Determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay

A surface plasmon resonance immunoassay has been developed to determine human growth hormone (hGH) directly and without pre-treatment in human serum samples. A binding inhibition immunoassay was employed. Antibody concentration, assay buffer and regeneration solution have been optimized in order to reach the best performance and the lower non-specific binding of the matrix components to the sensor surface. The lowest detection limit was 6 ng/mL, with a working range covering the physiological range. Reproducibility of the assay was excellent with both intra-assay and inter-assay relative standard deviations <5%, while a variation of 2.19% was obtained employing different sensor chips. Reutilization of the sensor surface allows its continuous use over 50 measurements with a signal drop <20%. The SPR immunoassay results were validated using enzyme-linked immunosorbent assay (ELISA) showing an excellent correlation (R² = 0.985). A portable and fully automated system (Sensia SL) was employed in this work. This is the first SPR biosensor assay capable of detecting relevant concentrations of a clinical analyte in serum. This study shows the potentials of this device as a diagnostic tool for the detection of multiple clinical analytes.     

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.     

A New Approach for Thermodynamic Studies on Binding of Some Metal Ions with Human Growth Hormone

A thermodynamic study on the interaction between Mg²⁺, Ni²⁺ and Co²⁺ ions (M²⁺) and human growth hormone (hGH) was made at 27 °C in aqueous NaCl solutions using isothermal titration calorimetry. Gholamreza Rezaei Behbehani’s solvation model (GRB) was used to model the enthalpies of M²⁺+hGH interactions over the studied range of metal ion concentrations. The solvation parameters derived from the solvation model were attributed to a structural change of hGH due to its interactions with the metal ion.     

Tracking growth hormone abuse in sport: Performance of marker proteins in a controlled setting

Human growth hormone (hGH) abuse in sport is a challenge at present. The current strategy used, known as direct method, is based on the quantification of hGH variants in serum. An alternative strategy, known as indirect method, focuses on serum markers such as insulin-like growth factor I (IGF-I) and procollagen type III N-terminal propeptide (P-III-NP). The indirect method allows a longer window of detection (WOO) of hGH abuse. To evaluate the performance of the indirect method, in parallel to the direct method, a clinical trial with recombinant hGH (rhGH) was conducted on healthy male subjects during 7 days (0.026 mg⁻¹ kg⁻¹ person⁻¹ day⁻¹). The data were fit to the discriminant formula proposed in the previously published GH-2000 project. The low sensitivity of the scores, judged from the high number of false negative outcomes, imposed a new discriminant analysis, standarised using local population subjects demographically similar to the ones of the study. The sensitivity of the method significantly increased, highlighting the importance of the standardisation. The indirect method allowed extended window of opportunity (WOO), although two false positive evaluations were observed derived from elevated basal IGF-I and P-III-NP concentrations stressing the need for an independent confirmation method. When direct and indirect methods were combined the best selectivity and sensitivity were achieved.     

Preparative alkaline urea gradient PAGE: application to purification of extraordinarily-stable disulfide-linked homodimer of human growth hormone

The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.     

An efficient expression of human growth hormone (hGH) in the milk of transgenic mice using rat Β-casein/hGH fusion genes

In order to produce human growth hormone (hGH) in the milk of transgenic mice, two expression vectors for hGH differing in their 3′ flanking sequences were constructed by placing the genomic sequences of hGH gene under the control of the rat Β-casein gene promotor. The 3′ flanking sequences of the expression constructs were derived from either the hGH gene (pBCN1GH) or the rat Β-casein gene (pBCN2GH). Transgenic lines bearing pBCN1GH expressed hGH more efficiently than those bearing pBCN2GH in the milk (19-5500 Μg/mL vs 0.7-2 Μg/mL). In particular, one of the BCN1GH lines expressed hGH as much as 5500 ±620 Μg/mL. Northern blot analysis showed that the transgene expression was specifically confined to the mammary gland and developmentally regulated like the endogenous mouse Β-casein gene in the mammary gland. However, a low level of nonmammary expression was also detected with more sensitive assay methods. In conclusion, the rat Β-casein/hGH fusion gene could direct an efficient production of hGH in a highly tissue-and stage-specific manner in the transgenic mice and the 3′ flanking sequences of hGH gene had an important role for the efficient expression.     

