- Assay range：62.5-4000nmol/L
- Sensitivity：31.25nmol/L
- Method：Competitive
Intended use:
This immunoassay kit allows for the in vitro quantitative determination of General HYP concentrations in serum, Plasma, tissue homogenates and Cell culture supernates and Other biological fluids.
Test principle:
The ELISA is based on the competitive binding enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antibody specific to General HYP, During the reaction, General HYP in the sample or standard competes with a fixed amount of biotin-labeled General HYP for sites on a pre-coated Monoclonal antibody specific to General HYP. Excess conjugate and unbound sample or standard are washed from the plate. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of General HYP in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Linearity
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Hydroxyproline and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample | 1:2 | 1:4 | 1:8 | 1:16 |
serum(n=5) | 111-120% | 102-112% | 85-94% | 100-111% |
EDTA plasma(n=5) | 109-118% | 92-104% | 113-123% | 106-115% |
heparin plasma(n=5) | 93-105%
| 101-111% | 99-110% | 96-108% |
General Annotation
Function:
Hydroxyproline is a major component of the protein collagen. Hydroxyproline and proline play key roles for collagen stability. They permit the sharp twisting of the collagen helix. Hydroxyproline is found in few proteins other than collagen. For this reason, hydroxyproline content has been used as an indicator to determine collagen and/or gelatin amount.
Location:
Hydroxyproline is produced by hydroxylation of the amino acid proline by the enzyme prolyl hydroxylase following protein synthesis (as a post-translational modification). The enzyme catalysed reaction takes place in the lumen of the endoplasmic reticulum. Although it is not directly incorporated into proteins, hydroxyproline comprises roughly 4% of all amino acids found in animal tissue, more than seven amino acids which are directly incorporated.
Sequence: C5H9NO3