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.     

A common epitope of beta-lactoglobulin and serum retinol-binding proteins: elucidation of its core sequence using synthetic peptides.

One of the monoclonal antibodies raised against bovine beta-lactoglobulin reacted with human serum retinol binding protein. The finding that this monoclonal antibody also reacted with the serum retinol binding proteins isolated from other animals, suggested that this epitopic conformation is conserved among these proteins. Using ELISA and various synthetic peptides of defined sequence, we show in this paper that the epitope defined by this monoclonal antibody comprises of the highly conserved core sequence of DTDY present in beta-lactoglobulin and retinol binding proteins.     

Comparison of the antigenicities of native human growth hormone (hGH) and three forms of recombinant hGHs using monoclonal antibodies

A panel of hybridomas producing antibodies specific for human growth hormone (hGH) were prepared by using a recombinant hGH [methionylsomatotropin (r-hGH)] as an immunogen. Thirteen representative monoclonal antibodies which showed different reactivity patterns were used to analyze the antigenicities of four different forms of hGHs by RIA inhibition studies. Native hGH and r-hGH showed almost the same antigenicities with these monoclonal antibodies. A Cys-substituted recombinant hGH (r-hGH-165) retained the epitopes recognized by 11 monoclonals but not those recognized by two monoclonals. All except one of the monoclonals showed little or no reactivity with a recombinant hGH fragment (r-hGH-AB). On the basis of these results, the differences in the structures and antigenicities of the recombinant hGH proteins were discussed.     

Properties of Cryptic Epitopes and Their Corresponding Antibodies as Indicated by the Study of Human and Ovine Growth Hormones

Antibodies (Ab) directed to hidden antigenic determinants (cryptotopes) are undesirable because they are not neutralizing. Additionally, we have previously demonstrated a close association between the extent of Ab to cryptic determinants and the expression of autoantibodies (autoAb) under some experimental conditions. Thus, the first objective of this work was to establish the physicochemical characteristics of Ab to cryptotopes and the second one was to examine the structural features of cryptic epitopes themselves. Using human and ovine growth hormones (hGH and oGH) as antigenic models and competition ELISA under different conditions of temperature, pH or ionic strength, we did not find any difference between the binding properties of anti-cryptic epitope antibodies (Ab) and anti-native epitope Ab. Then, using synthetic peptides and tryptic digests and direct and competition ELISAs we studied the structures of cryptic hGH and oGH epitopes. Isolated peptides either in solution or adsorbed on microplates failed to react. Partially digested hGH was recognized only when insolubilized on microplates, and anti-oGH Ab only reacted with a large fragment of the hormone either in solution or insolubilized. These results indicate that, at least in the case of hGH and oGH, cryptic epitopes are not simple linear sequences, as commonly referred without any evidence, but new exposed conformational structures different from those found in the native antigen.     

Properties of cryptic epitopes and their corresponding antibodies as indicated by the study of human and ovine growth hormones

Antibodies (Ab) directed to hidden antigenic determinants (cryptotopes) are undesirable because they are not neutralizing. Additionally, we have previously demonstrated a close association between the extent of Ab to cryptic determinants and the expression of autoantibodies (autoAb) under some experimental conditions. Thus, the first objective of this work was to establish the physicochemical characteristics of Ab to cryptotopes and the second one was to examine the structural features of cryptic epitopes themselves. Using human and ovine growth hormones (hGH and oGH) as antigenic models and competition ELISA under different conditions of temperature, pH or ionic strength, we did not find any difference between the binding properties of anti-cryptic epitope antibodies (Ab) and anti-native epitope Ab. Then, using synthetic peptides and tryptic digests and direct and competition ELISAs we studied the structures of cryptic hGH and oGH epitopes. Isolated peptides either in solution or adsorbed on microplates failed to react. Partially digested hGH was recognized only when insolubilized on microplates, and anti-oGH Ab only reacted with a large fragment of the hormone either in solution or insolubilized. These results indicate that, at least in the case of hGH and oGH, cryptic epitopes are not simple linear sequences, as commonly referred without any evidence, but new exposed conformational structures different from those found in the native antigen.     