Detection of recombinant growth hormone in human plasma by a 2‐D PAGE method

Human growth hormone (GH) has several central metabolic functions including bone growth in childhood, and its anabolic and lipolytic effects in particular are assumed reasons for the abuse of GH by athletes. Human endogenous GH consists of a main 22 kDa variant and several isoforms. In contrast, recombinant GH consists of only one variant being identical to the main endogenous isoform. The method presented here separates different isoforms by 2‐D PAGE after isolation of GH from plasma using an immunoaffinity purification system. While samples containing endogenous GH yield up to four isoforms, samples with recombinant GH contain the main 22 kDa spot only. Normalized spot volumes (NSV) are calculated after addition of an internal standard and a discrimination limit was determined at 0.52 for the NSV of the main 22 kDa spot. Above this value, samples containing endogenous GH show at least the main 22 kDa isoform and the 20 kDa splice variant. In contrast, samples with a NSV >0.52 and only one spot are suspicious to contain recombinant GH. This method detects discrete isoforms of GH from plasma and discriminates endogenous GH from its recombinant analog, which makes it useful for doping control purposes.     

Using a new solvation model for thermodynamic study on the interaction of nickel with human growth hormone

Thermodynamics of the interaction between Ni²⁺ and human growth hormone (hGH) were determined at 27 °C in Nail solution by isothermal titration calorimetry. A new method to predict protein penetration and the effect of metal ions on the stability of proteins is introduced. The new solvation model was used to reproduce the enthalpies of Ni²⁺-hGH interaction over the whole range of Ni²⁺ concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of hGH and its biological activity.     

Application of europium(III) chelates-bonded silica nanoparticle in time-resolved immunofluorometric detection assay for human thyroid stimulating hormone

Eu(III) chelate-bonded silica nanoparticle was used as a fluorescent label to develop a highly sensitive time-resolved immunofluorometric assay (TrIFA) for human thyroid stimulating hormone (hTSH). The limit of detection of the assay calculated according to the 2SD method was 0.0007 mIU L⁻¹ and became 0.003 mIU L⁻¹ when serum-based matrix was used for calibrators, indicating that this TrIFA is comparable with the most sensitive assays. The linear range was from 0.005 to 100 mIU L⁻¹ of hTSH with coefficient of variation between 1.9% and 8.3%. The correlation study using 204 blood spot samples from newborns showed that the results from this new method were coincident with that of the commercial dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) system, with a correlation coefficient of 0.938. The fluorescent nanoparticle label allows directly reading the fluorescent signal, omitting the signal development step required for the DELFIA system, and the whole procedure of this assay is fulfilled within 2 h. Thus, we developed a novel, sensitive, quantitative and simple nanoparticle label-based TrIFA assay, suitable for routine application in hTSH screening of neonatal hypothyroidism.     

Normalization using a tagged-internal standard assay for analysis of antibody arrays and the evaluation of serological biomarkers for liver disease

For minimizing systemic experimental variation in the analysis of antibody array data, we developed a novel median-centered/IgM-tagged-internal standard (TIS) assay normalization using median-centering and TIS assay-based determination of serum IgM concentrations. We evaluated five normalization methods by analyzing correlation coefficients and coefficients of variation for six serum proteins using human serum samples from normal controls (n = 25) and patients with liver cirrhosis (n = 25) or hepatocellular carcinoma (HCC; n = 29). Median-centered normalization improved correlation coefficients, while IgM-based normalizations improved coefficients of variation. The TIS assay was more efficient, economical, and reproducible for determining IgM concentrations than enzyme-linked immunosorbent assay. Additionally, we normalized antibody array data for six serum proteins using the median-centered/IgM-TIS assay, and evaluated serum biomarkers through distribution analysis of normalized fluorescence intensities and receiver operating characteristic analyses for the diagnosis of liver cirrhosis and HCC. Apolipoprotein A-1 and a combination of alpha-fetoprotein and C-reactive protein were determined to be potential serological biomarkers for liver cirrhosis and HCC, respectively. Thus, median-centered/IgM-TIS assay normalization is a useful approach for analyzing antibody array data and evaluating serological biomarkers for the diagnosis of liver disease or cancers.     

hGH release from directly compressed hGH-PLGA biodegradable implantable tablets: Influence of physicomechanical factors

The incidence of compression conditions, porosity and polymer degradation on human growth hormone (hGH) release from PLGA implantable tablets was evaluated with the aim of gaining insight in the mechanism involved in drug delivery from biodegradable matrices. Tablets elaborated by direct compression of hGH with PLGA, applying various compression forces for different times, kept the integrity and the stability of the hormone. Tablet dimensions, viscoelastic properties, glass to rubber transition temperature (T_g), PLGA degradation rate and water uptake were analyzed in the freshly prepared implantable tablets as well as at several times during release test in phosphate buffer pH 7.4. Placebo tablets were also prepared to evaluate the incidence of hGH on the physicomechanical properties of the device and PLGA degradation rate. Porosity remarkably determined the amount of hGH released, through an effect on the easiness of water penetration in the tablet and on the beginning of PLGA degradation. The decrease in PLGA molecular weight during the first days in the release medium, despite of being minor, significantly conditioned hGH release rate. The more dramatic changes in PLGA molecular weight observed after 20 days in the release medium notably reduced the T_g and the viscous and elastic moduli of the tablets. The overall analysis of the events underwent by the tablets in contact with the aqueous medium was used to explain the drug release profile and may help to optimize the design of the PLGA-based implantable tablets as peptidic drug delivery systems.     

