Hydroxyproline ELISA Kit

Catalog No. ABIN956238

The Human Hydroxyproline ELISA Kit (ABIN956238) is a Colorimetric ELISA Kit designed to quantify Human Hydroxyproline.

Quick Overview for Hydroxyproline ELISA Kit (ABIN956238)

Target: Hydroxyproline (Hyp)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Product Details Hydroxyproline ELISA Kit

Purpose: This HYP ELISA kit is intended for laboratory research use only and is not for use in diagnostic or therapeutic procedures.

Analytical Method: Quantitative

Characteristics: This HYP enzyme linked immunosorbent assay applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a polyclonal antibody specific for HYP. Calibrators or samples are then added to the microtiter plate wells and HYP if present, will bind to the antibody pre-coated wells. In order to quantitatively determine the amount of HYP present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody, specific for HYP are added to each well to sandwich the HYP immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, A and B substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain HYP and enzyme- conjugated antibody will exhibit a change in color. 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.

Components: Microtiter Plate: 96 wells
Calibrator 1 (0 µmol/L)
Calibrator 2 (0.5 µmol/L)
Calibrator 3 (1 µmol/L)
Calibrator 4 (2.5 µmol/L)
Calibrator 5 (5 µmol/L)
Calibrator 6 (10 µmol/L)
Enzyme Conjugate (1 x 6 mL)
Substrate A (1 x 6 mL)
Substrate B (1 x 6 mL)
Stop Solution (1 x 6 mL)
Wash Buffer (100X concentrate) (1 x 6 mL)
Lysis Buffer Solution (1 x 6 mL)
Note: The lysis buffer solution is used only when the sample is cell culture fluid & body fluid & tissue homogenate, If the sample is serum or blood plasma, then the lysis buffer solution is a superfluous reagent.

Material not included: 1. Microplate reader capable of measuring absorbance at 450 nm.
2. Pipettes and pipette tips.
3. 100 mL and 1 liter graduated cylinders.
4. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi- channel pipette is desirable for large assays.)
5. 37°C incubator.
6. Absorbent paper.
7. Distilled or de-ionized water.
8. Data analysis and graphing software. Graph paper: linear (Cartesian), log-log or semi-log, or log-logit as desired.
9. Tubes to prepare calibrator or sample dilutions.

Application Details

Plate: Pre-coated

Reagent Preparation:

Bring all kit components and samples to room temperature (18-25°C) before use. Dispense 5 µL of lysis buffer solution into 50 µL specimens, mix and stand for one hour (The proportion of lysis buffer and specimens shall be no less than 1:10). (NOTE: This step is required when the sample is cell culture fluid & body fluid & tissue homogenate, If the sample is serum or blood plasma, then this step should be skipped.) Wash Solution Dilute 10 mL of Wash Solution concentrate (100X) with 990 mL of de-ionized or distilled water to prepare 1,000 mL of Wash Solution (1X).

Assay Procedure:

It is recommended that all Calibrators and Samples be added in duplicate to the Microtiter Plate.
1. Secure the desired number of coated wells in the holder then add 50 µL of Calibrators or Samples to the appropriate well in the antibody pre-coated Microtiter Plate.
2. Add 100 µL of Conjugate to each well. Mix well. Mixing well in this step is important. Cover and incubate for 1 hour at 37°C.
3. Wash the microtiter plate using one of the specified methods indicated below:
4. Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Fill in each well completely with diluted wash solution, and then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure five times for a total of five washes. After washing, invert plate, and blot dry by hitting the plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame. Complete removal of liquid at each step is essential to good performance.
5. Automated Washing: Wash plate five times with diluted wash solution (350-400 µL/well/wash) using an auto washer. After washing, dry the plate as above. It is recommended that the washer be set for a soaking time of 10 seconds and shaking time of 5 seconds between each wash.
6. Add 50 µL substrate A and 50 µL substrate B to each well, subsequently. Cover and incubate for 15 minutes at 20-25°C. (Avoid sunlight.)
7. Add 50 µL of stop solution to each well. Mix well.
8. Read the optical density (OD) at 450 nm using a microtiter plate reader immediately.

Calculation of Results:

1. This calibration curve is used to determine the amount of an unknown sample. Construct a calibration curve by plotting the average OD (450 nm) for each calibrator on the vertical (Y) axis against the concentration on the horizontal (X) axis, and draw a best fit curve through the points on the graph.
   2. First, calculate the mean OD value for each calibrator and sample. All OD values are subtracted by the mean value of blank control before result interpretation. Construct the calibration curve using graph paper or statistical software.
   3. To determine the amount in each sample, first locate the OD value on the Y-axis and extend a horizontal line to the calibration curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
   4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own calibration curve.
   5. The sensitivity in this assay is 0.1 µmol/L.
   6. This assay has high sensitivity and excellent specificity for detection of HYP. No significant cross- reactivity or interference between HYP and analogues was observed. Note: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between HYP and all the analogues, therefore, cross reaction may still exist in some cases.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: All reagents provided are stored at 4°C. Refer to the expiration date on the label.

Expiry Date: The expiry date is stated on the label.

Target Details for Hydroxyproline

Target: Hydroxyproline (Hyp)

Alternative Name: Hydroxyproline

Target Type: Amino Acid