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.     

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.     

Evaluation of 29 monoclonal and polyclonal antibodies used in the diagnosis of pituitary adenomas. A collaborative study from pathologists of the Club Fran?ais de l'Hypophyse

Fifteen polyclonal antibodies (pAbs) and 14 monoclonal antibodies (mAbs) directed against hGH, hPRL, beta hFSH, beta hLH, beta hTSH and alpha-subunit were assessed by five different laboratories on normal and adenomatous pituitary tissues. This study aims at providing pathologists with a selected panel of antisera suitable for diagnosis, and appreciating the interest of the recently introduced mAbs. All the anti-hGH Abs proved to be specific (3 pAbs and 4 mAbs); three mAb out of four gave a few false-negative reactions. Three out of six polyclonal anti-hPRL showed cross-reactivity with hGH; anti-hPRL mAbs gave a strong staining with no false-negativity detected so far. MAbs proved to be more efficient for detecting glycoprotein hormones and alpha subunit than pAbs, which, in several cases, gave widespread cross-reactivity. This lack of specificity could explain the noticeable discrepancies reported so far in the appraisal of gonadotropic and somatoprolactinic adenomas.     

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.     

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.     

Reactivity of ACTH and synthetic ACTH peptides with antisera to human calcitonin.

We studied reactivity of highly purified pituitary hormones in our human calcitonin (hCT) radioimmunoassay (RIA) which can detect 1 pg of hCT. ACTH at doses of greater than 1 microgram of peptide per RIA tube reacted in the hCT assay, as did beta-endorphin (beta EPH) at a dose of 10 micrograms per tube. No reactivity was observed with comparable concentrations of all other known pituitary hormones. ACTH also reacted at doses greater than 1 microgram per tube with 7 other hCT antisera which recognized differing antigenic determinants in the calcitonin molecule but it was not reactive with 2 antisera against porcine calcitonin or 2 antisera against salmon calcitonin. This slight degree of cross-reactivity of hACTH and beta EPH in the hCT RIA cannot account for the presence of immunoreactive CT in pituitary glands. Nevertheless, antisera used for the localization of peptides must be rigorously tested for the existence of cross-reactivities with other possible substances, especially if such antisera detect the peptide in unexpected tissues.     

Analysis of human pituitary tumors by in situ hybridization

A procedure for performing in situ hybridization histochemistry (ISH) on frozen and paraffin sections of human pituitary tissues is described. The use of oligonucleotide probes for hPRL and hGH labeled with 35S allowed detection of a specific messenger RNA in frozen and paraffin sections. This technique can be combined with immunochemistry to localize both the gene product and the hormone(s) produced by specific cells and should be very helpful in the characterization of normal and neoplastic human pituitary cells.     

Reanalysis of Antisera Specificities and Binding Characteristics of Rat Pituitary Hormone Assays

Rat pituitary hormone radioimmunoassays (RIAs) are widely used in reproductive research, yet data on specificity and binding characteristics of many of the antisera are not widely available. This report characterizes one set of rat antisera supplied by the National Institutes of Health (USA). Rat follicle-stimulating hormone (FSH) and thyrotropin-stimulating hormone (TSH) antisera appear specific, but TSH exhibited significant competition in the rat luteinizing hormone (LH) assay. In addition, statistically significant nonparallelism was demonstrable in all three assay systems. This creates further problems in characterizing antisera cross-reactivity and may make potency estimates for pituitary standards inaccurate.     

人生长激素基因真核表达载体的构建及鉴定

目的 构建人生长激素的真核细胞表达载体,测定并分析目的 基因序列.方法 用PCR方法扩增出人生长激素基因,连接至T克隆载体,然后分别亚克隆至真核细胞表达载体pCDNA3.1、pCEP4中.使用DNA ssist软件分析序列.结果 所构建真核表达载体的酶切鉴定、PCR鉴定、测序鉴定均正确.结论 成功构建出多个人生长激素基因真核细胞表达载体,验证了基因序列的正确性,为下一步基因治疗研究打下基础.     

