results monitored and analysed by laser densitometry. ELISA analysis was performed using the same peroxidase labelled goat anti-mouse subclass antibody conjugates. In addition, goat anti-mouse IgG(H+L), IgG(H), IgM, and IgA conjugates were used for ELISA tests.

Authors : Thiele D, Willems H, Glas M,

(10) Secondary immunoglobulin responses of BALB/c mice previously stimulated with goat anti-mouse IgD.[TOP]

Pubmed ID :2026442
Publication Date : //
Intravenous injection of goat antibodies to mouse IgD (GAMD) into BALB/c mice has been shown to induce vigorous T-cell dependent immunoglobulin responses, particularly of the IgG1 and IgE isotypes. We have confirmed these findings and show that IgA responses are also triggered in this model. Since the study of IgE regulation in allergic individuals is concerned with secondary and subsequent T- and B-cell responses, we boosted GAMD-primed mice with goat antibodies to IgE or IgA in an attempt to specifically retrigger IgE- and IgA-bearing memory B cells. However, we found that secondary IgG1, IgE and IgA production could be elicited equally well by either antibody preparation or by normal goat IgG (GIg). As with the primary response, GIg primed and boosted mice produced very low or undetectable IgG1, IgE and IgA responses. These data suggest that GAMD is very efficient at priming T cells specific for GIg epitopes and that once primed they can be readily re-triggered by GIg. Spleen cells taken 7 days after boosting GAMD-primed mice were found to spontaneously produce much higher levels of interleukin-6 (IL-6) in culture than cells from unboosted or GIg primed and boosted mice. In contrast to primary responses, where IgE levels return to background (less than 40 ng/ml) very quickly, circulating IgE levels in boosted mice initially declined before reaching a plateau level (approximately 1 microgram/ml) which was maintained for at least 148 days. IgG1 and IgA levels continued to fall over this same time period. Mice which had been primed (but not boosted) 10 months earlier were all found to have detectable IgE in their blood, despite the fact that following priming IgE becomes undetectable within 2-3 weeks. Since only a part of the IgE response was directed towards the antigen (GIg), these observations suggest the possibility that B cells initially primed to make IgE can be non-specifically retriggered in vivo.

Authors : Champion B R, Buckham S, Page K, Obray H, Zanders E D,