Sensitive and simultaneous detection of different disease markers using multiplexed gold nanorods

A multiplexed bioanalytical assay is produced by incorporating two types of gold nanorods (GNRs). Besides retaining the desirable features of common GNRs LSPR sensors, this sensor is easy to fabricate and requires only a visible–NIR spectrometer for detection. This assay can simultaneously detect different acceptor–ligand pairs by choosing the proper GNRs possessing various LPWs in a wide detection wavelength range and can be developed into a high-throughput detection method. This bioanalytical assay allows easy detection of human serum specimens infected by S. japonicum and tuberculosis (TB) from human serum specimens (human serum/Tris–HCl buffer ratio = 1:10⁴) without the need for sample pretreatment. The technique is very sensitive compared to other standard methods such as indirect hemagglutination assays (IHA) that require a serum concentration ratio of larger than 1:20 and enzyme-linked immunosorbent assays (ELISA) requiring a ratio larger than 1:100. This methodology can be readily extended to other immunoassays to realize wider diagnostic applications.     

An optical surface plasmon resonance biosensor for determination of tetanus toxin

Surface plasmon resonance (SPR) has been successfully applied for the simple, rapid, and label-free assay of various biomolecules. This assay evaluates a novel wavelength modulation SPR biosensor for the detection of tetanus toxin. The wavelength modulation SPR biosensor is designed based on fixing the incident angle of light and measuring the reflected intensities in the resonance wavelength range spanning 400-800 nm simultaneously. Tetanus toxin (TeNT), one of the most potent toxins known, is synthesized as a 150 kDa single polypeptide chain. The SPR biosensor has been shown to be capable of directly detecting concentration of tetanus toxin as low as 0.028 Lf ml⁻¹. Under selected experimental conditions, the SPR biosensor has a good reproducibility, sensitivity and reversibility. The results illustrate how wavelength modulation SPR biosensor can be used to detect biomolecular interactions.     

Selective and sensitive homogenous assay of serum albumin with 1-anilinonaphthalene-8-sulphonate as a biosensor

Homogenous selective assay of albumin (ALB) in clinical sera was tested with 1-anilinonaphthalene-8-sulphonate (ANS) as Förster-resonance-energy-transfer (FRET) acceptor of tryptophan residues and biosensor of ALB. Between the excitation at 280 and 350 nm, the ratio of the fluorescence at 470 nm of free ANS in ethanol was about 1.9 while that of the complexes of ALB and ANS was about 3.9, supporting FRET in complexes of ANS and ALB. ANS below 1.0 mM saturated one site of ALB with K_d of about 0.13 μM in 20 mM sodium phosphate buffer at pH 7.0. For selective assay of ALB, 0.30 μM ANS was used to quantify fluorescence of the complexes at 470 nm under the excitation at 280 nm. ALB from 1.8 to 25 nM was quantified, whose lower limit was below 1% than that by bromocresol green assay while one-third than that by immunoturbidimetric assay. Globular proteins at comparable levels gave negligible signals. This new method showed reasonable resistance to other interfering substances in clinical sera. Quantities of ALB in clinical sera by this method were consistent with those by bromocresol green assay and immunoturbidimetric assay. Hence, homogenous assay of ALB with ANS as FRET biosensor was effective.     