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.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): I. Production,Specificity, and Intramolecular Binding Sites*

ABSTRACT: Thirty-nine monoclonal antibody (MCA) producing hybridoma cell lines derived from fusions of mouse myeloma cells with spleen cells from mice immunized with human chorionic gonadotropin (hCG) have been established. Their products have been tested in radioimmunoassays using ¹²⁵I-labeled hCG, luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), the alpha (α) and beta (β) subunits of hCG and LH, and the C-terminal peptide 109–145 (CTP) of CG. All MCA were, in addition, tested in indirect immunofluorescence (IIF) on paraffin sections of human pituitary glands. According to the intramolecular localization of the determinants recognized, three main groups of MCA can be distinguished: 1) MCA directed against epitopes on the β-chain (α-MCA), 2) MCA directed against (β-chain determinants (β-MCA), and 3) MCA reacting with a conformational determinant only present on the native hormone and not on either subunit (con-formational-MCA). All α-MCA cross-react with human LH, FSH, and TSH. The β-MCA do not react with FSH or TSH, but do react to a varying degree with LH. The conformational-MCA show no binding of labeled FSH or TSH and very little or no cross-reactivity with LH.     

Serological studies on rabbit antibodies to the rabbit anterior pituitary

Rabbits immunized with suspensions or extracts of rabbit anterior pituitary in Freund adjuvant may develop specific antibodies to components of the rabbit pituitary. Immunofluorescent staining with such antisera occurred in isolated cells of the anterior pituitary. These correspond to cells stained with acid fuchsin, i.e. acidophils or alpha cells. Some of the pituitary antisera fix complement with pituitary extracts. A tanned-cell haemagglutination test using pituitary extracts as coating antigen yielded positive reactions with some of the pituitary antisera. The rabbit antisera appeared to be specific for the anterior pituitary within the limits of the rabbit organs tested. Hog, guinea-pig, dog and beef pituitaries share the antigen, but monkey and human pituitaries fail to react.

Immunofluorescent staining revealed that antisera reacted with the pituitary of the antibody producing rabbit, i.e. the sera contain pituitary autoantibodies. No direct or indirect evidence for pathological changes in the autoantibody producing animals could be found.

     

Development and characterization of rabbit antisera to human MHC-linked transporters associated with antigen processing

The limited availability of sera to human MHC-linked transporters associated with antigen processing (TAP) has hampered the analysis of the role of these molecules in the reduced HLA Class I antigen expression by normal and transformed cells. To overcome these limitations, anti-human TAP1 and anti-human TAP2 xenoantisera have been generated and characterized. To this end rabbits have been immunized with TAP 1-specific or TAP2-specific peptides which correspond to nonhomologous, hydrophilic regions of each transporter subunit. The immunized rabbits developed high titer IgG antibodies which displayed specific reactivity with the immunizing peptides in ELISA. Both anti-TAPl and anti-TAP2 antisera immunoprecipitated the 70–76 kDa TAP complex from TAP1+-TAP2+ cell lines WALK and Colo 38, but precipitated no component from TAP1+-TAP2+ cell lines T2 and SK-MEL-19. Furthermore, in immunode-pletion experiments anti-TAPl and anti-TAP2 antisera removed the molecules recognized by each of them in a lymphoid cell extract. Lastly, in Western blotting assays anti-TAPl and anti-TAP2 antisera reacted specifically with isolated TAP1 and TAP2, respectively. The latter results in conjunction with those of the immunodepletion experiments indicate thatTAPl and TAP2 are not detectable as isolated subunits in a cell extract and that TAP heterocomplex is the major, if not the only detectable molecular species in cells. Anti-TAPl and anti-TAP2 antisera were evaluated in immunohistochemical staining of both frozen and formalin fixed sections of skin and primary malignant melanoma lesions. Both antisera stained the cytoplasm of keratinocytes in normal skin and of melanoma cells in malignant lesions. The antisera we have elicited with TAP1- and TAP2-specific peptides appear to be useful reagents to characterize the role of TAP in abnormalities of HLA Class I antigen expression.     

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.     

Polyreactivity of human monoclonal antibodies: Human IgM anti-rhesus monoclonal antibodies are frequently polyreactive

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.     

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.     

