Antibody-coated tube method for radioimmunoassay of human growth hormone (original) (raw)
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[Radioimmunoassay for human growth hormone]
Radiobiologia, radiotherapia
Detailedmethodsfor: (1) 1311 iodination of human growth hormone (HGH); (2) purification of labeled HGH (HGH-139); and (3) radioimmunoassay of HGH are given. Bio-GeI filtration is introduced as a rapid and reproducible method for purifying Solution B: 67.0 gin,
Molecular Immunology, 1982
The reactivities of five mouse monoclonal antibodies against human growth hormone (hCiH) were defined by either a c~)mpetitive radioimmunoassay with insolubilized antibodies or by an aggfutination-inhibition method with hGH-coated polystyrene particles, The five antibodies reacted sigmficantly 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 to 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 s&ructure cf a major ant&tic site of which various portions are recognized by the mono~lonal antibodies.
Time-resolved immunofluorometric assay (tr IFMA) of human growth hormone
Clinical Chemistry
We describe a time-resolved immunofluorometric assay (trIFMA) for human growth hormone (hGH), in which monoclonal antibody (mAb)-coated microtiter strip wells and a europium (Eu) chelate-labeled mAb are used. We compare the new trIFMA, in which two mAbs are used, with an immunoradiometric assay (IRMA) in which polyclonal antibodies are used. Serum samples (n = 185) from 36 childrenwith various diagnoses were analyzed. In addition, 24-h profile samples (72 per child) from 39 children were analyzed. The trIFMA was more sensitive (detection limit,0.03 mIU/L) than existing IRMAs. Boththe intra-and interassay CVs were 10.6% for hGH concentrations between 1 and 100 mlU/L The trIFMA is technically simple and rapid, requires no centrifugation or separation reagent, and has a counting time of only 1 s per sample. In addition, the Eu label is nontoxic, presents no waste-disposal problems, and has a long shelf-life.
Clinical chemistry, 1997
The impact of the adoption of the new biosynthetic growth hormone (GH) WHO International Reference Preparation (IRP 88/624), and the recommendation to report results in microgram/L instead of mU/L, is described. Conversion factors were determined by comparing both the linear and nonlinear relations of the GH values. The Pharmacia polyclonal IRMA (p-IRMA) and the DELFIA monoclonal time-resolved immunofluorometric assay (trIFMA) with kit calibrators calibrated either against the pituitary-derived WHO IRP 80/505 or the new 88/624 were evaluated. Conversion factors of 4.17 mU/L = 1 microgram/L for the p-IRMA and 4.31 mU/L = 1 microgram/L for the trIFMA were necessary. Different cross-reactivity patterns for the deaminated and dimer 22-kDa, 20-kDa, and 17-kDa GH isoforms were found. Expected GH recovery was similar when the measured values were adjusted according to the results of the cross-reactivity study.
Analytical Chemistry, 1991
The technique of high-performance affinity chromatography (HPAC) is applied to the quantltative determination of antibodies to human growth hormone (hGH) in serum from patients. An afflntty column consisting of covalently hnmobtlized protein G on a rigid support is used to capture the antibodies. Texas Red labeled hGH (hGH-TR) is used as a fluorescence probe for detectlng the anti-hGH antibodies. Callbration curves are established by uslng a well-characterized monoclonal antibody to hGH (GHC101). The minimum detectable concentration (MDC) of anti-hGH antibody in serum is 250 ng/mL (this represents 10 ng of anti-hGH injected onto the protein G column). Analytical recoveries are 92-110% for seven replicates with 250-4000 ng/mL of GHC101. A precision of 15% relative standard deviation (RSD) can be achieved at the MDC. The preclslon Is better above the detection limit. The linear dynamic range of the method is approximately 2 orders of magnitude. The total fluorescence recovery from the affinity column is 296%. Sample analysis times are on the order of 20 min. The HPAC technlque gives results in absolute units of concentration that correlate well with blnding capaclty values determlned by radioimmunoassay.
Specific monoclonal antibodies and ultrasensitive immunoassays for 20K and 22K human growth hormone
Growth Hormone & IGF Research, 2010
Objective: Generation of specific monoclonal antibodies (mAbs) against 20K and 22K human growth hormone (hGH) and development of ultrasensitive immunoassays to quantify 20K and 22K hGH. Design: Mice were immunized with recombinant 20K or 22K hGH. Hybridoma cells were screened with biotinylated 20K and 22K hGH simultaneously. The specific mAbs were further characterized and used for construction of isoform specific assays. The ultrasensitive chemiluminescent assays were developed with AMDEX™ streptavidin-HRP and a sensitive substrate. Results: The 20K hGH specific mAb 1G12 and the 22K hGH specific mAb 5E1 showed less than 0.1% crossreactivity to 22K or 20K hGH by competitive binding assay, respectively. Western blot analysis also confirmed the specificity of mAb 1G12 and mAb 5E1. Using mAb 1G12 and mAb 5E1, 20K and 22K specific assays with working range of 2-2000 pg/mL were constructed. The 22K hGH concentrations in 103 serum samples from different healthy subjects in the basal GH state were 343.7 ± 421.5 pg/mL (18.6-1820 pg/ mL). The 20K hGH concentrations were 30.7 ± 37.5 pg/mL (2.4-205 pg/mL). The ratios of 20K to 20K plus 22K hGH were 9.8 ± 4.4% (3.3-28.3%). Both 22K hGH and 20K hGH concentrations in women (465.9 ± 476.3 pg/mL and 43.7 ± 46.1 pg/mL, n = 47) were significantly higher than those (241.1 ± 337.0 pg/mL and 20K hGH 19.8 ± 23.0 pg/mL, n = 56, P < 0.01) in men. However, there was no difference in the proportion of 20K to 20K plus 22K between men and women (P > 0.05). The strong correlation between 20K and 22K hGH (R = 0.914, P < 0.01) indicated the constant proportion between 20K and 22K hGH in the basal GH state of healthy subjects. Conclusions: Specific monoclonal antibodies and ultrasensitive chemiluminescent immunoassays for 20K and 22K hGH were generated. The ultrasensitive immunoassays are essential for the determination of 20K and 22K hGH in the basal GH state. This universal ultrasensitive immunoassay form can be adapted to other immunoassays for broad application.
ELISA for Determination of Human Growth Hormone: Recognition of Helix 4 Epitopes
Journal of Biomedicine and Biotechnology, 2004
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.