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We have analysed and compared the fine specificity and behavior in various immunoassays of 10 mouse monoclonal antibodies, from three independent laboratories, directed against IgA1, IgA2 or non-IgA2m(2). The following observations were made. (1) Although all of the monoclonal antibodies were specific for a particular IgA subclass or isoallotype in a radioimmunoassay, three of them were not specific when tested in indirect immunofluorescence on plasma cells derived from pokeweed-activated peripheral blood lymphocytes. In this highly sensitive system, contrary to direct immunofluorescence previously performed using formalin-fixed lymphoid tissue, the anti-IgA1 69.114 reacted with some of the IgA2 plasma cells, the anti-IgA2 DLDB7 reacted with some of the IgA1 plasma cells and the anti-IgA2 16.512 dimly reacted with all IgM plasma cells. (2) Among the eight anti-IgA subclass antibodies, seven were directed against the CH2 domain of IgA whereas the anti-IgA1 1-155-1 recognised an epitope destroyed by Streptococcus sanguis IgA1 protease and localised in the hinge region of IgA1. The two anti-isoallotype antibodies were directed against epitope(s) probably localised in the 65 C-terminal amino acid residues of the alpha-CH3 domain. All of the 10 antibodies were able to react with endogeneously produced surface IgA on B-cells. (3) Using monoclonal anti-IgA subclass antibodies in radioimmunoassay may be hazardous in the absence of knowledge of their affinity constants and of careful control experiments: some of the antibodies were not sensitive in radioimmunoassays designed to measure the serum titer of specific IgA1 and IgA2 antibodies. Moreover, major differences were observed between the different monoclonal reagents with respect to the influence of the size of IgA on a solid-phase sandwich radioimmunoassay. While three of the anti-IgA1 underestimated dimeric IgA relative to monomeric IgA, the fourth anti-IgA1 and all the anti-IgA2 overestimated dimeric IgA relative to monomeric IgA, by a factor sometimes close to 7.
Mol Immunol 1986 Apr
PMID:Monoclonal antibodies against isotypic and isoallotypic determinants of human IgA1 and IgA2: fine specificities and binding properties. 242 48

Past work has shown that B lymphocytes from Peyer's patches (PP) and from spleen differ from each other in the following ways: (1) PP are enriched for IgA-producing precursor cells while splenic B-cells are a rich source of IgM-precursor cells; and (2) PP are a poor source of antibody-secreting cells when compared to the spleen. The reasons for these differences are unclear. In this work B-cells have been examined expressing newly regenerated surface immunoglobulin heavy chains in murine PP and spleens for immunoglobulin isotype expression. Dual color immunofluorescence demonstrated higher levels of B-cells regenerating two surface isotypes in the PP (IgM+ IgG+ = 14.9%; IgM+ IgA+ = 9.4%) than in the spleen (IgM+ IgG+ = 0.4%; IgM+ IgA+ = 0.2%). Poly A+ RNA was purified from these B-cells and compared for immunoglobulin heavy chain isotype expression by DNA-excess slot blot hybridization. Splenic B-cells contained higher amounts (at least two-fold) of Ig heavy chain-specific mRNA per microgram of polysomal RNA than did PP B-cells. Peyer's patches B-cells demonstrated slightly lower mu:alpha ratios than splenic B-cells. In vitro translation of the RNAs suggested higher levels of translatable alpha-specific Poly A+ RNA in PP B-cells than in B-cells from MLN or spleen; furthermore, splenic B-cell RNA contained higher levels of translatable mu-RNA than did the other tissues examined. Northern blot analysis of RNA derived from these tissues identified major mu-, gamma 2b-, and alpha-hybridizing sequences, though PP-derived B-cell preparations were shown to be enriched for the membrane forms of mRNA for gamma 2b and alpha when compared to the spleen-derived B-cell preparations. These results suggest that the level of differentiation of PP B-cells (that are capable of regeneration of surface Ig) may differ significantly from that of splenic B-cells.
Mol Immunol 1986 Jun
PMID:Comparison of immunoglobulin heavy chain isotype expression in Peyer's patch and splenic B-cells. 242 36