UV-treated polystyrene microtitre plates for use in an ELISA to measure antibodies against synthetic peptides.

Detection of peptide-specific antibodies by the conventional ELISA technique is sometimes hampered by the difficulties encountered in immobilizing stretches of amino acids on the solid support. To improve the attachment of synthetic peptides to the solid phase, we have developed a sensitive and rapid immunoassay based on the irradiation of polystyrene plates with UV light prior to coating the target peptide. This pretreatment increases the specific signal in a dose-dependent manner without augmenting the background or altering the specificity of the assay. This simple method was shown to be suitable for the quantitation of murine monoclonal antibodies as well as human and rabbit polyclonal antibodies. It should be applicable to a variety of synthetic peptides and polystyrene ELISA plates. Using this technique, we were able to localize the antigenic motifs recognized by neutralizing monoclonal antibodies generated against the envelope protein gp120 of the human immunodeficiency virus.     

Detection of circulating immune complexes with a Raji cell enzyme immunoassay

The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4 X 10(10) l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay.     

Antigenic sites of human growth hormone and related molecules detected by monoclonal antibodies to human growth hormone

Four major antigenic sites for human growth hormone (hGH) were identified by 27 mouse monoclonal antibodies to hGH. Sites 1 and 2 are spatially close whereas sites 3 and 4 are located in other parts of the molecule. There also appears to be a subdivision of antigenic sites. A panel of 10 monoclonal antibodies, which included representatives from each antigenic site group, were used to determine cross-reactivities between hGH and human placental lactogen (hPL), human prolactin (hPRL), the 20,000 mol. wt variant of hGH (hGH20K) and a disulfide-linked dimer of hGH (diS-dimer). The data suggest a high conformational dependence of antigenic sites in hGH. DiS-dimer retains all four antigenic sites of hGH, although all have been altered. hGH20K retains sites 2-4 but site 1 has been dramatically altered. hPL retains site 3, whereas sites 1 and 4 have been dramatically altered and site 2 may be lacking. The extremely low cross-reactivity observed for hPRL is consistent with the dissimilarity between hGH and hPRL. Antigenic site 3 is the most conserved of all sites. The lack of structural similarity compared with hGH of site 1 in hGH20K and of a portion of site 3 in diS-dimer suggests that it may be possible to develop specific radioimmunoassays for these structural variants of hGH.     

Persistent and enhanced expression of c-fos gene products in various kinds of human hematopoietic cell lines. Immunofluorescence study using prepared monoclonal antibodies

Two kinds of monoclonal antibodies recognizing fos proto-oncogene (c-fos) products were prepared using a synthetic oligopeptide corresponding to amino acids 127-152 of the fos oncogene products. These monoclonal antibodies (FO-120 & FO-145) detected fos gene products induced in a human monocyte cell line (U-937) by phorbol acetate (TPA) and induced in both human and mouse fibroblast cell lines (284, BALB/c 3T3) by serum-stimulation. One of the monoclonal antibodies (FO-120) reacted with 50-kDa and 42-kDa proteins and the other antibody (FO-145) reacted with a 30-33-kDa protein. The expression of the fos gene in various human hematopoietic cell lines was investigated using these prepared monoclonal antibodies. While almost all hematopoietic cell lines tested reacted with these monoclonal antibodies to various degrees, the majority of normal peripheral blood lymphocytes cultured with lectin (PHA) and interleukin 2 (IL-2) did not, suggesting that cells of some permanent hematopoietic cell lines, irrespective of their lineage specificity and growth factor dependency, continuously express the fos oncogene. These monoclonal antibodies may be useful for detecting early neoplastic changes in hematopoietic cells.     

Immunodominant structures of human growth hormone identified by homolog-scanning mutagenesis.

