Ingileif Jónsdóttir

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder

SummarySpleen cells from BALB/c mice immunized with cells derived from transitional cell carcinomas (TCC) of the human urinary bladder were fused with mouse myeloma Sp 2/0 Ag14 cells. Monoclonal antibodies from six established hybridomas were investigated for specificity in a cell ELISA and in indirect immunofluorescence against a large panel of fixed intact cells. Three of the antibodies reacted with half or more of the eight bladder tumors and with a few unrelated tumors. They did not react at all with malignant or normal cells of hematopoietic origin. A fourth antibody reacted with seven of eight bladder tumors. It also reacted weakly with a prostatic carcinoma, with five of six malignant or transformed B cell lines, and with a subpopulation of normal lymphocytes, but not with any of the other cells on the test panel. These four antibodies did not react with cells derived from normal urothelium. The results suggest that these antibodies might recognize cell-type-restricted antigens associated with malignancy. Another antibody reacted with almost all urothelium-derived cells. It also reacted with three of three melanomas but not with any other cells on the panel. The sixth antibody reacted with 32 of the 37 cells tested. The spectrum of reactivities displayed by the antibody suggested that it recognizes HLA antigens.

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.

Serum Levels and Molecular Sizes of Growth Hormone during Pregnancy in Relation to Levels of Lactogens, Insulin-Like Growth Factor I and Insulin-Like Growth Factor Binding Protein-1

Growth hormone (GH), placental lactogen (PL), prolactin (PRL), insulin-like growth factor I (IGF-I) and IGF binding protein-1 (IGFBP-1) were determined in serum by radioimmunoassays (RIAs) in 12 women during pregnancy. GH and PL were analyzed by two monoclonal antibodies (Mab 3 and Mab 1) raised against pituitary GH. Serum IGFBP-1 had reached maximum levels at midpregnancy while PRL, PL and IGF-I increased continuously during pregnancy. Mab 1, which cross-reacts with PL, measured consistently higher levels of PL in serum than a commercial PL RIA (p less than 0.01) due to interference of cross-reacting serum proteins in the Mab 1 RIA. The GH-specific Mab 3 showed decreasing GH levels in unfractionated serum throughout gestation, but detected GH-immunoreactive proteins of approximately 40-200 kD after molecular sieve chromatography of pooled serum from late pregnancy. It is suggested that the formation of GH complexes of large molecular mass account for the successive disappearance of monomeric GH during pregnancy.

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.

Monoclonal Antibodies Against Human Growth Hormone

Abstract Hybridoma lines secreting monoclonal antibodies (McAbs) against hGH were established, and specificity studies of antibodies from three such lines performed. One of the McAbs cross reacts with hCS whereas the others do not, indicating that the different McAbs are directed against at least two distinct antigenic determinants. The cross reacting McAb also possesses the highest relative affinity for hGH. These antibodies were used to compare pituitary hGH and biosynthetic hGH produced in bacteria by means of recombinant-DNA, technique. In an indirect competitive ELISA. and in an indirect passive hemagglutination inhibition assay none of the McAbs could discriminate biosynthetic from pituitary hGH. This provides evidence for idential amino acid sequence and conformation in the two hormone preparations within at least two antigenic regions.