Monoclonal antibodies to human growth hormone can distinguish between pituitary and genetically engineered forms

Monoclonal antibodies (MABs) prepared against human pituitary growth hormone (hGH) have been compared for their binding to pituitary-derived and genetically engineered methionyl growth hormone (met-hGH). The antibodies bind to four non-overlapping epitopes of which two are completely shared with human choronic somatomammotropin (hCS). The determinant defined by MAB NA27 was expressed on met-hGH to a lesser degree than on hGH of pituitary origin. However, another antibody, QA68, which binds to a determinant closely related to NA27, failed to discriminate between hGH and met-hGH. A further two MABs (EB1 and NA71) were similarly ineffective in distinguishing between the two forms of the hormone. The determinant recognized by antibody EB2 was equally represented on hGH and met-hGH when assessed by a liquid-phase radioimmunoassay: however, measurement of the binding in a solid-phase assay resulted in a two-four-fold lower binding to met-hGH. Bioactivity assessed by both an in vitro cell proliferation assay and an in vivo cartilage sulphation bioassay failed to distinguish between the two hormones. It is therefore concluded that the NH2-terminal methionine on bacterially derived growth hormone results in altered antigenicity of the hormone without any measurable effect on bioactivity.     

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.     

Antigenic structure of human chorionic somatomammotropin: four epitopes revealed by monoclonal antibodies

The characterization of epitope specificity of a panel of 15 monoclonal antibodies against human chorionic somatomammotropin (hCS), previously prepared in our laboratory, allowed us to distinguish 4 antigenic determinants on the hCS molecule. Experiments were carried out with competition and sandwich assays. The results allowed us to divide our MAbs into 4 groups, recognizing 4 different epitopes, and to select 4 MAbs each distinguishing a different epitope in order to pursue further studies on the antigenic structure of hCS.     

Determinants of ryegrass pollen cytochrome c recognized by human IgE and murine monoclonal antibodies

In attempts to produce fragments of an allergenic molecule which would retain allergenic and/or antigenic determinant(s), the cytochrome c of ryegrass (RG) pollen, which had been shown to be an allergenic constituent of this pollen, was digested with trypsin and chymotrypsin and the resulting fragments were separated by high performance liquid chromatography. Several of these fragments were shown, with the aid of the radioallergosorbent test and solid phase radioimmunoassays, to bind IgE antibodies present in a pool of six sera from grass-sensitive patients and three murine monoclonal antibodies, designated as Mab 41, Mab 42 and Mab 43, which had been originally produced against the crossreacting cytochrome c of Kentucky bluegrass (KBG). In summary, (i) fragments C-67 and C-74 reacted with all antibodies, (ii) fragments T-45, T-46 and C-69 bound to human IgE antibodies as well as to Mab 41 and Mab 42, but not to Mab 43, (iii) fragment T-44 reacted only with Mab 41 and Mab 42, and (iv) fragment C-83 bound only Mab 42 and Mab 43. On the basis of these results, it is concluded that (i) immunochemically active fragments of the RG cytochrome c can be readily produced by enzymatic degradation, (ii) there is significant crossreaction between the antigenic determinants of RG and KBG cytochromes c, (iii) whereas all fragments possessed at least two of the original antigenic determinants, fragments C-83 and T-44 were devoid of allergenic determinants, (iv) the antigenic determinants recognized by Mab 41 and Mab 42 were different from those reacting with human IgE antibodies and Mab 43, (v) each of the three monoclonal antibodies recognized a distinct antigenic determinant, (vi) fragments C-67 and C-74 possessed all determinants recognized by the human IgE and mouse antibodies used.     

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.     

Monoclonal antibodies to human growth hormone induce an allosteric conformational change in the antigen.

We re-investigated the properties of a monoclonal antibody (mAb), 4D11, to human growth hormone (hGH) that showed a very weak affinity, recognizing hGH only when the hormone was solubilized on a solid surface. MAb4D11 did not significantly bind 125I-hGH. It was found that three mAb directed to different hGH epitopes (mAb 3C11, 10C1 and NA71) were able to induce the binding of the soluble antigen to mAb 4D11. The co-operative effect could be demonstrated by the formation of binary complexes (Ag:Ab, 1:2) detected by high-performance liquid chromatography (HPLC) and by the increase of radioactivity found when the synergistic mAb were added to 125I-hGH incubated with mAb 4D11 immobilized on polyvinyl microplates. Other possible explanations, such as the formation of cyclic complexes or the generation of a new epitope in the Fc fragment of the first antibody (Ab), were dismissed because the Fab fragment of one of the enhancing mAb (3C11) gave the same effect as the intact Ab. The data suggest that the hGH molecule undergoes a localized conformational change after binding to mAb 3C11, NA71 or 10C1 and that mAb 4D11 binds with high affinity to the modified region of the hormone. The formation or not of ternary complexes (Ag:Ab, 1:3) was used to localize the 4D11 epitope on the surface of the Ag. It is suggested that mAb 4D11 recognizes a conformational change produced in the region defined by the AE5/AC8 epitopes, which is close to the hGH antigenic domain only expressed when the protein is immobilized on plastic surfaces.     