Homolog-scanning mutagenesis has been reported to be useful in elucidating the antigenic epitopes recognized by monoclonal antibodies and hGH binding to its receptor. However, little is known about which structures are recognized as immunodominant by murine serum antibodies. Therefore, the previously published series of hGH homologs and additional mutants of human placental lactogen (hPL), porcine growth hormone (pGH), and human prolactin (hPRL) were examined for their interaction with murine serum derived anti-hGH antibodies. As compared to wild-type hGH, nine of the nineteen segment substituted mutants tested showed a significant reduction in binding to anti-hGH sera. These disruptive substitutions mapped to 5 regions on a structural model of hGH: the length of helix 1 (residues 11-33), the loop between the first disulfide bond and helix 2 (residues 54-74), the beginning of helix 3 (residues 109-112), the carboxyl half of helix 4 (residues 167-182), and the final carboxyl terminus segment of the molecule (residues 184-191). In terms of the current structural model, three of the five immunodominant regions (the loop between residues 54-74, central portion of helix 4 to the carboxyl terminus and part of the amino terminus region of helix 1) closely overlaps the hGH receptor binding epitopes.     

PERSISTENT and ENHANCED EXPRESSION OF c-fos GENE PRODUCTS IN VARIOUS KINDS OF HUMAN HEMATOPOIETIC CELL LINES. Immunofluorescence Study Using Prepared Monoclonal Antibodies

Two kinds of monoclonal antibodies recognizing fos proto-oncogene (c-fos) products were prepared using a synthetic oligopeptide corresponding to amino acids 127-152 of the fos oncogene products. These monoclonal antibodies (FO-120 & FO-145) detected fos gene products induced in a human monocyte cell line (U-937) by phorbol acetate (TPA) and induced in both human and mouse fibroblast cell lines (284, BALB/c 3T3) by serum-stimulation. One of the monoclonal antibodies (FO-120) reacted with 50-kDa and 42-kDa proteins and the other antibody (FO-145) reacted with a 30-33-kDa protein. The expression of the fos gene in various human hematopoietic cell lines was investigated using these prepared monoclonal antibodies. While almost all hematopoietic cell lines tested reacted with these monoclonal antibodies to various degrees, the majority of normal peripheral blood lymphocytes cultured with lectin (PHA) and interleukin 2 (IL-2) did not, suggesting that cells of some permanent hematopoietic cell lines, irrespective of their lineage specificity and growth factor dependency, continuously express the fos oncogene. These monoclonal antibodies may be useful for detecting early neoplastic changes in hematopoietic cells. ACTA PATHOL JPN 38: 1523-1536, 1988.     

Production and characterization of monoclonal antibodies to anti-human chorionic somatomammotropin by immunization with two free synthetic peptides

The peptides corresponding to the fragments 135-140 and 166-174 of human chorionic somatomammotropin (hCS) were synthesized, and used to raise monoclonal antibodies to the native hCS molecule. The synthetic peptides were injected into BALB/c mice in the free form, i.e. not conjugated to a carrier, and the spleens were fused with Sp2/01Ag8 myeloma line to produce monoclonal antibodies. The antibodies produced belonged to the IgM and IgG classes and, once purified by affinity chromatography on hCS-Sepharose, they were covalently coupled to macroporous polystyrene beads and characterized by competitive radioimmunoassay. Their affinity constants were determined by elaborating the radioimmunoassay data by nonlinear regression analysis and they were found to range from 10(5) to 10(6) M-1. The evaluation of the affinity constant of the antibodies produced is always important as a measure of the immunogenicity of an antigen, particularly when synthetic peptides are used as immunogens.     

Study of antigenic structure and inhibition of activity of human growth hormone and chorionic somatomammotropin by monoclonal antibodies

