ELISA for Determination of Human Growth Hormone: Recognition of Helix 4 Epitopes

Moura, Juliana F.; DeLacerda, Luiz; Sandrini, Romolo; Borba, Fernanda M.; Castelo, Denise N.; Sade, Elis R.; Sella, Sandra; Minozzo, João C.; Callefe, Luis G.; Figueiredo, Bonald C.

doi:10.1155/S1110724304308090

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ELISA for Determination of Human Growth Hormone: Recognition of Helix 4 Epitopes

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Author(s): Juliana F. Moura ¹ , Luiz DeLacerda ² , Romolo Sandrini ² , Fernanda M. Borba ² , Denise N. Castelo ² , Elis R. Sade ¹ , Sandra Sella ³ , João C. Minozzo ³ , Luis G. Callefe ¹ , Bonald C. Figueiredo ¹ ^, ^*

Publication date (Print): 29 July 2004

Journal: Journal of Biomedicine and Biotechnology

Publisher: Hindawi Publishing Corporation

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Abstract

Human growth hormone (hGH) signal transduction initiates with a receptor dimerization in which one molecule binds to the receptor through sites 1 and 2. A sandwich enzyme-linked immunosorbent assay was developed for quantifying hGH molecules that present helix 4 from binding site 1. For this, horse anti-rhGH antibodies were eluted by an immunoaffinity column constituted by sepharose-rhGH. These antibodies were purified through a second column with synthetic peptide correspondent to hGH helix 4 , immobilized to sepharose, and used as capture antibodies. Those that did not recognize synthetic peptide were used as a marker antibody. The working range was of 1.95 to 31.25 ng/mL of hGH. The intra-assay coefficient of variation (CV) was between 4.53% and 6.33%, while the interassay CV was between 6.00% and 8.27%. The recovery range was between 96.0% to 103.8%. There was no cross-reactivity with human prolactin. These features show that our assay is an efficient method for the determination of hGH.

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Most cited references 29

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A hot spot of binding energy in a hormone-receptor interface.

T Clackson, Adam Wells (1995)

The x-ray crystal structure of the complex between human growth hormone (hGH) and the extracellular domian of its first bound receptor (hGHbp) shows that about 30 side chains from each protein make contact. Individual replacement of contact residues in the hGHbp with alanine showed that a central hydrophobic region, dominated by two tryptophan residues, accounts for more than three-quarters of the binding free energy. This "functional epitope" is surrounded by less important contact residues that are generally hydrophilic and partially hydrated, so that the interface resembles a cross section through a globular protein. The functionally important residues on the hGHbp directly contact those on hGH. Thus, only a small and complementary set of contact residues maintains binding affinity, a property that may be general to protein-protein interfaces.

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High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis.

B C Cunningham, J Wells (1989)

A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four.