Relative distribution of various antigenic determinants on the human growth hormone surface

The relative distribution of 12 antigenic determinants on the surface of the human growth hormone (hGH) molecule has been established. The necessary information was obtained by testing the ability of paired monoclonal antibodies (MAb) to bind simultaneously or not, to 125I-hGH which leads to the formation of 1:2 or 1:1, Ag-Ab complexes, respectively. The results obtained indicate that the epitopes occupy a large percentage of the total hGH molecular surface and revealed the existence of; an antigenic region specific for hGH; at least two independent domains of immunological identity between hGH and human placental lactogen (hPL), one of them also shared by heterologous GH; and other independent areas of partial cross reactivity with hPL. MAb competition experiments in a solid-phase RIA showed the unreliability of this technique for mapping purposes. The distribution of the hGH epitopes suggested in this work is in accord with present views on protein antigenicity and also explains data existing in the literature concerning the behavior of some of the MAb tested here.     

Topographic and functional assay of antigenic determinants of human prolactin with monoclonal antibodies

Six distinct antigenic determinants were identified on human prolactin (hPRL) by competition assays with murine monoclonal antibodies (MABs). The affinity of binding and the cross-reactivity of the antibodies with two non-primate prolactins was also determined. Binding of 125I-hPRL to MAB-coated microtitre plates in the presence of a second MAB resulted in either inhibition or enhancement of antigen binding to the plate. These results were interpreted in terms of conformational changes to epitopes, induced allosterically by the binding of a second MAB to the antigen. The topographic relationship of epitopes to the biologically active regions on the hormone was examined on the basis of the neutralizing potency of MABs in the proliferation of the prolactin-dependent cell line NB2. The NB2 growth-inhibitory activity was restricted to three distinct epitopes (NE02/6, 1208 and NE03) but absent from three other MABs tested (QB01, WC01/3 and 1200). On the basis of the competition and functional studies, an elemental scheme of the topographic localization of epitopes is presented. The experimental approach employed may contribute to studies on the allocation of biologically active sites on protein hormones.     

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.     

Changes in the Specificity of Antibodies in Mice Infected with Lactate Dehydrogenase-Elevating Virus

Infection with lactate dehydrogenase-elevating virus (LDV) modifies the isotypic distribution of antibodies (Ab) directed to several antigenic proteins with a preferential production of IgG2a. Because it was not known whether the virus could also affect the Ab specificity, the authors addressed this point using human growth hormone (hGH) as a model antigen. Anti-hGH monoclonal antibodies (MoAb) were used as probes to study the occurrence of Ab to three native hGH epitopes (3C11, F11 and 10D6) in sera from LDV-infected CBA/Ht and BALB/c mice immunized with hGH. Competition ELISA was used to determine the extent of Ab directed to cryptic hGH epitopes, i.e. antigenic determinants hidden in the native hormone. Results indicated that in LDV-infected CBA/Ht mice the titres of anti-hGH Ab were lower than in controls, although a consistent isotypic shift to IgG2a subclass was observed. Concurrently, the presence of Ab to epitopes 3C11, F11 and/or 10D6 were markedly reduced in infected animals and most anti-hGH Ab were directed to hGH cryptic epitopes. By contrast, LDV infection increased the amount of anti-KLH Ab elicited by CBA/Ht mice and did not affect Ab specificity, whilst control and LDV-infected BALB/c mice showed similar concentrations of anti-hGH Ab. Furthermore, the proportion of Ab to cryptic hGH epitopes did not change in infected animals even though an important shift to IgG2a was detected. Thus, data presented herein suggest that LDV infection modifies Ab specificity depending on the mice genetic background and on the antigenic characteristics of the immunogen.     

