Christopher Bird

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.

Development of immunoassays for human interleukin 3 and interleukin 4, some of which discriminate between different recombinant DNA-derived molecules

Two panels of hybridomas were produced that secreted monoclonal antibodies (MAbs) against recombinant DNA-derived human interleukin 3 and interleukin 4 (rhIL-3 and rhIL-4). From each panel, sensitive immunoradiometric assays (IRMAs) were developed which were capable of detecting the recombinant molecule used as the immunogen but were unable to recognize natural or other recombinant forms of the same cytokine. Subsequent studies using the MAbs from each panel showed that a number of the MAbs appeared only to recognize that particular recombinant molecule used as immunogen, with little or no binding to other recombinant forms of the molecule. By using MAbs that were found to be unrestricted in their recognition for different recombinant forms of the cytokines, it was possible to develop an IRMA for IL-4 that was capable of detecting natural IL-4 as well as all the recombinant forms equally. An IRMA was also developed for IL-3 but was not of equivalent sensitivity in detecting the different recombinant forms of IL-3 used in the study. The recombinant DNA-derived cytokine molecules used to raise the two panels of MAbs contained amino acid substitutions relative to the natural sequences, and these findings indicate that caution should be exercised when using immunoassays to estimate natural sequence molecules if antibodies raised to modified rDNA-derived molecules are used.

Nature of the endogenous pyrogen (EP) induced by influenza viruses: lack of correlation between EP levels and content of the known pyrogenic cytokines, interleukin 1, interleukin 6 and tumour necrosis factor

Fever in influenza results from the release of endogenous pyrogen (EP) following virus-phagocyte interaction and its level correlates with the differing virulence of virus strains. However, the different levels of fever produced in ferrets by intracardial inoculation of EP obtained from the interaction of different virus strains with ferret of human phagocytes did not correlate with the levels of interleukin 1 (IL-1), IL-6 or tumour necrosis factor in the same samples as assayed by conventional in vitro methods. Hence, the EP produced by influenza virus appears to be different to these cytokines.

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.

Production of polyclonal and monoclonal antibodies to human granulocyte colony-stimulating factor (GCSF) and development of immunoassays

Murine monoclonal antibodies and a sheep polyclonal antiserum against recombinant human granulocyte colony-stimulating factor (GCSF) have been produced. These have been used to develop an immunoassay which can detect 250 pg/ml (25 U) of both natural and recombinant human GCSF. The assay involves forming a complex between GCSF and a monoclonal anti-GCSF, binding of the complex to microtitre wells coated with sheep anti-GCSF and detection of the bound complex with 125I-labelled sheep anti-mouse IgG. Unlike the classical bone marrow assay and other cell line based bioassays for GCSF, the immunoassay was specific for the cytokine, showing no cross-reactivity with GM-CSF, IL-6, IL-3 or IL-1 alpha and -beta. The assay does not exhibit interfering matrix effects when used for the estimation of human GCSF in serum.

Freeze drying formulation using microscale and design of experiment approaches: a case study using granulocyte colony-stimulating factor.

The lyophilization of proteins in microplates, to assess and optimise formulations rapidly, has been applied for the first time to a therapeutic protein and, in particular, one that requires a cell-based biological assay, in order to demonstrate the broader usefulness of the approach. Factorial design of experiment methods were combined with lyophilization in microplates to identify optimum formulations that stabilised granulocyte colony-stimulating factor during freeze drying. An initial screen rapidly identified key excipients and potential interactions, which was then followed by a central composite face designed optimisation experiment. Human serum albumin and Tween 20 had significant effects on maintaining protein stability. As previously, the optimum formulation was then freeze-dried in stoppered vials to verify that the microscale data is relevant to pilot scales. However, to validate the approach further, the selected formulation was also assessed for solid-state shelf-life through the use of accelerated stability studies. This approach allows for a high-throughput assessment of excipient options early on in product development, while also reducing costs in terms of time and quantity of materials required.

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.