Total cellular Poly A+ RNA from TEPC15 myeloma and murine lymphoid tissues was analyzed by denaturing agarose gel electrophoresis and Northern blot hybridization to specific heavy chain constant region cDNAs; mu, gamma 2b and alpha RNA species were identified in each of the tissues and in the IgA producing TEPC15 myeloma. In total Poly A+ RNA from TEPC15 myeloma, alpha-cDNA hybridized predominantly to a 2.3 kb RNA species; 11.5 and 4.1 kb RNA species were evident as well. Successive hybridization of the same RNA to mu- and gamma 2b-specific cDNAs demonstrated the presence of both mu and gamma 2b specific RNA species with electrophoretic mobilities apparently identical to the 11.5 and 2.3 kb RNA species identified by alpha-specific hybridization. These data establish the presence of Poly A+ RNA species containing alpha, mu, and gamma 2b sequences in TEPC15 cells and suggest the transcription of RNA from both CH-containing chromosomes in TEPC15 myeloma (one chromosome in the TEPC15 cell line contains all the CH genes while the other chromosome has deleted all CH genes except alpha). In total Poly A+ RNA from normal mouse tissues (Peyer's patch and spleen) all three cDNA probes hybridized predominantly to an 11.5 kb RNA species. Primer extension experiments demonstrated that alpha cDNA could prime for the synthesis by reverse transcriptase of gamma 2b DNA when Peyer's patch Poly A+ RNA was used as the template. This suggests the existence of a single transcript containing alpha and gamma 2b sequences. Murine lymphoid tissues contained putative mRNAs for mu, gamma 2b and alpha heavy chain proteins, whereas TEPC15 myeloma polysomal Poly A+ RNA contained only alpha mRNA.
Mol Immunol 1986 Jun
PMID:Identification of the major immunoglobulin heavy chain poly A+ RNA in murine lymphoid tissue. 242 42

The specificity of 14 monoclonal antibodies has been determined by immunoblotting (IB) and haemagglutination-inhibition (HAI) analysis using IgA1 and IgA2 myeloma proteins and eight different IgA1 fragments. Two antibodies probably recognized epitopes on the CH1 domain of IgA. They reacted with all Fab-containing fragments irrespective of whether these originated from the same or different IgA proteins. Seven antibodies were directed against epitopes on the CH2 domain. These antibodies were reactive with F(abc)2 fragments. They failed to react with Fab, Fab' and F(ab')2 fragments. Two out of these seven antibodies did not react with two-chain IgA half-molecules and Fabc fragments containing a single heavy and a single light chain. This suggests that these two antibodies recognized an epitope whose structure is dependent on disulfide linked heavy chains. Five other antibodies showed specificity for the CH3 domain. They were reactive with all CH3-containing molecules, irrespective of whether they comprised one or two alpha chains. Our study demonstrates that IB is an appropriate technique to determine domain specificity of monoclonal anti-immunoglobulin reagents. Although the IB tests were performed on denatured proteins the results agreed surprisingly well with those of the HAI analyses. Moreover, the IB technique could be used on fragments which could not be purified well enough for HAI analyses.
Mol Immunol 1986 Jul
PMID:Monoclonal antibodies against different domains of human IgA: specificities determined by immunoblotting and haemagglutination-inhibition. 243 12

It has previously been shown that the AML-2-23 monoclonal antibody (MoAb) reacts with a glycoprotein on differentiated myeloid cells. The antigen, My23, is released from these cells in culture and is also detectable in normal human plasma. We have now raised a panel of MoAbs against the soluble form of the antigen which reacts with monocytes and calcitriol-treated U937 and HL-60 myeloid cell lines, but not with lymphocytes or undifferentiated U937 and HL-60 cells. As with the AML-2-23 MoAb, the anti-My23 MoAbs immunoprecipitated a 50,000-55,000 protein from calcitriol treated HL-60 cells. Besides binding to cell surface My23, all eight MoAbs as well as the 63D3 MoAb reacted with crude and purified forms of soluble My23. A novel ELISA epitope analysis assay was developed to identify four distinct antigenic determinants on My23. Thus, the soluble and cell surface forms of My23 share several antigenic determinants and are biochemically very similar. Pooled anti-My23 caused selective patching, capping and clearing of My23 from the cell surface. My23 was not associated with cell surface, HLA-DR, HLA-A,B,C, beta 2-microglobulin or the Fc receptors for IgG or IgA. These anti-My23 MoAbs should be of importance in antigen functional studies and in clinical treatment protocols for patients with acute myelogenous leukemia. Furthermore, we have shown that a soluble myeloid differentiation antigen can serve as an effective immunogen for the preparation of MoAbs against important cell surface molecules thus obviating many problems inherent in the use of whole cells or detergent lysate-derived immunoprecipitates as immunogens.
Mol Immunol 1987 Jan
PMID:Monoclonal antibodies that bind to the My23 human myeloid cell surface molecule: epitope analysis and antigen modulation studies. 244 Dec 45

