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Biomedical subjects

R Thorpe

Publications and source records attributed to R Thorpe.

At least 145 records · Page 8Linked to original sources
PubMed1987

The use of monoclonal antibodies raised against a human IgE myeloma paraprotein for the study of allergen extracts and sera from allergic patients.

Murine monoclonal antibodies have been produced against a single human IgE myeloma preparation. A single fusion yielded six different monoclonal antibodies which bound strongly to the immunogen. Two of these failed to react with 'normal' human IgE or with a second IgE paraprotein. The other four antibodies reacted with normal and myeloma-derived IgE and were found to be specific for antibodies of this class. Two of these antibodies were potent reagents for the detection of IgE by two site immunoradiometric assay, immunoblotting, and radioallergosorbent test. The other two antibodies were considerably less potent reagents when used in these assay systems. Our findings suggest that care should be taken in selection of suitable monoclonal antibodies for immunochemical study of allergens and sera from allergic patients.

Allergens↗
PubMed1986

Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex.

We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin. Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.

Actins↗
PubMed1986

The effect of primary immunization and concanavalin A on the production of monoclonal natural antibodies.

Hybridomas derived from mice sham-immunized with saline were found to secrete 'natural' antibodies with a wide range of specificities. A high response to bovine serum albumin (BSA) and in particular BSA conjugated with oxazolone was observed routinely. The oxazolone-BSA response was probably directed toward antigenic sites exposed as a result of the coupling procedure; only 2% of the oxazolone-BSA-binding supernatants also bound to oxazolone-ovalbumin. An unexpected cross-reactivity was seen between the oxazolone-BSA-binding supernatants and an 18-amino-acid peptide that forms part of the VP1 capsid protein of poliovirus serotype 3. Some supernatants were also found to react with all proteins tested, including the synthetic poliovirus peptide; this reactivity was maintained following cloning. Primary immunization resulting in the generation of antibodies to the injected antigen nevertheless had no effect on the repertoire of 'natural' antibodies produced. Injection of the T-cell mitogen concanavalin A increased the frequency of 'natural' antibodies. This effect was enhanced when ConA was injected together with antigen.

Animals↗
PubMed1985

Multiple proteins related to the soluble galactose-binding animal lectin revealed by a monoclonal anti-lectin antibody.

A monoclonal antibody (NIBy 142-36/8) raised against the soluble galactose-binding lectin of bovine heart muscle has been tested by solid-phase vinyl-plate radiobinding and nitrocellulose immunoblotting with homogenates of various bovine tissues, and the muscle tissues of pig, rabbit, chicken and rat. Muscle lectins of chicken, rabbit and rat differed from those of man and pig in their lack of reactivity with the 36/8 antibody. There was a good correlation of haemagglutinating activities and immunoreactivities of the bovine tissue homogenates, suggesting that the soluble galactose-binding protein is a major haemagglutinin in various tissues. Immunoblotting experiments revealed an array of antigenically active components in the homogenates in addition to the 13 and 26kDa proteins that were previously detected in preparations of purified lectin. These were in the range 36kDa to more than 200kDa, and a different spectrum of immunoreactive components was found in various cell types. Galactose-binding activity was demonstrable in 13, 26 and 36kDa components in certain bovine tissues, suggesting that the immunoreactive components of higher Mr may be inactive precursor forms of the lectin.

Animals↗
PubMed1985

Transformation and growth related changes in levels of nuclear and cytoplasmic proteins antigenically related to mammalian beta-galactoside-binding lectin.

Immunofluorescence and immunoblotting experiments, using a monoclonal antibody to the 13 kDa mammalian beta-galactoside-binding lectin have shown that human lymphocytes contain nuclear and cytoplasmic proteins of apparent molecular masses of 130, 80, 65 and 13 kDa that are antigenically related to the lectin and whose levels and patterns of expression change in association with transformation, or after stimulation with mitogens. These observations, together with the finding that the myeloid cell line K562 is also rich in the 130 kDa component, whereas the mature granulocytes of normal donors and of patients with chronic myeloid leukaemia are lacking in all of the immunoreactive forms, raise the possibility that this family of lectin-related proteins may be components of growth regulatory systems that are variously elicited in the transformed and stimulated cells.

Antibodies, Monoclonal↗
PubMed1985

Use of 'single shot' intrasplenic immunization for production of monoclonal antibodies specific for human IgM.

