Human PRCP (Prolylcarboxypeptidase) ELISA Kit (HUES02338)

SKU:
HUES02338
Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
€539

Description

ELISA Kit Technical ManualMSDS

Human PRCP (Prolylcarboxypeptidase) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human PRCP . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human PRCP and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human PRCP, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The OD value is proportional to the concentration of Human PRCP. The concentration of Human PRCP in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human PRCP in samples. No significant cross-reactivity or interference between Human PRCP and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(ng/mL)
O.D Average Corrected
102.37
2.398
2.3842.312
51.742
1.752
1.7471.675
2.50.937
0.919
0.9280.856
1.250.491
0.497
0.4940.422
0.630.286
0.282
0.2840.212
0.320.182
0.178
0.180.108
0.160.119
0.135
0.1270.055
00.067
0.077
0.072--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human PRCP were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PRCP were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.531.174.830.541.104.41
Standard deviation0.030.070.240.030.050.22
C V (%)5.665.984.975.564.554.99

Recovery

The recovery of Human PRCP spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)86-9992
EDTA plasma (n=5)87-10194
Cell culture media (n=5)88-10195

Linearity

Samples were spiked with high concentrations of Human PRCP and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)98-11195-10991-101
Average (%)10510096
1:4Range (%)90-10382-9488-99
Average (%)958893
1:8Range (%)88-10386-9785-100
Average (%)959292
1:16Range (%)89-10482-9687-99
Average (%)968894

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human PRCP (Prolylcarboxypeptidase) ELISA Kit (HUES02338) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37 °C.
    7. Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human PRCP (Prolylcarboxypeptidase) ELISA Kit (HUES02338) Protein Information

UniProt Protein Function:PRCP: Cleaves C-terminal amino acids linked to proline in peptides such as angiotensin II, III and des-Arg9-bradykinin. This cleavage occurs at acidic pH, but enzymatic activity is retained with some substrates at neutral pH. Belongs to the peptidase S28 family.
UniProt Protein Details:

Protein type:EC 3.4.16.2; Protease

Chromosomal Location of Human Ortholog: 11q14

Cellular Component: lysosome; plasma membrane

Molecular Function:serine carboxypeptidase activity; protein binding

Biological Process: plasma kallikrein-kinin cascade; negative regulation of systemic arterial blood pressure; regulation of blood vessel endothelial cell migration; glucose homeostasis; proteolysis; blood coagulation; angiogenesis involved in wound healing; blood coagulation, intrinsic pathway

NCBI Summary:This gene encodes a member of the peptidase S28 family of serine exopeptidases. The encoded preproprotein is proteolytically processed to generate the mature lysosomal prolylcarboxypeptidase. This enzyme cleaves C-terminal amino acids linked to proline in peptides such as angiotension II, III and des-Arg9-bradykinin. The cleavage occurs at acidic pH, but the enzyme activity is retained with some substrates at neutral pH. This enzyme has been shown to be an activator of the cell matrix-associated prekallikrein. The importance of angiotension II, one of the substrates of this enzyme, in regulating blood pressure and electrolyte balance suggests that this gene may be related to essential hypertension. A pseudogene of this gene has been identified on chromosome 2. Alternative splicing results in multiple transcript variants, at least one of which encodes an isoform that is proteolytically processed. [provided by RefSeq, Jan 2016]
UniProt Code:P42785
NCBI GenInfo Identifier:1172047
NCBI Gene ID:5547
NCBI Accession:P42785.1
UniProt Related Accession:P42785
Molecular Weight:
NCBI Full Name:Lysosomal Pro-X carboxypeptidase
NCBI Synonym Full Names:prolylcarboxypeptidase
NCBI Official Symbol:PRCP
NCBI Official Synonym Symbols:PCP; HUMPCP
NCBI Protein Information:lysosomal Pro-X carboxypeptidase
UniProt Protein Name:Lysosomal Pro-X carboxypeptidase
UniProt Synonym Protein Names:Angiotensinase C; Lysosomal carboxypeptidase C; Proline carboxypeptidase; Prolylcarboxypeptidase; PRCP
Protein Family:Paracaspase
UniProt Gene Name:PRCP
UniProt Entry Name:PCP_HUMAN
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