Ultrastructure,immunohistochemistry and hormone release of pituitary adenomas in relation to prolactin production

Summary Fifteen cases of pituitary adenoma, 14 of which were associated with hyperprolactinemia, were studied by observation and granule morphometry of electron micrographs, immunohistochemistry and sequential observation of in vitro release with regard to hormone production, storage and secretion. Adenoma cells of 6 cases with marked elevation of plasma prolactin were sparsely granulated, showed characteristic ultrastrucures including the presence of small secretory granules, well developed Golgi and rough membranes, misplaced exocytosis, and positive or negative immunostaining for prolactin. These adenomas also showed vigorous release of the hormone into the circulation and/or culture medium. In vitro studies showed that negative immunostaining of adenoma cells did not preclude the production and secretion of the hormone. One densely granulated adenoma containing cells with numerous lactotroph type granules showed moderate release of prolactin into the circulation. In an acromegalic case associated with both high plasma growth hormone and prolactin, some cells were shown by immunohistochemistry to store both hormones. There were 4 adenomas which could not be shown to produce, store and secrete prolactin by any method available.Abbreviations Used in this Paper ACTH adrenocorticotropic hormone - -MSH -melanocyte stimulating hormone - hGH human growth hormone - hPRL human prolactin - LH luteinizing hormone - FSH follicle stimulating hormone - TSH thyroid stimulating hormone - TRH Thyrotropin-releasing hormone This work was supported in part by Grants-in-Aid for Cancer Research (No. 50-14) and for Specific Diseases (Disorder of Hypothalamic and Pituitary System) from the Ministry of Health and Welfare, and for Cancer Research (No. 401034) from the Ministry of Education, Science and Culture, Japan     

细胞因子调节垂体MtT/S细胞中人生长激素基因启动子的活性

目的:探讨细胞因子白介素-11(IL-11)、睫状神经营养因子(CNTF)和转化生长因子(TGF-β)对大鼠垂体MtT/S细胞中人生长激素(hGH)基因启动子活性的影响及其与垂体特异性转录因子Pit-1蛋白的关系。方法:采用荧光素酶报告基因的方法。首先建立含hGH基因启动子(-484～30 bp)和荧光素酶融合基因的稳定转化MtT/S细胞系,然后用细胞因子刺激,检测细胞培养液和细胞裂解液中GH的含量,反映它们对GH分泌和合成的影响;检测MtT/S细胞内荧光素酶的变化,说明细胞因子对hGH基因启动子活性的作用。将Pit-1蛋白表达质粒(pcDNA-pit-1-cDNA)单独转染或与Pit-1反义寡核苷酸(Pit-1OND)共转染于稳定转化的MtT/S细胞中,观察加入细胞因子后荧光素酶的变化,探讨细胞因子的作用与Pit-1蛋白的关系。结果:IL-11(20 nmol/L)、CNTF(10 nmol/L)能刺激大鼠垂体MtT/S细胞中GH的分泌和合成,增强MtT/S细胞中荧光素酶的表达,分别增加到对照组的134%、122%。TGF-β(5 nmol/L)能减少GH的分泌和合成,抑制荧光素酶的表达到对照组的72%。Pit-1蛋白过表达和表达被抑制对细胞因子的调节作用没有影响。结论:IL-11、CNTF和TGF-β通过调节大鼠垂体MtT/S细胞中hGH基因启动子活性影响GH的合成,Pit-1蛋白可能不参与这一调节作用。     

Monoclonal antibodies to Ebola virus: isolation, characteristics, and study of cross reactivity with Marburg virus

Thirteen hybridoma strains producing monoclonal antibodies (Mabs) to Ebola virus were prepared by fusion of NS-O mouse myeloma cells with splenocytes of BALB/c mice immunized with purified and inactivated Ebola virus (Mayinga strain). Mabs directed against viral proteins were selected and tested by ELISA. Protein specificity of 13 Mabs was determined by immunoblotting with SDS-PAGE proteins of Ebola virus. Of these, 11 hybridoma Mabs reacted with 116 kDa protein (NP) and 2 with Ebola virus VP35. Antigenic cross-reactivity between Ebola and Marburg viruses was examined in ELISA and immunoblotting with polyclonal and monoclonal antibodies. In ELISA, polyclonal antibodies of immune sera to Ebola or Marburg viruses reacted with heterologous filoviruses, and two anti-NP Ebola antibodies (Mabs 7E1 and 6G8) cross-reacted with both viruses. Target proteins for cross-reactivity, Ebola NP (116 kDa) and Marburg NP (96 kDa), and VP35 of both filoviruses were detected by immunoblotting with polyclonal and monoclonal antibodies (6G8) to Ebola virus.     

Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay.

Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B.(ABSTRACT TRUNCATED AT 250 WORDS)