Search PubMed⌕ Search

Biomedical subjects

R Thorpe

Publications and source records attributed to R Thorpe.

At least 127 records · Page 7Linked to original sources
PubMed1989

The production and characterisation of monoclonal antibodies against human prolactin and the development of a two-site immunoradiometric assay.

Monoclonal antibodies against human prolactin (PRL) have been produced and characterised and used to develop a sensitive two-site immunoradiometric assay (IRMA). Nine anti-PRL monoclonal antibodies were assessed for reactivity in immunoblotting experiments with PRL, hPL, hGH and pituitary gland extract. There was no detectable crossreactivity with hPL or hGH. In liquid phase radioimmunoassay (RIA) studies using three of the antibodies there was no detectable crossreaction from hPL or hGH. Five antibodies were positive in immunocytochemical studies using sections of human pituitary gland. Using FPLC purified monoclonal antibodies, a two-site IRMA was developed that could assay PRL over the range 17.5-3500 mIU per litre and was readily adapted to assaying serum samples from patients. The two-site IRMA could be performed within one day without loss of sensitivity and has potential as a rapid and simple method for screening clinical samples.

Antibodies, Monoclonal↗
PubMed1989

Presence of the inflammatory cytokines IL-1, TNF, and IL-6 in preparations of monoclonal antibodies.

We have detected significant levels of IL-1, TNF and IL-6 in ascitic fluid from mice injected with different hybridomas. Each of these murine cytokines is active on a wide range of both human and murine target cells, and all are involved in inflammatory responses in vivo. We could not detect TNF or IL-1 in culture supernatant from the hybridoma line, used to generate the ascites; however, one out of ten did produce high levels of IL-6. These cytokines can interfere in many in vitro systems where monoclonal antibodies are used as diluted ascitic fluid to inhibit their ligands. A potentially more serious problem could arise with monoclonal antibodies used for therapy, in vivo diagnosis, or as ex vivo reagents. The use of monoclonal antibodies derived from culture supernatants is, therefore, obviously preferable to use of ascitic fluid for clinical applications, however, even with supernatant it cannot be assumed that cytokines are not present. Cytokine levels should, therefore, be monitored in all starting solutions and if present, their clearance during purification should be determined.

Animals↗
PubMed1989

HTLV-1 infection in Papua New Guinea: evidence for serologic false positivity.

Serum samples from 557 individuals participating in studies from four separate lowland and highland populations in Papua New Guinea exhibited consistently false-positive results for human T lymphotropic virus (HTLV) type 1 (10%) and human immunodeficiency virus (HIV) type 1 (5%) antibody in direct antiglobulin and agglutination assays. All serum samples were negative in competitive ELISAs and radioimmunoassays for HTLV-1 and HIV-1; selected samples of reactive sera were negative in an HTLV-2 competitive ELISA. Immunofluorescent antibody tests using HTLV-1 infected cells correlated poorly with ELISA results. None of the sera from Papua New Guinea neutralized vesicular stomatitis virus pseudotypes of HTLV-1. By Western blot analysis, only three serum samples were weakly reactive to HTLV-1 gag proteins. These studies suggest there is as yet no firm evidence of HTLV-1, HTLV-2, or HIV-1 infection in Papua New Guinea, although there may be a low prevalence.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Inhibition of interleukin-2 secretion by factor VIII concentrates: a possible cause of immunosuppression in haemophiliacs.

The inhibitory effect of factor VIII concentrate products on IL-2 secretion by human T-cells was investigated. The six products used widely in the U.K. showed very different activities varying from almost total inhibition to no significant effect. There appeared to be no obvious relationship between inhibitory activity and protein composition but factor VIII itself was not responsible for the effect as affinity purified products were entirely non-inhibitory. The two wet-heated products were most inhibitory whereas dry-heated products were less inhibitory or non-inhibitory. However, a wet-heated version of a non-inhibitory dry-heated product was also non-inhibitory, suggesting that the composition of the concentrate rather than anti-viral treatment is important for immunosuppressive activity. A product treated by the solvent/detergent procedure showed considerable inhibitory activity. Immunoglobulin and albumin products did not inhibit IL-2 secretion to any significant extent, but factor IX concentrates were inhibitory. We suggest that inhibition of IL-2 secretion by factor VIII concentrates may be related to the immunosuppression observed in haemophiliacs treated with high dose factor VIII products and that our results should be considered by clinicians and manufacturers of factor VIII products.

Antibodies, Monoclonal↗
PubMed1988

The international standard for human interleukin-2. Calibration by international collaborative study.