We report here the use of 'single shot' intrasplenic injection of human IgM for immunization of mice to obtain splenocytes for use in the production of hybridomas secreting antibodies against human IgM. Fusion was performed 3 days after intrasplenic injection of 20 micrograms of myeloma IgM. IgM-specific antibodies were found in 12% of the fusion wells; only 1 well contained antibodies which cross-reacted with other immunoglobulin classes. Two monoclonal antibodies (McAbs) have been fully characterized as specific for different epitopes on Fc mu. These antibodies can be used to detect IgM on the surface of human B cells by immunofluorescence and in solution by solid-phase radiobinding assay or single radial immunodiffusion. Both McAbs can also detect IgM fragments by immunoblotting from non-reducing SDS-polyacrylamide gels.

Animals↗
PubMed1985

Human B cell proliferation is stimulated by interleukin 2.

The proliferation of human B cells was studied for response to interleukin 2 (IL-2) produced in Escherichia coli using recombinant DNA technology. The IL-2 was found to be an homogenous preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the anti-IL-2 monoclonal antibody DMS-1. IL-2 was found to stimulate B cell proliferation. Activation of the B cells using anti-IgM antibodies increased this response. Resting T cells from the same donors were found to be less reactive to IL-2. The results suggest that human B cell proliferation can be stimulated by IL-2 alone.

Antibodies, Anti-Idiotypic↗
PubMed1985

Naturally occurring antibodies in human sera that react with the iron-regulated outer membrane proteins of Escherichia coli.

Sera from normal healthy human adults and infants, as well as sera from mice, rabbits, and guinea pigs, were examined by immunoblotting for naturally occurring antibodies reacting with outer membrane proteins of two Escherichia coli strains, O111 and O18. Some individuals had antibodies reacting very strongly with the iron-regulated outer membrane proteins, including the ferric-enterochelin receptor protein (Mr, 81,000), as well as with ompA. However, sera from infants contained predominantly antibodies to ompA; antibodies recognizing the iron-regulated outer membrane proteins were either absent or barely detectable. In human serum the antibodies were mainly of the immunoglobulin G class. No serotype-specific antibodies to the lipopolysaccharide of E. coli O111 or O18 were found in the sera tested.

Antibodies, Bacterial↗
PubMed1985

Interleukin-2 in vivo: production of and response to interleukin-2 in lymphoid organs undergoing a primary immune response to heterologous erythrocytes.

In this report, we describe experiments which demonstrate that antigenic stimulation in vivo causes the appearance of cells in both spleen and lymph node which secrete interleukin-2 (IL-2). Cells also appear in these organs which proliferate in response to IL-2. For these experiments, sheep red cells (SRBC) were injected into the spleens or footpads of mice, and cell suspensions from spleens or popliteal lymph nodes prepared at various times after antigenic stimulation. These cells were assayed for their ability to respond to IL-2, and their cell culture supernatants for secreted IL-2. The proliferative response to IL-2 steadily increased following SRBC injection to reach a peak at Day 2 for spleen cells and at Day 3 for lymph node cells. Maximal production of IL-2 was displaced from the maximal response to the lymphokine by peaking one day later for both organs. Our results strongly implicate the participation of IL-2 in the in vivo immune response and suggest the existence of in vivo regulatory mechanisms, which can control the time of IL-2 production and also the appearance of cells with receptors for IL-2.

Animals↗
PubMed1984

Immunoblotting with monoclonal antibodies: loss of immunoreactivity with human immunoglobulins arises from polypeptide chain separation.

Immunoblotting has been used to study the antigen binding characteristics of 5 monoclonal antibodies (Mc/Abs) against human Ig (1 anti-kappa, 2 anti-gamma and 2 anti-delta chain. Of the 4 Mc/Abs only 1 (the anti-kappa chain Mc/Ab) reacted with its antigen when blotted from reducing SDS polyacrylamide gels. However, the 4 Mc/Abs which recognise immunoglobulin heavy chains were able to bind their antigens when blotted from native or non-reducing SDS gels. The lack of reactivity of the latter Mc/Abs in blots from reduced SDS gels may be attributed to the separation of Ig which occurs during electrophoresis after the -S-S- bonds are broken. It may be concluded that the conformation of Ig heavy chains is considerably altered when Ig molecules are disrupted and Ig chains separated, and several heavy chain determinants are lost during this process. Therefore determinants recognised by the anti-heavy chain Mc/Abs are most likely to be of the 'conformational' type whereas the anti-light chain Mc/Ab may well recognise a purely sequential determinant.

