Human Hydroxyproline, HYP ELISA Kit

Catalog No.: BZEK2395

Size: 96T

Range: 2.5 μg/L - 40 μg/L

Sensitivity: 0.2 μg/L

Storage and Expiration: Store at 2-8℃ for 6 months. 

Application: For quantitative detection of HYP in human serum, plasma, tissue homogenate or cell culture supernatant.

Introduction

(2S,4R)-4-Hydroxyproline, or L-hydroxyproline (C5H9O3N), is an amino acid, abbreviated as Hyp or O, e.g., in Protein Data Bank. 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 catalyzed 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, an amount greater than seven other amino acids that are translationally incorporated.

Principle of the Assay  

This kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. The purified anti- HYP antibody was pre-coated onto 96-well plates. And the HRP conjugated anti- HYP antibody was used as detection antibodies. The standards, test samples and HRP conjugated detection antibody were added to the wells subsequently, mixed and incubated, then, unbound conjugates were washed away with wash buffer. TMB substrates (A & B) were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the HYP amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of HYP can be calculated.

Kit components

1. One 96-well plate pre-coated with anti- human HYP antibody

2. Standard: 0.5ml (80 μg/L)

3. Standard diluent buffer: 1.5 ml

4. Wash buffer (30×): 20 ml. Dilution: 1:30

5. Sample diluent buffer: 6 ml

6. HRP conjugated anti- human HYP antibody (RTU): 6ml

7. Stop solution: 6 ml

8. TMB substrate A:  6ml

9. TMB substrate B:  6ml

10. Plate sealer:   2

11. Hermetic bag:  1

Material Required But Not Provided

1. 37℃ incubator
2. Microplate reader (wavelength: 450nm)
3. Precise pipette and disposable pipette tips
4. Automated plate washer
5. ELISA shaker
6. 1.5ml of Eppendorf tubes
7. Absorbent filter papers
8. Plastic or glass container with volume of above 1L