Method for rapid detection of membrane antigens by immunofluorescence and its application to screening monoclonal antibodies.
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Animals↗
Biomedical subjects
M Mirshahi
Publications and source records attributed to M Mirshahi.
Pineal-retinal molecular relationships: distribution of "S-antigen" in the pineal complex.
The immunocytochemical localization of S-antigen, a specific protein first discovered in retinal photoreceptors, was studied in the pineal complex of vertebrates (eel, pike, frog, lizard, passerines, mouse, hamster) using monoclonal antibody immunofluorescence. S-antigen immunoreactivity was demonstrated concurrently in retinal photoreceptors and in most pineal phototransducers of all species, i.e. in pineal cells of the receptor series (cone-like, modified photoreceptor cells, pinealocytes) and in cone-like photoreceptors of the frog frontal organ and lizard parietal eye. The labelling was distributed either in all compartments of these cells, or restricted to outer segments. The functional significance of the S-antigen as well as some phylogenetic and ontogenic implication of this marker are discussed.
Anguilla↗
Screening monoclonal antibodies against cell-surface antigens. High frequency of natural antibodies.
Two commonly used methods for screening hybridoma supernatants against cell-surface antigens were performed simultaneously on the supernatant culture fluids of different hybridizations, and compared with the detection of Ig secretion. Supernatants reacting with glutaraldehyde-fixed cells (ELISA (Enzyme-linked immunosorbent assay) on fixed cells) are mostly non-specific for the immunogen; in addition, with this method, only half of the antibodies detected by immunofluorescence are identified. These results can be explained by the frequent occurrence of hybridomas secreting antibodies displaying 'natural antibody' properties, which are strongly reactive with intracellular antigens, and apparently made accessible by the fixation procedure. Since artefacts impair the interpretation of results with ELISA on fixed cells, and complement-mediated cytotoxicity usually as a low yield, membrane immunofluorescence remains the best method for screening hybridoma antibodies.
Animals↗
S-antigen is coded by [poly A+] mRNA from bovine retina.
Total RNA, [poly (A)-] mRNA and [poly (A)+] mRNA purified from bovine retina were translated in vitro in a rabbit reticulocyte lysate system. Immunoprecipitation of translation products with antibodies to the retinal S-antigen (a photoreceptor specific protein involved in autoimmune retinal disease) revealed this protein as a 50,000 daltons band comigrating with purified S-antigen. This indicates that the S-antigen is synthesized in the retina and is not a maturation or degradation product of a larger protein. Its messenger RNA is the polyadenylated RNA, as for some other proteins expressed in nervous tissue.
Animals↗
Possible involvement of two proteins (phosphoprotein and CD9 (p24)) in regulation of platelet calcium fluxes.
The monoclonal antibody ALB6 directed against the leukocyte differentiation antigen CD9 (p24) increases the calcium incorporation into isolated platelet membrane vesicles enriched in internal membranes. The similarities of the effects of both the monoclonal antibody and the catalytic subunit of the cAMP-dependent protein kinase (C, subunit), which phosphorylates a protein of an apparent molecular mass of 23 kDa, led us to investigate the relationship between CD9 (p24) and the 23-kDa phosphoprotein (p23). ALB6IgG does not inhibit the C.subunit-induced phosphorylation of p23 and the immunoadsorption by ALB6IgG of p24 associated to membrane vesicles does not alter the phosphorylation pattern. Thus, proteins of similar molecular mass appear to be involved in calcium fluxes: one is recognized by the ALB6 antibody while the other can be phosphorylated by the C-subunit.
Antibodies, Monoclonal↗
Inhibition of experimental autoimmune uveoretinitis in rats by S-antigen-specific monoclonal antibodies.
Mouse monoclonal antibodies (mAb), of either IgG2a or IgG2b isotypes, specific for the retinal S-autoantigen (S-Ag) or a pool of rat anti-S-Ag sera prevented experimental autoimmune uveoretinitis in Lewis rats when injected i.p. at the time of immunization. Control mAb of the same isotypes, irrelevant to S-Ag, had no inhibitory effect. The humoral response to S-Ag, as studied by enzyme-linked immunosorbent assay using a mouse mAb specific for rat kappa chain, was moderately but significantly reduced in suppressed animals. The rapid disappearance of the injected mAb from rat sera, as measured using a rat mAb specific for mouse kappa chain, could be explained by its complexing with either autologous antigen released from the retina at the site of inflammation, or anti-idiotypic antibodies.