Antibodies, Anti-Idiotypic↗
PubMed1984

Production and characterization of monoclonal antibodies to beta-galactoside-binding lectin of bovine heart muscle. Direct evidence that haemagglutinating activity is associated with a 13kDa protein.

With the aim of obtaining monospecific antibodies against the beta-galactoside-binding lectin of bovine heart muscle, spleen cells from Lou rats immunized with lectin were fused with the rat myeloma line Y3.Ag1.2.3. Two immunoglobulin M (IgM)-producing clones, designated NIBy 142-36/8 and NIBy 143-9/5, derived from separate fusions, were used to generate ascites containing high-titre binding activity against the 13kDa component in preparations of lectin. Direct evidence that haemagglutinating activity is associated with the 13kDa protein was obtained by the specific elution of 13kDa polypeptides with haemagglutinating activity from an immobilized antibody adsorbent. Solid-phase radiobinding assays and immunoblotting of isolated lectins and/or muscle homogenates confirmed the earlier indications with conventional antisera that the beta-galactoside-binding lectins of bovine, human and monkey muscle tissue are antigenically related.

Animals↗
PubMed1984

Intrasplenic primary immunization for the production of monoclonal antibodies.

A novel immunization procedure for eliciting monoclonal antibodies ( McAbs ) is described. With intrasplenic inoculation only small amounts of immunogen are required. As little as 20 micrograms of protein antigen or 2.5 X 10(5) cells have been found sufficient to immunize mice or rat spleen cells for the production of specific McAbs . A high proportion of hybridomas secreting McAbs against cell surface antigens and soluble proteins has been obtained with this immunization procedure. The system could facilitate McAb production in many instances in which only small quantities of immunogen are available.

Animals↗
PubMed1984

Molecular identity, distribution and heterogeneity of glial fibrillary acidic protein: an immunoblotting and immunohistochemical study of Schwann cells, satellite cells, enteric glia and astrocytes.

Glial fibrillary acidic protein has been firmly established as the predominant component of astrocyte intermediate filaments. It has also been detected immunohistochemically in the glial cells of the enteric nervous system and some Schwann cells in the P.N.S. The molecular identity of this GFAP immunoreactivity in the P.N.S. has so far not been investigated. This study compares GFAP in the C.N.S. and P.N.S. of adult rats both immunochemically and immunohistochemically. Using SDS polyacrylamide gel electrophoresis combined with immunoblotting, and a polyclonal antiserum to brain GFAP, we show that the peripheral GFAP immunoreactivity resides in a polypeptide with a molecular weight of 49 kd, which is identical to that of rat brain GFAP. Furthermore, we find that this GFAP reactivity can be detected immunohistochemically in Schwann cells in a wide variety of nerves in the P.N.S. and in some satellite cells in both sensory and sympathetic ganglia, in addition to enteric glia. The pattern of distribution of GFAP filaments in Schwann cells suggests that, in the nerves surveyed, they may be expressed by most or all non-myelin forming Schwann cells but not by myelin-forming Schwann cells. We also show, using a monoclonal antibody to GFAP (anti-GFAP-3) in both immunohistochemical and immunoblotting studies, that the GFAP found in most peripheral glia is not identical to that of astrocytes since it lacks an antigenic determinant, defined by this monoclonal antibody, which is present in astrocytes. An exception to this finding is seen in the myenteric plexuses where immunohistochemically detectable GFAP is found in some, but not all, of the enteric glia, using the monoclonal antibody. Thus, the results suggest that GFA polypeptides may be a heterogeneous group, that share some common determinants and a common molecular weight, and show a widespread and complex distribution in the glia of both the C.N.S. and P.N.S.

Animals↗
PubMed1984

Single shot intrasplenic immunization: an advantageous procedure for production of monoclonal antibodies specific for human fibrin fragments.

We describe the use of the single shot intrasplenic immunization technique as a particularly effective procedure for the production of specific monoclonal antibodies against a high molecular weight antigen. We found that with this technique several different, completely specific monoclonal antibodies could be produced against high molecular weight crosslinked fibrin degradation products. These results contrasted with those obtained using conventional multidose immunization, which only produced monoclonal antibodies that were cross-reactive with fibrinogen and/or noncrosslinked fibrin degradation products.

Animals↗