Hans-Peter Ekre

Monoclonal antibody two-site ELISA for human IFN-γ: Adaptation for determinations in human serum or plasma

Mouse monoclonal antibodies (mAbs) against human interferon-gamma (IFN-gamma) were produced after immunization with recombinant IFN-gamma. Two mAbs (1-D1K and 7-B6-1) recognizing distinct epitopes on natural IFN-gamma were selected for the development of a two-site ELISA. The sensitivity was similar for IFN-gamma diluted in PBS with 1% bovine albumin, spent culture medium or fetal calf serum but reduced to approximately 50% when diluted in normal human serum. Individual normal human sera were tested and three of 14 gave false reactivities in the ELISA. One serum factor with major impact on the individual variation and the decreased sensitivity could be adsorbed to and eluted from protein A-Sepharose. Based on these observations we established a new ELISA protocol which made it possible to test for low levels of IFN-gamma in human serum and plasma samples. The modifications in this protocol are easy to apply with basic laboratory equipment.

Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.

Purification of pituitary and biosynthetic human growth hormoneusing monoclonal antibody immunoadsorbent

This report describes the purification of human growth hormone from crude pituitary extract and lysate of recombinant E. coli by an immunoadsorbent purification with monoclonal antibody coupled to solid phase. By a single-immunoaffinity chromatography step pure hGH can be obtained from either origin as revealed by SDS-PAGE followed by silver staining or immunoblotting. An additional ion-exchange chromatography step results in homogeneous 22 kDa hGH preparations. Furthermore, this method may be used for isolation of a pituitary hGH variant which has higher binding affinity for this monoclonal antibody than the major 22 kDa form. This study clearly illustrates the potential of monoclonal antibody immunoadsorbents for purification of different molecular forms of hGH.

Monoclonal antibodies recognize conformational alterations leading to decreased bioactivity of human growth hormone.

By means of monoclonal antibodies to five different antigenic determinants on human growth hormone (hGH) and polyclonal antisera from mice and rabbits, the immunoactivity of native hGH was compared with that of reduced and S-carboxymethylated hGH, which has an unstable conformation. Native and reduced and S-carboxymethylated hGH were also tested in a radioreceptor assay which reflects the bioactivity of the hormone. The IM-9 cell line was used as a receptor source in this assay. All five monoclonal antibodies were superior in discriminating between the native active form of hGH and its reduced and S-carboxymethylated form, which has a markedly reduced receptor binding activity.

High Molecular Weight Growth Hormone ( > 160 kD) in Human Serum Characterized with Monoclonal Antibodies

Human growth hormone (hGH) was analyzed by six monoclonal antibodies (Mabs) and a polyclonal antiserum (Pas) before and after molecular sieve chromatography of sera from healthy subjects. Their hGH levels were between < 0.2 and 0.4 ng/ml as determined with Pas. The six Mabs reacted with five distinct epitopes and bound to a hGH fragment corresponding to the amino acid sequence 15-125. Two of the Mabs showed reduced binding to 20-kD hGH. The binding of Mabs to dimeric forms of hGH varied. Human GH levels in unfractionated sera as determined with Mabs were < 3.1-390 ng/ml. After molecular sieve chromatography of the sera, one peak of hGH-immunoreactive material of high molecular weight (> 160 kD) and one at the elution volume of monomeric hGH were determined with Pas and Mabs. The major part of the high molecular weight hGH (> 160 kD) seemed to consist of 22-kD hGH molecules, since Pas and all Mabs detected the hGH immunoreactivity (> 160 kD) in a similar manner. This high molecular weight hGH (> 160 kD) was distinguishable from the identified, receptor-like hGH-binding protein in serum.

Immunochemical characterization of charge isomers of bacteria-derived human growth hormone with monoclonal antibodies.

Monoclonal antibodies were used to study the immunochemical nature of charge isomers of bacterially produced methionyl human growth hormone. After isoelectric focusing of the hormone the 12 monoclonal antibodies reacted similarly in immunoblotting experiments and none of them could discriminate between the two isolated charge isomers in ELISA. This indicates that the generation of charge isomers of met‐hGH does not result is loss of the determinants recognized by the monoclonal antibodies and that the conformation of the two main charge isomers is identical within these determinants.

Interferon-α: A gene family in therapeutic use☆

Abstract Several variants of interferon-α (IFN-α) were isolated and purified to homogeneity. They differed to various degrees in biological properties. However, three IFN-α2 variants showed only minor differences from a variant called IFN-α88 with regard to their ability to inhibit growth and to bind to specific receptors, tested on Daudi cells. Two monoclonal antibodies were studied, which showed overlapping specificity for at least one peptide obtained after HPLC separation of tryptic digests. The monoclonal antibodies could discriminate between sequence differences to a much higher degree than the receptor on Daudi cells. It is concluded that the receptor is degenerate and binds well to different structural variants of IFN and that for therapeutic use, several of the variants will probably have the same biological potency.