促红细胞生成素和人生长激素兴奋剂检测方法的研究进展

在2008年世界反兴奋剂组织颁布的兴奋剂目录中,S2项肽类激素及相关品种均属于内源性生物大分子物质,如何区分所检测物质属于外界摄入还是机体分泌是此项检测的重点与难点。本文针对其中应用最为普遍、研究较为深入的促红细胞生成素(EPO)和人生长激素(hGH)的检测,从间接血液指标检测、直接检测途径等方面进行了分类评述,侧重于从理化分析方法、免疫分析方法角度阐述识别及区分重组蛋白与内源性蛋白的新途径。     

Affinity-based microdialysis sampling using heparin for in vitro collection of human cytokines

Microdialysis sampling is a widely used method to sample from complex biological matrices. Cytokines are important signaling proteins that are typically recovered with low relative recovery values during microdialysis sampling. Heparin was included in the microdialysis perfusion fluid as an affinity agent to increase in vitro recovery of different cytokines through polyethersulfone (PES) microdialysis membranes with 100 kDa molecular weight cutoff. No change in fluid volumes collected from the microdialysis probes occurred when heparin was included in the perfusion fluid up to concentrations of 10 μM. The loss of heparin (10 μM) across the dialysis membrane was minimal (2.7 ± 0.9%, n = 3). Additionally, heparin at these concentrations did not interfere with the cytokine immunoassays. The control and heparin-enhanced relative recoveries for five human cytokines using 0.1 μM heparin in the microdialysis perfusion fluid flowing at 0.5 μL min⁻¹ were (n = 3): interleukin-4 (IL-4), 4.2 ± 0.5% and 7.2 ± 3.1%; interleukin-6 (IL-6), 1.4 ± 0.8% and 3.6 ± 1.3%; interleukin-7 (IL-7), 1.3 ± 0.8% and 4.8 ± 1.8%; monocyte chemoattractant protein-1 (MCP-1), 9.0 ± 1.6% and 19.5 ± 2.7%; and tumor necrosis factor-α (TNF-α), 7.4 ± 1.3% and 16.9 ± 1.6%, respectively. Heparin increased the microdialysis sampling relative recovery of several human cytokines in vitro.     

Radioiodination of recombinant human growth hormone and its use in radioimmunoassay

Radioimmunoassay (RIA) of human growth hormone (hGH) using¹²⁵I-labeled tracer prepared from DNA recombinant hGH (r-hGH) and characterization of the tracer in the assay system are described. The radioiodination of r-hGH resulted in high yield of immunoreactive tracer. The immunoreactive fraction could be purified by gel-filtration on sephadex G-75. The quality of radioiodinated tracer of r-hGH has been found to be same as that of the tracer obtained from pituitary hGH (p-hGH) with respect to immunoreactivity, assay sensitivity and RIA standard curve parameters.     

Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media

The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5 kDa, 10 kDa and 30 kDa) using lysozyme as a model system. A stable binding via reduction of a Schiff base was formed during random PEGylation on lysine residues with methoxy-PEG-aldehyde. A purification method for PEGylated proteins using cation exchange chromatography was developed, and different isoforms of mono-PEGylated lysozyme were isolated. TSKgel SP-5PW and Toyopearl GigaCap S-650M showed the best performance of all tested cation exchange resins, and the separation of PEGylated lysozyme could be also scaled up to semi-preparative level. Size-exclusion chromatography, SDS-PAGE and MALDI-TOF mass spectrometry were used for analysis. Separated mono-PEGylated lysozyme of different sizes was used to determine dynamic binding capacities (DBC) and selectivity of cation exchange chromatography resins. An optimization of binding conditions resulted in a more than 20-fold increase of DBC for Toyopearl GigaCap S-650M with 30 kDa mono-PEGylated lysozyme.     

Level of Human Growth Hormone (hGH) in Urine Determined by A Highly Specific and Sensitive Sandwich Enzyme Immunoassay

Abstract

Levels of human growth hormone (hGH) in urine samples were determined by a highly specific and sensitive sandwich enzyme immunoassay, which had little cross-reaction with human luteinizing hormone, human follicle-stimulating hormone, human thyroid-stimulating hormone and human prolactin. The volume of urine samples used was 0.05 ml. Urine hGH levels in normal subjects, aged less than 2 yr, 2-5 yr, 6-15 yr and 28-35 yr (male) were 78-113, 19-51, 7.8-25 and 1.1-5.2 ng/g of creatinine, respectively. In dwarfism patients aged 6-15 yr, urine hGH levels were < 0.2-6.0 ng/g of creatinine.     

Monitoring the production of biosynthetic human growth hormone by micro enzyme-linked immunosorbent assay

A micro enzyme-linked immunosorbent assay (e.l.i.s.a.) has been developed for monitoring the production of human growth hormone (hGH) in E. coli. The method is unusually flexible, as it is possible to modify its conditions to give a sensitive (detection limit 4 ng l^?1) or a fast (6 h) version. The assay is reproducible; the between-assay relative standard deviation is 6%. The effects of temperature, incubation time and the concentrations of protein and the most important reagents (the solid-phase antibody and the labelled antibody) are described. The utility of the method is demonstrated in experiments optimizing the conditions for production of hGH in E. coli.