Animals↗
A new set of monoclonal antibodies against acute lymphoblastic leukemia.
Six monoclonal antibodies produced by immunization of Balb/c mice with common acute lymphoblastic leukemia (cALL) cells were tested against various types of normal and malignant tissues. ALB1 and ALB2 are directed to the cALL antigen (CALLA gp100); ALB6 recognizes a determinant of p24; ALB7, ALB8 and ALB9 have a pattern of reactivity similar to Ba1. None of these antibodies specifically identify cALL but they should be useful tools for diagnosis or depletion of bone marrow in autologous therapy in transplantation. In addition, the example of ALB6 which acts as a platelet aggregating agent, suggests that the study of other cell systems expressing the antigens associated with cALL may shed light on the function of these antigens and subsequently on the physiopathology of the leukemic cells.
Animals↗
Experimental autoimmune uveoretinitis in athymic rats: specific IgE response to retinal S-antigen and disease.
Experimental autoimmune uveoretinitis (EAU) is an ocular autoimmune disease induced in rats by immunization with retinal S-antigen. Athymic nude rats (rnu/rnu) have been previously shown to be refractory to EAU induction and antibody production to S-antigen, while heterozygous (rnu/+) are good responders. Increasing the antigen dose and adding pertussis adjuvant produced ocular disease in some nude rats, and antibody response in most of them. Specific IgE antibodies were demonstrated by ELI-SA only in the serum of nude rats presenting the disease. However, most immunized nude rats had evidence of mast cell sensitization to S-antigen (direct degranulation test) and of circulating specific IgE detected by passive sensitization of normal mast cells (indirect degranulation test). This positive response could be explained by an incomplete depletion of the different T lymphocyte subsets.
Animals↗
Markers of fibrinogen derivatives used in clinical investigation.
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Antibodies, Monoclonal↗
Platelet aggregation occurs in congenital afibrinogenaemia despite the absence of fibrinogen or its fragments in plasma and platelets, as demonstrated by immunoenzymology.
Platelet aggregation of an afibrinogenaemic patient's platelet rich plasma (PRP) was greatly decreased when ADP was used for stimulation. In the presence of collagen or arachidonic acid the changes in light transmission recorded during platelet aggregation of patient's PRP were similar to those observed with normal PRP but the size of aggregates appeared to be smaller in comparison with those observed with normal platelets. In addition, thrombin-induced aggregation of washed platelets was similar to normal platelets. The interpretation was made possible because the fibrinogen level in plasma and in platelets was found to be almost nil as demonstrated by both an Elisa procedure described here and the determination of fibrinopeptide A (fpA). Furthermore, fibrinogen fragments, which could result from abnormal synthesis and therefore replace fibrinogen in platelet aggregation, were undetectable by immunological analysis using specific antibodies against A alpha, B beta and gamma chains and 10 different monoclonal antibodies against fibrin degradation products.
Adenosine Diphosphate↗
Retinal S-antigen epitopes in vertebrate and invertebrate photoreceptors.
Monoclonal antibodies specific for the retinal S-antigen were obtained by hybridization of spleen cells from a BALB/c mouse immunized with bovine S-antigen and NS-1 myeloma cells. Five selected antibodies specifically labeled the photoreceptor cells of the retina by immunofluorescence. Whereas antibody S9E2 only reacted with bovine S-antigen, the other antibodies showed interspecies cross-reactivity. They were used for the characterization of specific epitopes of S-antigen in photoreceptors from a wide range of species representative of various classes of vertebrates and invertebrates. The presence of S-antigen in distant species (vertebrates, Amphioxus, nemerteans, annelids, molluscs) indicates a high phylogenetic stability and suggests an important role for this protein in photoreceptor function.
Animals↗
S-antigen immunoreactivity in retinal rods and cones and pineal photosensitive cells.
The S-antigen is a protein of photoreceptors, mainly known for its autoantigenic properties in mammals, which is widely distributed in the retina of vertebrates and in photoreceptor organs of invertebrates. Using three monoclonal antibodies specific for different epitopes of S-antigen, this study complements our previous data on retinal rods and cones and presents new results on the photosensory cells of the pineal complex. Immunoreactivity was found in (i) retinal rods and cones, (ii) cone-like and modified photoreceptor cells, and pinealocytes of the pineal organ of vertebrates, (iii) cone-like photoreceptors of the frontal organ of the frog and of the third eye of the lizard. According to the species and the antibody used, some differences were found at the level of the cellular compartments of the pineal photoreceptor cells.