Distinct antigenic determinants were identified on native molecules of human growth hormone (hGH) and chorionic somatomammotropin (hCS) on the basis of competitive inhibition assays with eight murine monoclonal antibodies. Effective competition for antigen binding within a pair of antibodies indicated overlapping combining site specificities whereas a lack of competition suggested binding to sterically distinct structural moieties. An antigenic determinant, specific for hGH was detected by antibodies QA68 and NA27. whilst another marginally hCS-cross-reactive site was bound by NA71. Two distinct determinants fully expressed by either hGH or hCS were bound by antibody pairs NA39/EB2 and EBI/EB3 respectively, whereas a single hCS-specific determinant was recognized by antibody EB4. An unexpected reciprocal cross-inhibition of soluble antigen-antibody complex binding was observed between antibodies reacting to distinct determinants, i.e. for NA27 towards NA39/EB2 and for NA71 towards EBI/EB3. These results were tentatively interpreted in terms of conformational changes of antigen when bound in soluble immune complexes, but an alternative explanation of steric hindrance cannot yet be excluded. The effect of monoclonal antibodies on the hormonal biological activity was investigated in a dose-response study of the hormone-dependent growth stimulation of NB2 lymphoma cells in tissue culture. Although all eight antibodies were specifically growth-inhibitory, major quantitative differences in their efficacies have been observed. At limiting hormone doses antibodies EB2/NA39 were most effective whereas QA68 and NA71 were the most potent at excess hormone input. Various mechanisms operating through inhibition of hormone binding and/or modulation of cell receptor-bound complexes have been considered.     

Two monoclonal antibodies to two different epitopes of human growth hormone form a precipitin line when counterdiffused as soluble immune complexes

We have tested whether soluble immune complexes obtained by mixing human growth hormone (hGH) with one anti-hGH monoclonal antibody (MAb) can form a precipitin line when diffused against another MAb in a polyethylene glycol containing gel. By testing seven anti-hGH MAbs one against the other in this assay, we have found that 10 pairs of MAbs out of the 21 possible combinations formed a line. Apparently, the first MAb formed soluble hGH dimers that were linked by the second MAb into precipitating linear complexes. Since each precipitin line was formed by the cooperative reaction of two MAbs, this sequential reaction of MAbs may be used in methods for the positive selection of MAbs that are suitable for two-site immunoassays.     

T-cell receptors of man and mouse studied with antibodies against synthetic peptides.

We used polyclonal rabbit antibodies directed against synthetic peptides predicted from the gene sequence of the human T-cell receptor (TCR) beta-chain YT35 to study the antigen receptor on human helper T-cell leukemia lines and on normal mouse thymocytes. Antibodies were raised to peptides corresponding to joining segment (J beta) and to a conserved stretch of sequence around the first cysteine in the constant region (C beta). These peptides were selected on the basis of homology with corresponding segments of immunoglobulin light chains. The specificity of the antibodies was established using synthetic overlapping peptides that modelled the complete TCR beta-chain. Western blot analysis was performed against detergent lysates of T cells. Both of the antibodies reacted strongly with 2-3 polypeptides in the mass range 40-45 kDa in mouse and human cells. Clearance experiments using monoclonal antibodies against murine TCR alpha- and beta-chains and against human TCR beta-chain and immunoprecipitations with monoclonal antibody to the murine T3 complex established that these components represented the alpha/beta heterodimer. An additional component around 31 kDa was detected by anti-J beta antibodies in murine thymus extracts. The use of the affinity-purified antipeptide antibody in two-dimensional Western blot analyses allows the clear discrimination between the characteristic individual receptors of monoclonal neoplastic T cells and the polydisperse patterns representative of heterogeneous normal populations. Antigenic cross-reactions between T-cell receptor beta-chains of man and mouse observed with monoclonal antibodies and rabbit antisera to peptides are consistent with the homology in gene sequence between the two species.     

Analysis of human antibody epitopes on the 65-kilodalton protein of Mycobacterium leprae by using synthetic peptides.

In order to study antibody reactivity to the Mycobacterium leprae 65-kilodalton (kDa) antigen, peptides representing overlapping sequences of the 65-kDa protein were synthesized, and a recombinant protein expression system for r65-kDa was constructed. Mouse monoclonal antibodies and leprosy patient seroreactivity to peptides and r65-kDa were tested by an enzyme-linked immunosorbent assay. All seven of the monoclonal antibodies used in this study reacted with their previously defined epitopes when tested against peptides. All monoclonal antibodies also reacted with r65-kDa. Leprosy patient seroreactivity to peptides and r65-kDa was seen in about one-third of active multibacillary cases. Specimens from patients positive for antibodies to peptides were seen to recognize different epitopes than did mouse monoclonal antibodies used in this study. It is concluded that substantial differences exist between mouse monoclonal antibodies and human leprosy patient reactivity to the 65-kDa antigen and that human seroreactivity to the 65-kDa antigen is indicative of a highly elevated bacillary load.     

