Biomedical subjects

L A Retegui

Publications and source records attributed to L A Retegui.

At least 19 recordsLinked to original sources

PubMed1995

Monoclonal antibodies to human growth hormone modulate its biological properties.

Previous results indicated that monoclonal antibodies (mAbs) termed mAb AE5, mAb AC8 and mAb F11, recognizing the human growth hormone (hGH) region left exposed after binding to lactogenic, somatogenic and hGH-specific receptors, produce allosteric changes in the hormone which modify its binding properties. To study whether these mAbs could also influence hGH biological activity, experiments were carried out with Nb2 cells, a rat lymphoma cell line which proliferates in the presence of lactogenic hormones. Experiments involving previous binding of the hormone to receptors before adding 125I-mAbs indicated that the hGH domain defined by overlapped epitopes AE5, AC8 and F11 is uncovered in hGH when it is bound to the cell membranes. To reveal any alteration in the hGH molecule induced by the mAbs, preformed 125I-mAb:hGH complexes were added to the cell membranes. Data showed that 125I-mAb AE5:hGH complexes bound better to the receptors than free hormone. On the contrary, hGH previously bound to 125I-mAb AC8 or 125I-mAb F11 was poorly recognized by Nb2 receptors. Furthermore, both mAbs AC8 and F11 strongly inhibited 125I-hGH binding to Nb2 cell membranes and hGH-induced Nb2 cell proliferation whereas mAb AE5 enhanced both hormone binding and hGH mitogenic effect. Additionally, since mAb AC8 is directed towards an epitope shared by hGH and human placental lactogen (hPL), it was also shown that this mAb could impair hPL biological activity even though it recognizes the hPL region left exposed in hPL:Nb2 cell receptor complexes. Data presented in this work suggest that mAbs directed to the hGH or hPL regions unmasked after binding to Nb2 cell receptors produce allosteric alterations in the binding properties of these hormones leading to either enhancement or decrease of their biological activities.

Allosteric Regulation↗

PubMed1994

Allosteric effects of monoclonal antibodies on human growth hormone.

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors. The effect of MAbAE5,AC8 and F11 on hGH binding was measured by determining the formation of 125I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding 125I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes. Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case performed 125I-MAb:hGH complexes were added to microsomes. Data showed that 125I-MAb AE5:hGH complexes bound better to the various receptors than 125I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to 125I-MAb AC8 or 125I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32-46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding. Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗

PubMed1994

Synergistic monoclonal antibodies' interactions and their use for determination of antibody specificities.

Three monoclonal antibodies (MAb 3C11, F11 and 10D6) to human growth hormone (hGH) recognize independent epitopes and show mutually enhancing properties. Thus, 125I-hGH binding to each of these MAb augmented significantly in the presence of each one of the other two MAb. Moreover, preincubation of the hormone with paired MAb gave rise to ternary complexes (Ag:Ab1:Ab2) which bound better than the free tracer to the third MAb previously captured on a solid-phase. Highly stable quaternary complexes (Ag:Ab1:Ab2:Ab3) were thus formed. Since Fab fragments from the three MAb displayed the same behavior as the whole Ab molecule, neither the formation of multimolecular cyclic complexes nor the occurrence of interactions through Fc fragments could explain the reciprocal MAb binding enhancement. Therefore, the results obtained suggest that MAb 3C11, F11 and 10D6 produce some modification in the Ag, each one improving the binding of the two other MAb. Additionally, the inhibition of the formation of quaternary complexes between the MAb and hGH was used to evaluate specific Ab populations in polyclonal antisera, avoiding the masking effect of enhancing Ab. The results obtained indicate that Ab directed to the hGH antigenic domains defined by MAb 3C11, F11 and 10D6 could be detected in spite of the presence of enhancing Ab to all three MAb.

Antibodies, Monoclonal↗

PubMed1990

Equine growth hormone. Detection of immunoreactive sequences using poly- and monoclonal antibodies.

The immunochemical behavior of several fragments of equine growth hormone (eGH) was examined using competitive binding assays with antibodies (Abs) to eGH obtained from different sources. Antigenicity was detected within the sequences 5-72 and 73-123 by rabbit Abs to eGH and by three mouse monoclonal antibodies (MAbs) produced by using bovine growth hormone as immunogen, but showing heteroclitic properties towards eGH. The polyclonal Abs to eGH also recognized as immunoreactive two smaller peptides corresponding to the amino acid residues 52-72 and 110-123. By contrast, the heteroclitic Abs to eGH developed by hypopituitary patients therapeutically injected with human growth hormone failed to react with any eGH-derived fragment. The rabbit polyclonal Abs and the mouse MAbs scarely discriminated between native and S-carbamidomethylated eGH, while the heteroclitic human Abs detected a clear difference between the native and the modified hormone.

Amino Acids↗

PubMed1990

Relationship between the antigenic topography and the structure of human growth hormone.