Three lyophilized preparations of interleukin-2 coded 86/500, 86/564 and 86/504 have been evaluated in an international collaborative study for their suitability as an international standard. All of the preparations performed well in the different bioassay systems included in the study, and showed excellent stability on accelerated temperature degradation. Material similar to that in preparation 86/504 has served well as an interim reference reagent for interleukin-2 for 3 years. Therefore with the agreement of the study participants and the authorization of the Expert Committee on Biological Standardization of the World Health Organization, the preparation coded 86/504 was established in 1987 as the 1st international standard for interleukin-2, with a defined potency of 100 IU/ampoule.

Animals↗
PubMed1988

The use of Tween 20 alone as a blocking agent for immunoblotting can cause artefactual results.

The use of Tween 20 as a suitable blocking agent in immunoblotting studies was evaluated by screening a panel of monoclonal antibodies (MoAbs) against a selection of blotted proteins which were unrelated to the antigens used to raise the MoAbs. Using Tween 20 alone to block the nitrocellulose membranes clear reactions were observed between the panel of MoAbs and several components of the blotted protein mixture. In contrast, when haemoglobin was used to block the membranes such reactions were not observed. In the absence of added protein the use of Tween 20 alone as a blocking agent for immunoblotting appears to lead to false positive reactions by non-specific antigen-antibody complexes.

Antibodies, Monoclonal↗
PubMed1988

HTLV-1 infection in tropical spastic paraparesis: lymphocyte culture and serologic response.

All 17 patients with tropical spastic paraparesis (TSP) in a series seen in the United Kingdom have antibodies to the human T cell leukemia virus type 1 (HTLV-1). Cultured peripheral blood lymphocytes from these patients formed multinucleated giant cells and reacted with sera and monoclonal antibodies to HTLV-1 in a manner identical to adult T cell leukemia-lymphoma (ATLL) patient lymphocytes. Western blot analysis failed to reveal any marked difference in the antigens recognized by sera from TSP and ATLL patients. The sera from TSP patients, their asymptomatic relatives and ATLL patients were titrated using the following assays: enzyme-linked immunosorbent assays (ELISA), particle agglutination, antibody-dependent cell-mediated cytotoxicity, and pseudotype neutralization. There were significantly stronger serologic responses in the TSP patients than in their relatives or ATLL patients. High antibody titers in the presence of replicating virus often reflect the antigen load; however, these data are also consistent with the suggestion that neurologic damage in TSP may be immunologically mediated.

Adult↗
PubMed1988

Monoclonal antibodies to crosslinked fibrin degradation products (XL-FDP). I. Characterization and preliminary evaluation in plasma.

Monoclonal antibodies (mabs) were raised against X-oligomers, the earliest soluble fragments released from crosslinked fibrin (XL-FN), by the action of plasmin. Two of the mabs (NIBn 52 and NIBn 123) were monospecific for X-oligomers in that they showed no binding to fibrinogen, the plasmic fragments of fibrinogen (D and E) and non-crosslinked fibrin (X, Y, D and E), or the terminal digestion product of XL-FN, fragment DD-E. One other mab (NIBn 178) was panspecific for X-oligomers in that it exhibited a weak affinity for fibrinogen. The mabs were used to develop a two-site immunoradiometric assay (IRMA) and an enzyme-linked immunospecific assay (ELISA) which permitted the specific measurement of X-oligomers directly in plasma, rather than in serum. This immunoassay is a true assay of fibrinolysis as distinct from fibrinogenolysis and may be a potential aid in the diagnosis and evaluation of thrombosis. In preliminary studies, the assay detected low levels of X-oligomers in normal plasma and elevated levels in patients with disseminated intravascular coagulation.

Antibodies, Monoclonal↗
PubMed1988

Monoclonal antibodies to crosslinked fibrin degradation products (XL-FDP). II. Evaluation in a variety of clinical conditions.

Plasmas from patients with a wide variety of thrombotic and presumed prethrombotic conditions were examined for high molecular weight crosslinked fibrin degradation products (known as X-oligomers) using a two-site enzyme-linked immunospecific assay (ELISA). This assay employed a catcher-tag principle using two monoclonal antibodies (mabs) directed towards different epitopes on the complex X-oligomer fraction. In general, thrombotic events (pulmonary embolism, PE, myocardial infarction, MI, peripheral vascular disease, PVD, and disseminated intravascular coagulation, DIC) were accompanied by elevated levels of X-oligomers in the plasma. During pregnancy the value of X-oligomer assays was demonstrated to be a clear-cut marker for pre-eclampsia. Patients following a variety of forms of surgery present with heterogeneous plasma levels of X-oligomers and this may merely reflect the formation and lysis of the fibrin formed during and after surgery. The possible value of this ELISA procedure in monitoring thrombolytic therapy is discussed with a critical analysis of the data presented herein. While the assay of X-oligomer was demonstrated to be a valuable marker of fibrinolysis in plasma, more extensive data are required in order to assess whether such an assay is of diagnostic value in thrombosis-related conditions.