Six hybridomas, five from C58/J and one from C57BL/10 nu/nu, immunized with stearyl-isomaltotetraose (S-IM4) were established. One produced IgG3, one IgM and four IgA. The specificities and sizes of the antibody combining sites were determined by quantitative precipitin and ELISA quantitative inhibition assays. All cross-react with alpha(1----6)dextran B512. Their combining sites were complementary to five to seven glucose residues. Association constants of hybridoma antibodies for dextran B512 ranged from 10(3) to 10(5) ml/g, and for isomaltoheptaose (IM7) from 10(3) to 10(4)M-1. Two hybridoma antibodies, one IgA and another IgM derived from different fusions have identical inhibition curves and combining sites, and very close association constants. It will be important to establish whether they have very similar or identical nucleotide sequences in their variable regions. These studies provide further insight into the specificity and repertoire of antidextran combining sites.
Mol Immunol 1987 Apr
PMID:Immunochemical studies on monoclonal antibodies to stearyl-isomaltotetraose from C58/J and a C57BL/10 nude mouse. 244 31

More than 180 monoclonal antibodies (McAbs) to the cyclic undecapeptide cyclosporine (Cs) have been prepared. Several immunization protocols and antibody screening processes were compared. Two main groups of McAbs recognizing different "sides" of the Cs molecule could be differentiated. The antibodies belonged to the IgG and IgA classes and showed high affinity for Cs (up to 10(-10) -10(-11) mol/l). Based on their ability to discriminate Cs-derivatives modified singly at each of the 11 residues of the Cs molecule, the antigenic recognition pattern of different McAbs was studied at the level of individual residues. Closely related recognition patterns were found in each of the two main McAb groups. The apparent size of the Cs antigenic sites recognized by different McAbs varied from four to ten residues and did not correlate with antibody affinity.
Mol Immunol 1987 Nov
PMID:Fine specificity and cross-reactivity of monoclonal antibodies to cyclosporine. 244 93

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.
Mol Immunol 1988 Feb
PMID:Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture. 245 91

Fusion between the thioguanine resistant myeloma cell line MOPC-315 [which produces alpha, lambda-2 antibodies specific to the 2,4-dinitrophenyl (DNP) hapten] and a long term in vivo maintained hybridoma 6100.15 [which produces mu, lambda-1 antibodies specific to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten] resulted in the generation of 12 hybridomas. These hybridomas secrete a mixed family of immunoglobulins (Ig) that bind both DNP and NP and express both IgM and IgA serological determinants. Affinity purified molecules from NP, DNP, anti-mu, or anti-alpha immunosorbents react with both anti-mu and anti-alpha antisera, suggesting that these Ig represent IgM-IgA hybrid molecules. This conclusion was supported by idiotypic analyses. To determine the roles of individual immunoglobulin chains in determining antibody specificity this IgM-IgA hybridoma was used for immunoselection. Following lysis with specific anti-mu and anti-idiotype antibodies, an alpha+, mu- variant clone (A12) was identified, which secreted Ig that binds DNP but not NP. The DNP binding proteins were shown to express alpha, lambda-1 and lambda-2 chains. In contrast, the Ig which lack DNP binding activity only expressed alpha and lambda-1 determinants. The combined results demonstrate that the lambda-1 chain from 6100.15 hybridoma cannot replace lambda-2 of MOPC-315 for DNP binding activity. These data imply that these closely related lambda chains carry sites critical for antigen binding activity. An IgM-IgA hybridoma variant (MA2) which secretes Ig that binds to NP and DNP and expresses mu, alpha and lambda-2 chains was also characterized. This molecule lacked a lambda-1 chain. To determine if the Ig prepared with heterologous mu and lambda-2 chains had NP binding activity required immunoselection of a fourth clone (M2). M2 secretes homogeneous Ig bearing only mu and lambda-2 chains. In contrast to either parental Ig, the M2 antibody molecules express dual binding activity to both NP and DNP. Thus, critical amino acid substitutions in the MOPC-315 lambda-2 sequence are required for DNA binding specificity.
Mol Immunol 1989 Mar
PMID:Production and immunoselection of IgM-IgA hybridomas: preparing immunoglobulins with dual binding specificity. 249 37

A novel murine B lymphoma expressing membrane-associated IgA was isolated and used to compare mechanisms of signal transduction by sIgM and sIgA. Like other isotypes so far studied, crosslinking of sIgA by anti-immunoglobulin antibodies stimulates hydrolysis of inositol phospholipids and causes elevation of intracellular free calcium. Furthermore, signals generated through sIgA are coupled to elevation of c-fos proto-oncogene expression. Coupling appears to be through the protein kinase C rather than through the Ca2+ component of sIg signalling as phorbol diester, but the Ca2+ ionophore cannot mediate this effect. Thus these results, coupled with those from earlier studies, show that early signal transduction through surface immunoglobulin appears to be similar regardless of the particular isotype involved in binding ligand.
Mol Immunol 1989 Jul
PMID:Signalling through sIgA on a novel murine B lymphoma. 255 Aug 16


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