Animals↗
Production and specificity of monoclonal antibodies to retinal S antigen.
Hybridomas producing monoclonal antibodies to the retinal S antigen were obtained by fusion of spleen cells from a BALB/c mouse immunized with purified bovine S antigen and NS-1 myeloma cells. Six cloned hybridomas were selected and expanded as large scale cultures and as ascites in mice. The specificity of the antibodies produced by these hybridomas was assessed by ELISA and immunofluorescence. All were specific for S antigen, except one which showed slight reactivity with other proteins. One antibody was specific for bovine S antigen, whereas the others showed cross reactivity with purified S antigens from various mammals. Immunofluorescence allowed to demonstrate the presence of common epitopes of S antigen in the retinal photoreceptor cells of species representative of every class of Vertebrates.
Animals↗
[Identification of the so-called 48 K protein that interacts with illuminated rhodopsin in retinal rods, and the retinal S antigen, inductor of experimental autoimmune uveoretinitis].
In Vertebrate retinal rod outer segments, a soluble "48 K" protein binds to disk membranes upon illumination in presence of ATP or GTP (H. Kühn, Biochemistry, 17, 1978, p. 4389). Its binding to photoexcited rhodopsin implies a probable role of the "48 K" protein in the ATP dependent regulation of the photoinduced enzymatic cascade which controls the hydrolysis of cGMP. The "retinal S antigen" is also a soluble protein located in photoreceptor cells which is known to be an organ-specific auto-antigen inducing experimental autoimmune uveoretinitis. Using extracts of purified cattle and frog rod outer segments, purified bovine S antigen, and monoclonal antibodies against S antigen, we found that both proteins exhibit identical characteristics with respect to: their migration in SD S-gel electrophoresis; their binding to rod disc membranes upon illumination in presence of ATP or GTP; their immunological reactivity with monoclonal antibodies.
Animals↗
[Monoclonal antibodies against surface antigens of lymphoblasts and blood cells or bone marrow recognize constituents of the human nephron].
By immunizing mice with bone marrow cells of a patient with acute lymphoblastic leukemia, monoclonal antibodies have been produced against differentiation antigens of hemopoietic cells. These antibodies also recognize cells in different parts of the normal human kidney. Two antibodies, ALB1 et ALB2, which recognize the common acute lymphoblastic leukemia antigen, (CALLA), label the podocytes and the epithelial cells of the proximal tubules of the kidney; ALB6, which recognizes the "p 24" antigen of lymphocytes, labels the distal tubules; ALB9, which recognizes lymphoblasts and granulocytes, labels the thick ascending loop of Henle and the collecting tubules; PM1, which recognizes immature granulocytic cells, labels the thick ascending loop of Henle.
Animals↗
Characteristics of platelet aggregation induced by the monoclonal antibody ALB6 (acute lymphoblastic leukemia antigen p 24). Inhibition of aggregation by ALB6Fab.
The leukemia-associated cell surface antigen p 24 is found on normal platelets as well as on Bernard Soulier syndrome and thrombasthenia type I platelets. ALB6 IgG (a monoclonal antibody against p 24) induces the aggregation of platelets from normal donors but not from thrombasthenia. In contrast, ALB6 Fab inhibits platelet aggregation induced by collagen, ADP, thrombin, ionophore A 23187 and ALB6 IgG. The results suggest that ALB6 interferes with a mechanism common to all aggregation pathways; the possible mechanisms are discussed.
Antibodies, Monoclonal↗
A rapid method for detection of membrane antigens by immunofluorescence and its application to screening hybridoma antibodies.
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Antibodies, Monoclonal↗
Effect of interferon gamma and TNF alpha on the differentiation/activation of human glial cells: implication for the TNF alpha receptor 1.
This study focused on the activation/differentiation of human microglial cells and astrocytes, which is a prerequisite for HIV1 replication. In vitro, IFN gamma induced a differentiation-like morphological change in embryonic microglia and astrocytes, in both primary and in purified culture. This effect was enhanced by TNF alpha which in itself had no effect. IFN gamma did not increase the low level of endogenous TNF alpha, which is not secreted by embryonic cells, but stimulated the expression of TNF alpha R1, although to a relatively low extent. IFN gamma facilitated TNF alpha response through an increase in TNF alpha R1 expression, but probably also through interaction with different intracellular signal transduction pathways.
Astrocytes↗