The antigenic topography of human growth hormone

Murine monoclonal antibodies (MAb) have been used as tools to probe the antigenic topography of human growth hormone (hGH). Mapping experiments were carried out by testing the ability of paired MAb to bind simultaneously or separately to 125I-hGH. A putative three-dimensional model of the relative distribution of 20 hGH epitopes indicated that they covered the entire molecular surface, showing the following essential characteristics. A domain of unique hGH specificity representing approximately 20% of the whole area was detected, as well as the presence of a discontinuous band of immunological identity between hGH and human placental lactogen (hPL) occupying 30% of the molecular surface. The rest of the surface (about 50%) displayed only partial cross-reactivity with hPL. Three restricted antigenic areas were also recognized. One of them appeared to correlate with a conformational change induced by the adsorption of the protein to plastic surfaces and the other two showed cross-reactivity with human prolactin and heterologous GH, respectively.     

Multiple epitope interactions in the two-step sandwich immunoassay.

The 'hook' effect as related to the two-step sandwich immunoassay has been investigated experimentally and theoretically. The multiple epitope interactions between the analyte and the labeled antibody cause a 'hook' in the two-step sandwich immunoassay. Three different analytes and monoclonal antibodies were chosen to carefully demonstrate the effect of the analyte characteristics on this immunoassay. Two monoclonal antibodies against two different epitopes of biosynthetic human growth hormone (hGH) was the simplest model for this study. The sandwich immunoassay for hGH shows no 'hook' effect. The non-covalent dimeric form of hGH (D-hGH) possesses two repeating epitopes which is the simplest model for an analyte having a discrete number of repeating epitopes. The D-hGH assay demonstrated a 'hook' effect in the two-step sandwich immunoassay if the labeled antibody was allowed to interact with more than one epitope. In a third system multiple epitope interactions with the labeled antibody were observed using ferritin. The effect of the analyte concentration and the liquid-phase antibody have been examined to elucidate the nature of these various interactions. The cause of the 'hook' effect in the two-step sandwich immunoassay is attributed to the desorption of the bound analyte due to a conformational change after the labeled antibody interacts with several epitopes of the adsorbed analyte.     

A relevant antigenic site in human growth hormone localized in sequence 98–128

The immunoreactivity of six synthetic peptides covering different lengths of human growth hormone antigenic region 73–128 has been studied using antisera from rabbits and guinea pigs and sera from two familial prenatal growth hormone-deficient patients submitted to replacement therapy. This survey disclosed that peptide 98–128 was responsible for the total antigenicity of human growth hormone, strongly indicating that a relevant epitope(s) is located in this region of the molecule.     

Production and characterization of monoclonal antibodies to human growth hormone

Three BIOZZI-HR mice were immunized with human growth hormone (hGH). From the determination of the titer, the average equilibrium association constant and the heterogeneity index of the antisera, it was possible to select the most suitable mouse for production of monoclonal antibodies (Mabs). Resulting from a single fusion, eight Mabs were produced, purified and characterized. The equilibrium association constant of the Mabs ranged from 5.10(8) M-1 to 9.109 M-1 at physiological pH. Four areas on hGH are recognized by the Mabs (the topology of the Mabs was investigated by two-site immunoradiometric assays). The Mabs, which recognize a same area, show similar cross-reactivities between hGH and human Placental Lactogen (hPL). No selected Mabs bound human Prolactin (hPRL), equine Growth Hormone (eGH) and porcine Growth Hormone (pGH). Two complementary Mabs enable a two-site immunometric assay of pituitary and E. Coli derived hGH.