Heteroclitic behaviour of some monoclonal antibodies against bovine growth hormone

Five monoclonal antibodies (MAbs) prepared against bovine growth hormone (bGH) were found to be directed against an immunodominant antigenic domain in bGH. MAbs D8 and H3 reacted equally well with bGH and ovine growth hormone and to a lesser extent with equine (eGH) or porcine (pGH) growth hormones while MAbs H1, H2 and C12 behaved as heteroclitic antibodies, i.e. they bound better a cross-reacting antigen (eGH on pGH) than the immunogen. The reactivity of bGH with the heteroclitic MAbs increased proportionally to the time that the native protein was kept frozen. Deamidation of bGH by treatment with alkali also increased its reactivity. Circular dichroism measurements indicated the occurrence of changes in the conformation of the bGH molecule by these treatments which presumably uncover normally buried or non-accesible epitopes. bGH shares epitopes with eGH and pGH which are immunologically expressed by bGH only when its native conformation is modified.     

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.     

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.     

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.     

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.     

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.     

Generation of monoclonal antibodies to bovine follicle-stimulating hormone and application in an enzyme immunoassay

Twenty monoclonal antibodies (MAbs) to bovine follicle-stimulating hormone (bFSH) were generated by using the hybridoma technique. Twelve of the MAbs have been shown to bind specifically to bFSH and seven of them were purified from the respective ascitic fluids with the methods of caprylic acid precipitation and hydroxylapatite column chromatography. The purity of the MAbs was in the range of 86-95%. Three of the purified MAbs have been coupled to horseradish peroxidase (HRP). By using a matrix cross-matching procedure, two MAbs reacted with discrete antigenic determinants are selected for a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The designed ELISA procedure could be performed within 60 min in a two-stage incubation and has a minimum sensitivity of 1ngFSH/ml. For serum samples, its mean intra- and inter-assay coefficients of variation were 3.4% and 7.3% respectively. The DAS-ELISA can be used to establish a complete FSH profile in the field of in vitro diagnosis.     

Antigenic regions of bovine somatotropin as defined by monoclonal antibodies

Three distinct antigenic regions of bovine somatotropin (bST) were identified on the basis of the ability of a set of monoclonal antibodies to bind to proteolytic fragments and deletion variants of recombinant bST (rbST) in Western blot analyses. One of the regions is further subdivided into two epitopes on the basis of the cross-reaction of somatotropins from several species with the same set of antibodies in solid-phase RIA. The RIA and Western blot results suggest that amino acids 134–150, 181–190 and the amino terminus may be involved in the binding specificity of antibodies to the bovine somatotropin molecule. The total of four antigenic regions on the bST molecule parallels results described for human somatotropin. Labeled antibody competition tests were used to show that the epitope involving amino acids 134–150 is spatially separated from the other three epitopes.     

Immunomodulating IL-6 activity by murine monoclonal antibodies

The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injection of murine monoclonal antibodies (MAb) directed against cellular targets, has been reported extensively in several studies. We analysed here HAMA in 12 patients (six with multiple myeloma, MM, and six with metastatic renal cell carcinoma, MRCC) who were treated with B-E8, an IgG1 MAb against interleukin-6 (IL-6). Efficiency of the treatment was evidenced by the drop in the serum levels of C reactive protein (CRP), of which the in vivo production is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All of immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment efficiency was not affected by the presence of HAMA. Circulating B-E8 MAb were still able to bind to IL-6 and to inhibit IL-6-independent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA against MAb directed against cellular targets, HAMA against anti-IL-6 MAb idiotopes led neither to clearance nor to functional inactivation of the injected MAb. This was further shown by resuming the B-E8 treatment with success in a patient who still had anti-idiotypic HAMA.     

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.     

Antigenic variations in bovine viral diarrhea viruses detected by monoclonal antibodies.

Five murine monoclonal antibodies (MAbs) against the NADL strain of bovine viral diarrhea (BVD) virus were developed, identified, and characterized. Four of the MAbs were directed against a 53-kilodalton (kDa) viral protein, and one was specific to a 47-kDa polypeptide. Competitive radioimmunoassay showed that two MAbs were specific to related epitopes of the 53-kDa protein, and the other three MAbs were each specific to a different epitope. The MAbs were used to study heterogeneity among BVD virus strains. Various degrees of reactivity of cytopathic and noncytopathic virus isolates were detected by virus neutralization and immunofluorescence assays. The virus isolates were divided into six groups based on the neutralization test. The results indicated that the 53-kDa glycoprotein of BVD virus is the major protein involved in virus neutralization and that only a few epitopes of the protein contribute to the neutralization. None of the MAbs neutralized all the BVD virus isolates tested in this study, suggesting antigenic variations among BVD virus isolates.     

