Mouse EPO (Erythropoietin) ELISA Kit (MOES00647)

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MOES00647
€549

Description

ELISA Kit Technical ManualMSDS

Mouse EPO (Erythropoietin) 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 Mouse EPO . 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 Mouse EPO 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 Mouse EPO, 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 Mouse EPO. The concentration of Mouse EPO in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection MethodColormetric
Detection Range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Mouse EPO in samples. No significant cross-reactivity or interference between Mouse EPO 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
(pg/mL)
O.D Average Corrected
50002.543
2.569
2.5562.482
25001.685
1.729
1.7071.633
12501.049
1.035
1.0420.968
6250.469
0.497
0.4830.409
312.50.259
0.257
0.2580.184
156.250.189
0.177
0.1830.109
78.130.123
0.135
0.1290.055
00.073
0.075
0.074--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)245.10434.002393.20221.90454.002268.20
Standard deviation14.7019.50124.4012.0025.90111.10
C V (%)6.004.495.205.415.704.90

Recovery

The recovery of Mouse EPO 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)86-9791
EDTA plasma (n=5)86-10293
Cell culture media (n=5)90-10696

Linearity

Samples were spiked with high concentrations of Mouse EPO 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 (%)97-11288-10093-107
Average (%)10394101
1:4Range (%)99-11284-9697-113
Average (%)10590104
1:8Range (%)95-11083-9594-105
Average (%)10289100
1:16Range (%)100-11483-9692-105
Average (%)1068898

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

Mouse EPO (Erythropoietin) ELISA Kit (MOES00647) 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 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl 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 100µL 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 350µL 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 100µL 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 90µL 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.

Mouse EPO (Erythropoietin) ELISA Kit (MOES00647) Protein Information

UniProt Protein Function:EPO: Erythropoietin is the principal hormone involved in the regulation of erythrocyte differentiation and the maintenance of a physiological level of circulating erythrocyte mass. Genetic variation in EPO is associated with susceptbility to microvascular complications of diabetes type 2 (MVCD2). These are pathological conditions that develop in numerous tissues and organs as a consequence of diabetes mellitus. They include diabetic retinopathy, diabetic nephropathy leading to end-stage renal disease, and diabetic neuropathy. Diabetic retinopathy remains the major cause of new-onset blindness among diabetic adults. It is characterized by vascular permeability and increased tissue ischemia and angiogenesis. Belongs to the EPO/TPO family.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide

Cellular Component: extracellular space; cell surface; extracellular region

Molecular Function:erythropoietin receptor binding; hormone activity; protein kinase activator activity

Biological Process: hemoglobin biosynthetic process; heart morphogenesis; erythrocyte maturation; apoptosis; positive regulation of transcription, DNA-dependent; regulation of transcription from RNA polymerase II promoter; negative regulation of myeloid cell apoptosis; peptidyl-serine phosphorylation; positive regulation of tyrosine phosphorylation of Stat5 protein; positive regulation of protein kinase activity; positive regulation of cell proliferation; cardiac muscle morphogensis; response to hypoxia; erythrocyte differentiation; ventricular cardiac muscle morphogenesis; angiogenesis; positive regulation of Ras protein signal transduction; positive regulation of neuron differentiation; embryo implantation; positive regulation of DNA replication; negative regulation of apoptosis

NCBI Summary:This gene encodes the glycoprotein hormone erythropoietin that regulates the production of red blood cells and biosynthesis of hemoglobin. The predominant expression of this gene shifts from the liver during fetal development to kidney in adults. A complete lack of the encoded protein causes embryonic lethal anemia in mice. The conditional inactivation of this gene in adult mice results in a chronic, normocytic and normochromic anemia. Transgenic mice expressing the human ortholog of this gene exhibit polycythemia. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Aug 2015]
UniProt Code:P07321
NCBI GenInfo Identifier:119528
NCBI Gene ID:13856
NCBI Accession:P07321.1
UniProt Related Accession:P07321
Molecular Weight:
NCBI Full Name:Erythropoietin
NCBI Synonym Full Names:erythropoietin
NCBI Official Symbol:Epo  
NCBI Protein Information:erythropoietin
UniProt Protein Name:Erythropoietin
Protein Family:Erythropoietin
UniProt Gene Name:Epo  
UniProt Entry Name:EPO_MOUSE
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Additional Information

Reactivity:
Mouse
ELISA Type:
Sandwich
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