Antibodies, Monoclonal↗
PubMed1988

Experimental studies on red cell-based assays for total IgE and allergen-specific IgE.

The potential of red cell-based assays for IgE and allergen-specific IgE has been examined using mouse monoclonal anti-human IgE antibodies and chimaeric human IgE anti-4-hydroxy-3-iodo-5-nitrophenacetyl. Experiments concerned with developing a red cell IgE antibody-capture assay for allergen-specific IgE have pointed to the advantages of presenting the allergen on a second agglutinable red cell.

Allergens↗
PubMed1988

Sensitive and specific immunoradiometric assays for human interleukin-1 alpha.

In order to estimate human IL-1 alpha in physiological samples we have established sensitive and specific immunoradiometric assays using monoclonal and polyclonal antibodies. Solid phase assays employing two different monoclonal antibodies were suitable for estimating rDNA derived IL-1 alpha but were relatively insensitive. Sensitivity was increased by using a monoclonal antibody to capture antigen and 125I-labelled polyclonal sheep antibodies to develop the assay. This assay could detect as little as 20pg/ml IL-1 alpha and could be used for assaying antigen in monocyte supernatants, in human skin extracts and in joint fluid from patients with inflammatory disease. It was entirely specific for IL-1 alpha and was unaffected by non-IL-1 alpha components present in serum. It is therefore suitable for monitoring IL-1 alpha concentrations in the circulation of patients undergoing therapy with the monokine.

Antibodies, Monoclonal↗
PubMed1988

Assay of pyrogenic contamination in pharmaceuticals by cytokine release from monocytes.

In 1984 it was reported that a batch of human recombinant growth hormone which had passed the rabbit pyrogen test and a Limulus amoebocyte lysate (LAL) test was pyrogenic in man (1). Further, it was shown that the growth hormone caused human monocytes to release leukocyte pyrogen which evoked fevers in rabbits (1). Leukocyte pyrogen is believed to comprise of a mixture of (at least) 2 lymphokines with potent pyrogenic and inflammatory activities, interleukin-1 (IL-1) and Tumour Necrosis Factor (TNF), which are synthesized and released by activated peripheral blood monocytes. The development at NIBSC of sensitive and specific bioassays and immunoassays of IL-1 (2) and TNF (3) have permitted a new type of pyrogen test based on the in vitro release by human monocytes of the lymphokines IL-1 and TNF. Studies are underway to evaluate the suitability of monocytic cell lines, rather than human monocytes, since these require a time-consuming and labour intensive preparation. Investigations are in progress to evaluate the suitability of the above "monocyte test" for pyrogen to replace or complement the rabbit pyrogen test which is routinely applied to biological medicines. The new method has already proved useful in detecting pyrogenic contamination of a large volume parenteral medicinal product which passed the rabbit pyrogen test but caused adverse reactions in patients.

Cells, Cultured↗
PubMed1987

A simple sensitive bioassay for interleukin-1 which is unresponsive to 10(3) U/ml of interleukin-2.

A subclone, NOB-1, of the mouse EL-4 line constitutively produces very little interleukin-2 but in response to interleukin-1 produces high concentrations of interleukin-2. Co-stimulation with mitogen, phorbol esters or calcium ionophores was not required. NOB-1 is not responsive to tumour necrosis factor alpha, tumour necrosis factor beta, interferon gamma and lipopolysaccharide. The NOB-1 line was used in conjunction with a CTLL line to detect less than 1 pg/ml interleukin-1. Rapid assay was performed by co-culturing the EL-4 cells with CTLL cells. By incorporating a pre-incubation step, followed by thorough washing of the EL-4 cells, responses to interleukin-1 were maintained, but interleukin-2 had no effect. The assay was used to detect interleukin-1 in serum samples and to evaluate neutralizing antisera to interleukin-1.

Animals↗
PubMed1987

An improved immunoblotting procedure for the detection of antibodies against HIV.

Immunoblotting ('Western blotting') is routinely used for detection of antibodies against HIV in the diagnosis of HIV infection. We describe an improved procedure, which does not require virus purification and is easy to control for 'false-positive' results. The technique also does not produce erroneous results due to reactivity of the developing system with residual cellular proteins or viral antigens and does not give high nonspecific background staining. The technique can be applied to the detection of antibodies to HIV in serum, plasma, and blood products.

Acquired Immunodeficiency Syndrome↗