Human Parathyroid Hormone ELISA Kit 96 Wells High Precision and
Sensitivity
Cat.No E1055Hu
Standard Curve Range: 3pg/ml - 1000pg/ml
Sensitivity: 1.51pg/ml
Size: 96 wells
Storage: Store the reagents at 2-8°C. For over 6-month storage refer to
the expiration date keep it at -20°C. Avoid repeated thaw cycles.
If individual reagents are opened it is recommended that the kit be
used within 1 month.
*This product is for research use only, not for use in diagnosis
procedures. It’s highly recommend to read this instruction entirely
before use.
Intended Use
This sandwich kit is for the accurate quantitative detection of
Human Parathyroid Hormone (also known as PTH) in serum, plasma,
cell culture supernates, cell lysates, tissue homogenates.
Reagent Provided
| Components | Quantity |
| Standard Solution (1200pg/ml) | 0.5ml x1 |
| Pre-coated ELISA Plate | 12 * 8 well strips x1 |
| Standard Diluent | 3ml x1 |
| Streptavidin-HRP | 6ml x1 |
| Stop Solution | 6ml x1 |
| Substrate Solution A | 6ml x1 |
| Substrate Solution B | 6ml x1 |
| Wash Buffer Concentrate (30x) | 20ml x1 |
| Biotinylated Human PTH Antibody | 1ml x1 |
| User Instruction | 1 |
| Plate Sealer | 2 pics |
| Zipper bag | 1 pic |
Material Required But Not Supplied
- 37°C±0.5°C incubator
- Absorbent paper
- Precision pipettes and disposable pipette tips
- Clean tubes
- Deionized or distilled water
- Microplate reader with 450 ± 10nm wavelength filter
Specimen Collection
Serum Allow serum to clot for 10-20 minutes at room temperature.
Centrifuge at 2000-3000 RPM for 20 minutes.
Plasma Collect plasma using EDTA or heparin as an anticoagulant.
Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C
within 30 minutes of collection.
Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for
approximately 20 minutes. When collecting pleuroperitoneal fluid
and cerebrospinal fluid, please follow the procedures
above-mentioned.
Cell Culture Supernatant Collect by sterile tubes when examining secrete components.
Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect
the supernatants carefully. When examining the components within
the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the
cell concentration of approximately 1 million/ml. Damage cells
through repeated freeze-thaw cycles to let out the inside
components. Centrifuge at 2000-3000 RPM for approximately 20
minutes.
Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly
and weigh before homogenization. Mince tissues and homogenize them
in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or
freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20
minutes.
Reagent Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (1200pg/ml) with 120μl of
standard diluent to generate a 600pg/ml standard stock solution.
Allow the standard to sit for 15 mins with gentle agitation prior
to making dilutions. Prepare duplicate standard points by serially
diluting the standard stock solution (600pg/ml) 1:2 with standard
diluent to produce 300pg/ml, 150pg/ml, 75pg/ml and 37.5pg/ml
solutions. Standard diluent serves as the zero standard(0 pg/ml).
Any remaining solution should be frozen at -20°C and used within
one month. Dilution of standard solutions suggested are as follows:
| 600pg/ml | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
| 300pg/ml | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
| 150pg/ml | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
| 75pg/ml | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
| 37.5pg/ml | Standard No.1 | 120μl Standard No.2 + 120μl Standard Diluent |
| Standard Concentration | Standard No.5 | Standard No.4 | Standard No.3 | Standard No.2 | Standard No.1 |
| 1200pg/ml | 600pg/ml | 300pg/ml | 150pg/ml | 75pg/ml | 37.5pg/ml |
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or
distilled water to yield 500 ml of 1x Wash Buffer. If crystals have
formed in the concentrate, mix gently until the crystals have
completely dissolved.
Summary
1. Prepare all reagents, samples and standards.
2. Add sample and ELISA reagent into each well. Incubate for 1 hour
at 37°C.
3. Wash the plate 5 times.
4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.
5. Add stop solution and color develops.
6. Read the OD value within 10 minutes.
Calculation of Result
Construct a standard curve by plotting the average OD for each
standard 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. These calculations can be best performed with
computer-based curve-fitting software and the best fit line can be
determined by regression analysis.
References
"Structural basis for parathyroid hormone-related protein binding
to the parathyroid hormone receptor and design of
conformation-selective peptides."
Pioszak A.A., Parker N.R., Gardella T.J., Xu H.E.
J. Biol. Chem. 284:28382-28391(2009)
