N- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells.

Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt. Muteins expressed in stable cell lines showed similar differences in expression levels. Also each mutein could be affinity-purified from culture supernatants, and was biologically active in vivo. Based on secretion rates from BHK-21 cells, the most potent erythropoietin was rhuEpoGln24. This mutein is also considered to have biologic activities that are superior to rhuEpowt.

Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization.

Recombinant human erythropoietin (rhuEpo)-specific mouse monoclonal antibodies (MoAbs) have been produced and characterized. All antibodies were specifically reactive with rhuEpo in enzyme-linked immunosorbent assay (ELISA). Epitope exclusion studies showed three distinct epitope regions, A, B, and C, recognized by neutralizing MoAbs. An additional epitope region D was recognized by non-neutralizing MoAbs. Antibodies defining an epitope region competed with each other for binding sites, but did not compete with antibodies defining a different epitope region. Group B antibodies were able to compete for the receptor binding site on rhuEpo with a soluble human Epo-receptor-lg fusion protein. No single peptide sequences were found to specifically interact either with group B MoAbs or with the rhuEpo-receptor. Therefore, it is suggested that epitope region B and the receptor binding site share binding determinants that are primarily composed of conformational epitopes. Because group A and group C antibodies did not compete with the receptor for binding to the receptor binding site of the rhuEpo molecule, it is suggested that neutralization via epitope regions A and C is mediated through binding inhibition caused by conformational changes, transmuting the binding site(s) for the receptor. Conversely, binding to the receptor seems to induce conformational changes in the hormone molecule, eliminating epitopes for group A and C antibodies.

An enzyme linked immunosorbent assay for the detection of human interleukin 3 in serum.

A sandwich enzyme immunoassay was developed for measuring human Interleukin 3 (IL-3) in human and animal sera. Polyclonal and monoclonal antibodies were raised against the recombinant human protein. These have been used to develop an immunoassay which can detect down to 10 pg/ml of human IL-3. The assay involves a polyclonal rabbit antibody coupled to a solid phase and a mouse monoclonal antibody-horseradish peroxidase conjugate as the detection antibody. Unlike the classical bone marrow assay and other cell line based bioassays for IL-3, the immunoassay was specific for the cytokine showing no or only negligible cross-reactivity with IL-1, IL-2, IL-6, erythropoietin, granulocyte colony-stimulating factor (G-CSF) and GM-CSF. The assay does not exhibit interfering matrix effects when used for the estimation of human IL-3 in serum samples.

Highly specific and highly sensitive enzyme immunoassays for antibodies to human interleukin 3 (IL-3) and human erythropoietin (EPO) in serum.

Two versatile, rapid, easy-to-perform and highly sensitive enzyme immunoassays (ELISAs) were developed for the detection and quantification of antibodies to human IL-3 and human EPO in serum samples from both man and laboratory animals. These one-step assays are based on the "inverse sandwich" principle, where antibodies present in the sample linked both the solid phase antigen bound to a microtiter plate and the free antigen, which had been covalently coupled to horseradish peroxidase. The limits of detection are lower than those of the neutralization bioassays; antibodies to IL-3 and to EPO were detected at concentrations as low as 2 ng/ml and 5 ng/ml, respectively. No cross-reactivity with related proteins and no interfering matrix effects were observed when used for the estimation of antibodies against rhu IL-3 and rhu EPO in serum samples of various species. Thus both assays were used without modification for the screening of antibodies in serum samples from both man and animal during treatment with these hematopoietic hormones.

Development of an ELISA for the detection and determination of contaminating proteins in recombinant DNA derived human erythropoietin.

An enzyme linked immunosorbent assay (ELISA) has been developed for the quantitative determination of the most probable contaminating proteins (MCP) of recombinant human Erythropoietin produced in the mouse fibroblast cell line C-127. The developed ELISA is a double polyclonal sandwich type immunoassay, which allows a quantitation of the MCPs in the range of parts per million. The polyclonal antibody, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference MCPs (a collection of the most probable protein contaminants) by immunoblot analysis and immunoabsorption of radiolabeled MCPs. Affinity purification of this antibody preparation using the immobilized MCPs resulted in an assay with higher signal-to-noise ratio. The assay was demonstrated to be very specific for the MCPs obtained from the rhu EPO purification process. Since the purification of each recombinant DNA derived protein expressed in mammalian cells requires its own unique process, no generic assay for contaminating proteins can be developed. There are only a few common criteria for the development of such multi-antigen ELISA, which will be discussed in this paper.

Production and characterization of a monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol.

A monoclonal antibody was obtained by the fusion of mouse myeloma cells with splenocytes isolated from Balb/c mice, which had been immunized with diacetoxyscirpenol-hemiglutarate (DAS-hemiglutarate) and verrucarol-hemiglutarates covalently bound to ethylenediamine-modified bovine serum albumin. The anti-DAS-antibody that could be induced was of the IgM type with kappa-chains. The titer of the monoclonal anti-DAS-antibody in ascites fluid obtained from mice injected the selected cell line was much higher than those of conventional antisera. An enzyme-linked immunosorbent assay based on the competitive binding principle in which the antibody was applied had a sensitivity of 1 ng DAS per assay. The relative cross-reactivity of the monoclonal antibody in the CI-ELISA with the related trichothecenes such as triacetoxyscirpenol, 15-monoacetoxyscirpenol, diacetylverrucarol, 4-monoacetoxyscirpenol and scirpentriol were found to be 1.8, 0.8, 0.15, 0.02 and less than 0.001, respectively. The trichothecenes verrucarol, T-2 toxin, T-2 tetraol, deoxynivalenol, 3-acetyldeoxynivalenol and trichothecin showed no cross-reactivity.