Binding characteristics of monoclonal antibodies raised against bovine growth hormone

Monoclonal antibodies have been raised against pituitary bovine growth hormone using the hybridoma procedure. The binding characteristics of the seven selected monoclonal antibodies toward the antigen molecule in its native, chemically or enzymatically treated form have been studied. The reactivities of the monoclonal antibodies with growth hormones from other species and bovine prolactin have also been investigated. The epitopes recognized by four of the produced monoclonal antibodies are conformational, whereas two other monoclonal antibodies bind to sequential determinants. Three antibodies define immunological sites located between residues 6-124 of the bovine growth hormone molecule, and one of this antibody shows higher affinity to human than bovine growth hormone. The immunoreactivity of one monoclonal antibody is enhanced by the previous binding of the antigen to polyclonal antibodies, probably because of a localized conformational change of the bovine growth hormone molecule. This antibody also shows cross-reactivity with all the homologous hormones tested, indicating to recognize a highly conserved antigenic determinant.     

Autoantigenic determinants on human thyroglobulin. I. Determinant specificities of murine monoclonal antibodies.

To map the antigenic determinants, a panel of 20 mouse monoclonal antibodies (mAbs) to native human thyroglobulin (Tg) was generated. Four criteria were established for distinguishing the determinants recognized by the various mAbs: (i) reactivity to Tgs from eight different species; (ii) reactivity to oxidized and reduced human Tg; (iii) the ability of thyroxine to inhibit binding of mAbs; and (iv) the pattern of antigenic determinant reactivity as determined in reciprocal competitive inhibition binding assays. Of 20 mAbs examined, 12 bound only to human Tg, 3 bound to all eight species tested, and 5 bound to human Tg and to Tg from at least one of the other seven species. Eight mAbs bound oxidized/reduced and native human Tg equally well, 2 bound to the oxidized/reduced protein better than to the native, 7 showed virtually no reactivity to oxidized/reduced human Tg, and 3 others reacted to the oxidized/reduced protein significantly less than they bound to the native. The binding of 5 mAbs to native human Tg was inhibited by thyroxine. The pattern of shared determinant specificities revealed that at least 12 determinant clusters were defined by the panel of mAbs. Among the 12 determinant clusters were 3 designated as immunodominant; 8 of 20 mAbs defined these immunodominant clusters. The four criteria taken together indicate that at least 19 different epitopes on human Tg could be distinguished by this panel of mAbs. These mAbs are useful for the study of determinant specificities of Tg autoantibodies in humans.     

Mapping of antigenic determinants within peptides of a variant surface glycoprotein of Trypanosoma brucei

The location of antigenic determinants in the primary amino acid sequence of the variant surface glycoprotein of Trypanosoma brucei MITat 1.6 was investigated using monoclonal antibodies in conjunction with the known cyanogen bromide and tryptic cleavage patterns of this antigen. The cyanogen bromide digestion fragments of the antigen were purified and used to raise polyclonal antisera, which were specific for the appropriate cyanogen bromide fragment and partial digestion products, as well as recognising the intact variant surface glycoprotein. Competition radioimmunoassays were carried out between these antisera and nine monoclonal antibodies specific for MITat 1.6 variant surface glycoprotein, which have previously been characterised and shown to recognise five antigenic determinants of which only one is exposed on the surface of the living trypanosome. The binding of the monoclonal antibodies to the major tryptic peptide of MITat 1.6 variant surface glycoprotein was investigated by immunoblotting and by competition radioimmunoassay, and revealed that the five antigenic determinants recognised by the nine monoclonal antibodies are all located in the N-terminal two thirds of the MITat 1.6 variant surface glycoprotein molecule. Three of the determinants are located in an immunodominant region apparently formed by the folding together of two of the cyanogen bromide peptides. The other two determinants appear to be more conformationally labile; one of these is the determinant which is exposed on the surface of the living trypanosome, which is located in the N-terminal one third of the molecule.     

Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay

Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the beta-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125iodine. This assay was highly specific for HCG and there was no cross-reactivity with alpha, beta-subunit of HCG, luteinizing hormone and follicle stimulating hormone.     

Specificities of monoclonal antibodies against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis.

Eight hybrid cell lines secreting monoclonal antibodies directed against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis were grown in BALB/c mice. Ascites fluids were collected, and the antibodies were purified by antigen-affinity chromatography. The specificity of each monoclonal antibody for the toxin and protoxin was established by the indirect enzyme-linked immunosorbent assay. All the antibodies consisted of gamma 1 heavy and kappa light chains. They were reactive with both the native toxin and the protoxin. In contrast to specific goat antiserum, they failed, however, to bind to heat and sodium dodecyl sulfate denatured antigen. These eight cloned cell lines gave rise to five kinds of antibodies distinguished by isoelectric focusing. Competitive antibody binding studies revealed that these five antibodies recognize at least four distinct antigenic determinants of the native toxin and the protoxin. Two of the epitopes are unrelated, whereas three antibodies compete for binding to their antigenic determinants. In the bioassay with larvae of Pieris brassicae, one antibody was found to block the toxin and protoxin activity completely. A second inhibited it partially, whereas the other three antibodies did not affect it at all.     

