Human Growth Hormone Binding Protein (GHBP) ELISA Kit

Sample

Serum and plasma

Species Reactivity

Human

Detection Method

sELISA

Intended Use

The Human GHBP ELISA Kit is intended to be used for quantitative measurement of GHBP in human serum and plasma samples.

Contents of Kit

1. Microtiter plate: ready for use, coated with rabbit-anti-GHBP-antibody. Wells are separately breakable. (8x12) wells
2. Sample Buffer (PP): ready for use, please shake before use! 1 x 120 mL
3. Standards (A-F): lyophilized, (recombinant GHBP in rabbit serum), concentrations are given on vial labels and on quality certificate. 6 x 750 μL
4. Control Serum 1 (KS1): lyophilized, (human serum), concentration is given on quality certificate. 1 x 250 μL
5. Control Serum 2 (KS2): lyophilized, (human serum), concentration is given on quality certificate. 1 x 250 μL
6. Antibody Conjugate (AK): ready for use, contains rabbit biotinylated anti-GHBP antibody. 1 x 12 mL
7. Enzyme Conjugate (EK): ready for use, contains Streptavidin-Peroxidase Conjugate. 1 x 12 mL
8. Washing Buffer (WP): 20-fold concentrated solution. 1 x 50 mL
9. Substrate (S): ready for use, horseradish-peroxidase-(HRP) substrate, stabilised Tetramethylbencidine. 1 x 12 mL
10. Stopping Solution (SL): ready for use, 0.2 M sulfuric acid. 1 x 12 mL
11. Sealing Tape: for covering the microtiter plate. 3

Storage

Store the kit at 2-8°C after receipt until its expiry date. The lyophilized reagents should be stored at –20 °C after reconstitution. Avoid repeated thawing and freezing.
The shelf life of the components after initial opening is warranted for 4 weeks at 2-8°C, store the unused strips and microtiter wells airtight together with the desiccant at 2-8°C in the clip-lock bag, use in the frame provided. The reconstituted components standards A-F and Control Sera KS1 and KS2 must be stored at –20°C (max. 4 weeks). For further use, thaw quickly but gently (avoid temperature increase above room temperature and avoid excessive vortexing). Up to 3 of the freeze-thaw cycles did not influence the assay. The 1:20 diluted Washing Buffer WP is 4 weeks stable at 2-8°C

Precision

Intra-Assay-Variation
A native serum sample has been measured 16 times on different positions on the plate and at a mean concentration of 14.89 ng/mL GHBP (SD 0.65) an intra-assay variability of 4.38% was detected.
Inter-Assay-Variation
Serum samples were measured in independent assays. On average the coefficient of variation was 7.72% (Range 3.08 – 10.67%).

Sensitivity

The analytical sensitivity (LoD) was assessed by measuring the blank and calculating the theoretical concentration of the blank + 2SD. The analytical sensitivity of the Human GHBP ELISA Kit is 0.009 ng/mL as mean, in 3 independent determinations values ranging from 0.007 to 0.010 ng/mL were found. The theoretical limit of quantification (LoQ) (10 times the standard deviation of the blank value) is 0.043 ng/mL.

General Description

Growth Hormone Binding Protein (GHBP) consists of 238 amino acids and includes four sides for glycosylation and three disulphide bounds. In humans GHBP is formed by receptor shedding of the growth hormone receptor by a metalloprotease (ADAM17). In equilibrium about 50% of circulating growth hormone (GH) is bound to GHBP but only 2% of the circulating GHBP bound a GH molecule with a stoichiometry of 1:1. Only in case of supraphysiological GHBP levels a 2:1 ratio appears. The complex of GH and GHBP has an approximate molecular weight of 80 kDa (GHBP 60 kDa). In an animal model (guinea pig) the complex formation increases half-life from 11-20 minutes up to about 100 minutes and in general binding to GHBP inhibits GH cellular action.
GHBP Physiology:
GHBP concentration is independent of GH pulsatility and does not show a circadian rhythm. GHBP levels are low until 2-6 months of life, increase steeply in the first two years and continue to increase slowly until early adulthood. From the 4th decade the GHBP serum concentration declines slowly. GHBP correlates positively with the intraabdominal fat mass and is increased in type II diabetics with hyperinsulinemia. It is not known whether the tight relationship between fat mass and circulating GHBP results from GHBP expression in adipocytes or any other mechanism. From a scientific point of view undetectable GHBP levels could point to a GH insensitivity, caused by a deletion in the GH-receptor gene. Further, the IGF-I/GHBP ratio might be an indicator for GH-deficiency in adults, in particular in women. It could also be predictive for GH treatment response. The strong positive relationship with intraabdominal fat mass might be a hint, that GHBP is a possible biomarker for the amount of visceral adipose tissue.

Standard Curve

The exemplary data and the standard curve in Figure below cannot be used for the calculation of the test results. You have to establish a standard curve for each test you conduct.