Monoclonal antibodies (MAb) to human GH (hGH) were used to correlate the antigenic topography of the hormone with its structure. Competition experiments performed in a solid phase RIA system allowed us to measure the reactivity toward the MAb of the following hGH derivatives: hGH 20K (a natural variant lacking residues 32-46), hGH selectively modified in His or Met residues, hGH with the C and/or N-terminal disulfide bond reduced and carbamidomethylated, and hGH cleaved between residues 142-143. Results indicated that fragment 32-46 participates in the structure of epitopes EB1/EB3 and that the C-terminal bridge is located in epitope 10D6, whereas opening of both disulfide bridges alters the entire hGH antigenic surface. His-151 and Met-170 were placed in epitopes NA71 and AC8, respectively, whereas His-18 and Met-14 would be involved in the hGH antigenic domain formed by overlapping epitopes 3C11, 10C1, and HG3. MAb AE5, AE12, and AC3 define a flexible hGH region related to sequence 134-150; the respective epitopes show high conformational mobility induced by modifications in other regions of the molecule. Binding of the different hGH derivatives to lactogenic receptors from female rat liver gave some insights on the localization of the hormone-binding site. Epitopes EB1/EB3 and 10D6 were discarded because there was not a direct correlation between their drastic immunological alterations and the binding properties of the respective hGH derivatives. In the same way, epitopes AE5, AE12, and AC3 were excluded from the hGH-binding domain because a disruption in those sites did not affect the hGH interaction with receptors. We conclude that the hGH structure defined by epitopes 3C11, 10C1, and HG3 is probably related to the binding properties of the hormone.

Animals↗

PubMed1990

Monoclonal antibodies as probes to study the human growth hormone-binding domain to lactogenic rat liver receptors.

A set of monoclonal antibodies (MAb) to human GH (hGH) was used to study the hormone binding orientation to its receptors (R) from female rat liver. The hGH antigenic region left exposed after its binding to liver microsomes was detected by measuring the ability of various [125I]MAb to bind to the preformed hGH-R complexes. Results indicated that a cluster of epitopes defined by the MAb, termed AE5, AC8, and AE12, remains accessible in the hGH-R complex whereas overlapping epitopes 3C11 and HG3 would define a hGH region involved in the binding site. Supporting these findings, solubilization and HPLC gel filtration of [125I]MAb-hGH-R complexes showed a radioactive peak of about 450,000 mol wt for MAb AE5 or AC8, but not for MAb 3C11 or HG3. [125I]MAb AE12 behaved differently, suggesting that epitope AE12 may be masked or altered in hGH-R-solubilized complexes. MAb directed to the putative hGH-binding site (MAb 3C11, HG3, and the closely related MAb 10C1 and NA71) failed to inhibit binding of the preformed [125I]MAb AE5-hGH complex to the receptors, suggesting a hormone modification after MAb AE5 binding. Accordingly competition experiments indicated an increase in the affinity of hGH for its receptors induced by this MAb. A higher hGH concentration was required to obtain 50% [125I]hGH binding to liver microsomes in the presence of MAb AE5 than in its absence. As the MAb used define epitopes that were previously correlated with the hGH structure, we concluded that a high flexible region (sequences 134-150) is exposed in the hGH-R complex. Furthermore, some MAb directed to this region enhance the hormone affinity for its rat liver receptors, probably through an induced conformational change.

Animals↗

PubMed1989

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.

Antibodies, Monoclonal↗

PubMed1989

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.

Allosteric Site↗

PubMed1989

[The antigenic topography of human growth hormone and the humoral expression of its various epitopes].

Twenty monoclonal antibodies (MAb) against human growth hormone (hGH) were used to establish the antigenic topography of this protein. Mapping experiments were carried out by testing the ability of paired MAb to bind simultaneously or separately to 126I-hGH. Since the specificity of the MAb versus hGH, human placental lactogen, animal prolactins and growth hormones was known, consideration of the whole set of results obtained supported the proposal of a tridimensional model for the antigenic structure of hGH. Further work correlated some of the mapped epitopes with particular regions within the primary structure of the hormone. In addition, it could be demonstrated that certain MAb may induce conformational changes in the antigen as a result of its binding. The humoral expression of various hGH epitopes during the maturation of the immune response was studied in human patients therapeutically treated with hGH and in mice and hamsters submitted to different immunization schemes: chronic administration of the antigen, secondary response and conventional hyperimmunization. The results indicated temporal and individual variations in the titers of each class of Ab as well as the existence of enhancer and heteroclitic Ab. Hence, the expression of the various epitopes of a protein antigen during the immune response is quite probably a stochastic process which depends on individual variations.

Antibodies, Monoclonal↗

PubMed1988

Stochastic humoral expression of human growth hormone epitopes.

Competition experiments between insolubilized monoclonal antibodies (mAb) and polyclonal antisera has led to the description of the humoral expression of human growth hormone (hGH) epitopes. This study was carried out with sera from mice and hamsters submitted to different immunization schedules: chronic administration of the antigen, secondary response and conventional hyperimmunization. The results indicated the absence of a unique immunodominant epitope in hGH; a significant individual variation of antibody (Ab) population titres with time; changes with time in the relative proportion of one Ab population with respect to the others; and the occurrence of Ab enhancing the 125I-hGH binding to five mAb depending upon the individuals and the time of immunization. Heterocliticity towards non-human GH was also detected. Although most of the animals showed cross-reacting Ab, two out of 12 mice, chronically injected, developed heteroclitic Ab. The data suggest that the humoral response to different epitopes of a protein antigen during the maturation of the immune response is a stochastic process leading to transient humoral immunodominance, enhancing Ab populations and heterocliticity, depending upon individual characteristics, either in outbred or inbred populations.