A F(ab'')2 fragment blocking study of the HLA-B14 antigen

The HLA-B14 antigen exists in two serologically distinct forms termed Bw64 and Bw65. These two B14 subgroups were studied using the ability of F(ab')2 fragments from HLA-B14 and Bw6 antisera to block the binding of cytotoxic HLA antibodies directed towards the same or an adjacent antigenic site. The findings suggest that the B14 antigen consists of several antigenic determinants some of which are spatially distinct and others which are closely associated. A "common" determinant possessed by both B14 subtypes is proposed which is separate from a Bw64 (or Bw65) site. Additional determinants that B14 shares with B8, B18 and B38/B39 appear to be distinct from the Bw64/Bw65 site, variably associated with the B14 "common" determinant but closely adjoining the Bw6 antigen.     

Immunohistochemical localization of placental-like alkaline phosphatase in testis and germ-cell tumors using monoclonal antibodies.

Six monoclonal antibodies raised against the human placental alkaline phosphatase (ALP) recognizing distinct antigenic determinants on the surface of this isozyme were used for immunohistochemical studies of adult and fetal human testes and testicular germ-cell tumors. ALP reacting with all six antibodies was defined as placental, whereas ALP reacting with some but not all antibodies was labeled as placental-like. ALP reacting with one of the monoclonal antibodies that recognizes a determinant common to intestinal and placental ALP was tentatively considered probably intestinal, unless it reacted with any other monoclonal placental specific antibody. Using this approach, the authors have identified placental ALP in 4 of 7 seminomas, 3 of 7 tumors composed in part or fully of embryonal carcinoma, and 1 yolk sac carcinoma. Placental-like ALP was identified in 2 additional seminomas and 4 embryonal carcinoma-containing tumors, whereas 1 seminoma and 1 benign teratoma were devoid of either placental or placental-like ALP. Trophoblastic giant cells in 2 seminomas and 3 teratocarcinomas expressed only the antigenic determinant common to placental and intestinal ALP. The authors thus show that testicular tumor cells may express either placental or placental-like ALP and that in some instances, the tumor isozyme is antigenically different from ALP found on either fetal or adult testicular germ cells.     

Specificity analysis of monoclonal anti-DNA antibodies.

The specificity of a panel of murine monoclonal anti-DNA antibodies for DNA antigenic determinants was evaluated by testing their relative binding to various animal and bacterial DNAs. The antibody panel consisted of six monoclonal anti-DNAs of MRL-lpr/lpr and B6-lpr/lpr origin, while the antigens tested were calf thymus (CT), salmon testes (ST), E. coli (EC) and Micrococcus (MC) DNA. While all antibodies bound to CT, ST, and EC DNA to a similar extent by direct ELISA, only one showed an equivalent level of interaction with MC DNA. The relationship of antigenic sites recognized by the antibodies was evaluated further by competition ELISA, assessing the ability of the anti-DNAs to block the interaction of a biotinylated anti-DNA with solid-phase DNA antigen. For each of the DNAs tested, two patterns of DNA interaction could be distinguished on the basis of the relative inhibitory activity of the different monoclonals. These results suggest that anti-DNA antibodies can be characterized using naturally occurring DNAs, with the observed patterns of binding suggesting recognition of unique antigenic sites, some of which are discrete and non-overlapping.     

The recognition by monoclonal antibodies of various portions of a major antigenic site of human growth hormone

The reactivities of five mouse monoclonal antibodies against human growth hormone (hGH) were defined by either a competitive radioimmunoassay with insolubilized antibodies or by an agglutination-inhibition method with hGH-coated polystyrene particles. The five antibodies reacted significantly but to various degrees with human placental lactogen and at least three antibodies reacted with human prolactin and three synthetic peptides extending from residues 19 to 128, 73 to 128 and 98 and 128 of hGH. Four tested monoclonal antibodies failed to react with bovine growth hormone and with hGH oxidized by performic acid. The antibodies were further distinguished by their different reactions with hGH modified by reduction and alkylation or by adsorption on a polystyrene surface. The unique specificity of each antibody was confirmed for most of them by an agglutination method in which the agglutinating activity of hGH was tested on latex particles coated with various paired combinations of the monoclonal antibodies. The lack of agglutination with certain combinations suggested that the specificities of such a pair of antibodies overlapped each other. These results suggest that the sequences corresponding to the synthetic peptides participate in the structure of a major antigenic site of which various portions are recognized by the monoclonal antibodies.