Investigation of mast cell differentiation in vivo by use of monoclonal antibodies

In the present study mast cell differentiation/maturation was studied in vivo after depletion of mature mast cells from the peritoneal cavity by injection of distilled water. The reconstituting cell population was characterized by use of different staining methods. Additionally, the monoclonal antibody (MAb) IWF F2, which recognizes a membrane antigen of rat mast cells, was used to follow up mast cell differentiation/maturation in the course of the experiment. The antigen expression was studied both by immunofluorescence of antigen-bearing cells and by MAb inhibition of compound 48/80-stimulated histamine release from mast cells. In the course of the experiment the amount of antigen-positive cells increased continuously from less than 5% to control level (1st and 22nd days, respectively). The expression of the membrane antigen detectable by the MAb precedes the appearance of cytochemically identifiable mast cells for several days. The mast cells mature morphologically and functionally as indicated by increasing size, histamine content and MAb inhibition of compound 48/80-stimulated histamine release. The results obtained suggest the MAb IWF F2 to be a useful methodical tool for additional characterization of mast cell differentiation/maturation processes.     

Enhancement of antibody activity to bovine leukemia virus by complement

Summary Cocultivation of fetal lamb kidney cells infected with bovine leukemia virus (BLV) and the cat cells containing murine sarcoma virus genome resulted in the rapid production of syncytia. This syncytia formation was inhibited by serum containing antibodies to glycoprotein antigen of BLV. When rabbit complement was added to the antiserum for early syncytia inhibition (ESI) test, a significant enhancement of ESI activity of the antiserum was observed. This enhancement was associated with IgG fraction but not with IgM fraction of the antiserum. The results of the comparative serological tests showed that the ESI test with complement was much more sensitive than either immunodiffusion or complement fixation tests in the detection of BLV antibodies.With 1 Figure     

Cross-reactivity of mouse monoclonal antibodies produced by in vitro or in vivo immunization

The effect of different immunization schemes on the resulting antibody specificity was investigated. The cross-reactivity of monoclonal antibodies produced by in vitro vs. in vivo immunization was tested, using a solid phase enzyme immunoassay. Ten different monoclonal antibodies were tested against 15 different antigens. There was no difference in cross-reactivity between the two types of antibodies when tested against antigens coated onto the plastic wells in a buffer solution.When the antigens were dried onto the plastic wells the IgM monoclonal antibodies produced by in vitro immunization exhibited a somewhat different reactivity pattern. However, the assay design was shown to be of more importance than the immunization procedure when determining antibody specificities.     

Enhancement of neutralizing efficacy by combining three monoclonal antibodies to human interferon-alpha.

Three murine monoclonal antibodies (mAb) directed to distinct epitopes on recombinant human interferon (IFN)-alpha 1, and three mAb recognizing distinct epitopes on recombinant human interferon (IFN) alpha 1, and three mAb recognizing distinct epitopes on recombinant human IFN-alpha sc, were studied by IFN-neutralizing assays. The efficacy of neutralization of the anti-viral and the anti-proliferative activities of IFN-alpha 1, or IFN-alpha 2c, by the specific antibodies used, individually or in combination, were evaluated. In comparison with single mAb, the mixtures of three mAb against IFN-alpha 1 or three mAb against IFN-alpha 2c were capable of neutralizing more than 10-times larger amounts of IFN-alpha 1 and alpha 2c, respectively. The strong potentiation of the neutralization efficacy resulting from mixing different mAb was demonstrated by neutralization of the anti-viral as well as the anti-proliferative activities of both recombinant IFN. The neutralization experiments support the interpretation that the observed potentiation results from simultaneous interaction of anti-IFN mAb with different epitope specificity.     

Characterization of the in vitro and in vivo activity of monoclonal antibodies to human IL-18

IL-18 is a cytokine with potent IFN-gamma inducing activities as well as an important mediator of Th1 polarized immune responses. In this study we demonstrated that IL-18 induces the concentration-dependent production of the proinflammatory mediators IFN-gamma, IL-6, and GM-CSF, but not the anti-inflammatory cytokine, IL-10 from peripheral blood lymphocytes in the presence of mitogen. Three neutralizing IL-18 monoclonal antibodies (MAbs) were investigated, one of which (2C10) inhibited IL-18 bioactivity with an IC50 of 0.1 nM and had a K(D) of 3.9 x 10(-11) M. A NOD/SCID mouse model engrafted with human peripheral blood lymphocytes was developed to test the in vivo efficacy of this MAb. The IFN-gamma production induced by LPS administration was inhibited approximately 90% by prior dosing of MAb 2C10. The therapeutic utility of a high-affinity IL-18 MAb may be of benefit in Th1-driven autoimmune diseases such as rheumatoid arthritis and Crohn's Disease, where elevated levels of IL-18 have been observed.     

Anti-idiotypic sera against monoclonal anti-porcine growth hormone antibodies: production in rabbits and characterization of specificity

Antisera were raised in rabbits against two mouse monoclonal anti-porcine growth hormone (pGH) antibodies. Anti-idiotypic antibodies were isolated from rabbit sera by successive passage over immunoadsorbent columns of normal mouse Ig (mIg), followed by the specific immunizing monoclonal and elution from the latter. Enzyme linked immunoadsorbent assay (ELISA) for anti-idiotype and free idiotype were established. While showing specificity for their respective inducing monoclonals, anti-idiotypes also cross reacted in varying degrees with other anti-pGH monoclonals regardless of specificity differences between the antibodies, demonstrating the presence of a cross-reactive idiotype.