Animals↗

PubMed1987

Proteolytic modification of growth hormone by a rat kidney lysosomal protease.

A protease activity has been detected in rat kidney which has some degree of specificity to cleave native bovine growth hormone (bGH). The enzyme has an optimum pH of about 5.0, is dependent on thiol reagents and non-inhibited by EDTA or Aprotinin. Controlled digestion of bGH leads to a derivative retaining growth promoting activity, while showing a different physicochemical behavior from that of the native hormone. The data support the hypothesis of the kidney, among other tissues, originating active forms of growth hormones.

Animals↗

PubMed1986

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.

Animals↗

PubMed1986

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.

Antibodies, Monoclonal↗

PubMed1985

Comparison of human lactoferrins from milk and neutrophilic leucocytes. Relative molecular mass, isoelectric point, iron-binding properties and uptake by the liver.

Human lactoferrins isolated from neutrophilic leucocytes and milk by CM-Sephadex chromatography were similar in Mr (76000) and pI (8.7). Upon acidification, both proteins released their two Fe3+ ions/molecule in a similar biphasic way. Both proteins intravenously injected into mice were cleared from plasma at the same rate. The maximal uptakes by the liver, which occurred 5 min after injection, were inhibited to the same extent by milk lactoferrin used as a competitor.

Animals↗

PubMed1985

Specificities of antibodies to human growth hormone (hGH) in patients treated with hGH: longitudinal study and comparison with the specificities of animal antisera.

The specificities of human and animal antibodies (Abs) against human GH (hGH) were analyzed using competition experiments with five monoclonal antibodies to hGH (MAbs). The results indicate that 1) the Abs produced by patients receiving long term hGH therapy as well as Abs of goat, rabbit, and mouse origin recognized the various hGH epitopes defined by the MAbs; 2) the proportion of each Ab population, with a given specificity, differed markedly in different patients and also with time in the same patient; 3) the titer of certain Ab populations was very low in some patients, and 4) polyclonal mouse Abs and some human Abs enhanced the binding of [125I] hGH to insolubilized MAbs.

Adolescent↗

PubMed1984

Monoclonal antibodies and specific cell surface receptors do not discriminate between human growth hormone prepared by DNA recombinant techniques and the native hormone.

Native and E. coli-derived human growth hormone (hGH) have been compared in their ability to react with four different monoclonal antibodies (MAbs) as well as with the specific cell surface receptors of cultured human IM-9 lymphocytes and liver membranes of pregnant rabbit. For all the tests used, the competition curves obtained using both hormone preparations were superimposable. The results suggest a close similarity in the immunological and biological (binding reaction) properties of native and recombinant hGH.

Antibodies, Monoclonal↗

PubMed1984

Uptake of lactoferrin by the liver. I. Role of the reticuloendothelial system as indicated by blockade experiments.

When 125I-labeled human lactoferrin was injected intravenously into mice it was rapidly taken up by the liver, where 75% of the dose was recovered after 15 minutes. The inhibition curve with unlabeled lactoferrin showed that the fates of labeled and unlabeled protein were similar and that the uptake capacity was saturable by milligram quantities of protein per gram of liver, suggesting the existence of numerous specific binding sites. The presence of these sites on the reticuloendothelial system was indicated by the blocking effect of dextran sulfate and latex particles. Fucoidin (fucan sulfate), which is used in the study of receptors specific for fucose, was found to inhibit the uptake of goat red blood cells as well as lactoferrin. Therefore, the inhibition exerted by fucoidin on the uptake of lactoferrin could be mediated by blockade of the reticuloendothelial system and not necessarily by competition for fucose receptors. These data indicate that the report by others that lactoferrin was taken up by a fucosyl receptor on hepatocytes is incorrect. The competition curve obtained when mouse lactoferrin was injected with its human homologue indicated that both proteins reacted with the same binding sites. However, a significant part of mouse lactoferrin was found to be taken up by the kidneys. Hepatic and renal uptakes were both reduced by prior injection of dextran sulfate.

Animals↗

PubMed1984

Particle-counting immunoassay of human somatotropin.

Human somatotropin was assayed by a novel automated nonradioisotopic technique, "particle-counting immunoassay," that requires a 45-min incubation and only 60 microL of 10-fold diluted sample. The principle of the assay is agglutination of antibody-coated latex particle by somatotropin, the reaction being measured by the (instrumented) counting of residual non-agglutinated particles. The dynamic range in serum extends from 0.2 to 40 micrograms/L. The between-assay CV was 12% for a concentration of 3.3 micrograms/L and 8.5% for 31.9 micrograms/L. The coefficient of correlation (r) with radioimmunoassay was 0.97. Curves for various dilutions of the macromolecular and monomeric forms of the hormone were not parallel.

Agglutination Tests↗