Rat Thrombopoietin ELISA Kit (A80372)

Name: Rat Thrombopoietin ELISA Kit
Brand: Antibodies.com
SKU: A80372
Price: 410.0 GBP
Availability: InStock

1 Citation

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This product is discontinued

Rat Thrombopoietin ELISA Kit (A80372) has been discontinued and is no longer available.

View all Thrombopoietin ELISA Kits.

Name

Rat Thrombopoietin ELISA Kit

Description

Rat Thrombopoietin ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of rat Thrombopoietin in serum, plasma, tissue homogenates, and other biological fluids.

Assay Type

Sandwich (quantitative)

Principle of Assay

Rat Thrombopoietin ELISA Kit (A80372) employs the sandwich enzyme immunoassay technique for the quantitative measurement of rat Thrombopoietin in serum, plasma, tissue homogenates, and other biological fluids. An antibody specific for Thrombopoietin has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the Thrombopoietin present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-Thrombopoietin Antibody, which binds the captured Thrombopoietin present in each well. Following incubation, unbound biotinylated detection antibody is removed by washing, and an HRP-Streptavidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is proportional to the amount of Thrombopoietin captured in each well. The concentration of Thrombopoietin can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.

Platform

Pre-coated Microplate (12 x 8 well strips)

Detection Type

Colorimetric

Instrument

Colorimetric Microplate Reader

Sample Type

Serum, plasma, tissue homogenates, and other biological fluids.

Sensitivity

18.75 pg/ml

Product Range

31.25-2000 pg/ml

Assay Time

4 hours

Reactivity

Rat

Recovery

Sample Type	n	Range	Average
Serum	5	87% - 105%	95%
EDTA Plasma	5	87% - 102%	94%
Heparin Plasma	5	87% - 104%	95%

Linearity

Sample Type	n	1:2	1:4	1:8
Serum	5	88-105%	85-96%	87-98%
EDTA Plasma	5	90-101%	84-94%	82-100%
Heparin Plasma	5	84-97%	82-96%	84-88%

Precision

Intra-assay CV: < 8%, Inter-assay CV: < 10%

Storage

Store at +4°C. Do not use past expiration date!

Synonyms

C-mpl ligand, Megakaryocyte colony-stimulating factor, Megakaryocyte growth and development factor, MGDF, ML, Myeloproliferative leukemia virus oncogene ligand, THPO

Product Note

Information online is representative. Always refer to the Product Manual inside the kit.

Disclaimer

This product is for research use only. It is not intended for diagnostic or therapeutic use.

Kit Components

Item	Quantity	Storage
Pre-Coated 96 Well Microplate	12 x 8 Well Strips	+4°C
Lyopholized Standard	2 Vials	+4°C
Sample Dilution Buffer	20ml	+4°C
Biotinylated Detection Antibody	120µl	+4°C
Antibody Dilution Buffer	10ml	+4°C
HRP-Streptavidin Conjugate	120µl	+4°C
SABC Dilution Buffer	10ml	+4°C
TMB Substrate	10ml	+4°C
Stop Solution	10ml	+4°C
Wash Buffer (25X)	30ml	+4°C
Plate Sealers	5 Adhesive Strips	-
Foil Pouch	1 Zip-Sealed Pouch	-

Required Materials

The following materials are not included in the kit, but are required to run this assay:

Microplate reader capable of measuring absorbance at 450nm ± 10nm.
Squirt bottle, multichannel pipette reservoir, or automated microplate washer.
Graph paper or computer software capable of generating logarithmic functions.
Test tubes and Eppendorf tubes to prepare standards and samples.
Multi- and single-channel pipettes and sterile pipette tips.
Deionized or distilled water.
Absorbent paper towels.
37°C incubator.

Important Notes

Sample concentrations should be estimated before being used in the assay and a proper dilution factor should be selected to make the diluted target protein concentration fall in the optimal detection range of this kit.
Sample matrix components will interfere with the test results. All samples must be diluted at least 1:2 with Sample Dilution Buffer.
Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.
This ELISA kit is intended for the analysis of biological samples and has not been validated for purified/recombinant proteins, industrial, or commercial materials. Performance with such samples cannot be guaranteed, as differences in expression, sequence, tags, folding, post-translational modifications, purity, activity, or buffer composition may affect assay results. For further guidance, please contact our technical support team.

Bring all reagents and samples to room temperature before use.

We recommend that all standards and samples be assayed in duplicate.

Step 1

Prepare all reagents, samples, and working standards as directed in the previous sections.

Step 2

Determine the number of microplate strips required for the assay and insert them into the plate frame. Unused strips should be stored in the foil pouch at +4°C.

Step 3

Add 100μl of standard solutions to the standard wells. Note: A plate layout is provided to record standards and samples assayed.

Step 4

Add 100μl of samples to the sample wells. Note: All samples must be properly diluted in order to produce sample values within the dynamic range of the assay. All samples must be diluted at least 1:2 with Sample Dilution Buffer.

Step 5

Cover with an adhesive plate sealer provided and incubate at 37°C for 90 minutes.

Step 6

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate two times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time. Note: Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Blot the plate onto paper towels or other absorbent material. Do not let the wells dry completely at any time!

Step 7

Add 100μl of Biotinylated Detection Antibody working solution to each well.

Step 8

Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

Step 9

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate three times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time.

Step 10

Add 100μl of HRP-Streptavidin Conjugate (SABC) working solution to each well.

Step 11

Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

Step 12

Remove the plate sealer, aspirate the liquid from the plate, and wash the plate five times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time.

Step 13

Add 90μl of TMB Substrate to each well. Cover with a new adhesive plate sealer and incubate at 37°C in the dark for 10-20 minutes. Note: This incubation time is for reference only, the optimal reaction time should be determined by the end-user and can be shortened or extended according to the actual color change. The reaction may be terminated when a gradient is apparent in the standard wells. Do not exceed 30 minutes!

Step 14

Add 50μl of Stop Solution to each well. The color in the wells should change from blue to yellow immediately. Note: If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

Step 15

Determine the optical density (O.D. value) of each well immediately using a microplate reader set to 450 nm.

Scientific Validation DataValidation Data (2)

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Standard Curve - Rat Thrombopoietin ELISA Kit (A80372)

Representative standard curve for Rat Thrombopoietin ELISA Kit (A80372).

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Rat Thrombopoietin ELISA Kit (A80372)

Quantification of thrombopoietin, IL-6 and CXCL12 in bone marrow extracellular fluid and serum from WKY and SHR rats using Rat Thrombopoietin ELISA Kit (A80372). The data summarise cytokine alterations underlying enhanced marrow thrombopoiesis in hypertensive rats, including IL-6-driven megakaryocyte expansion and CXCL12-dependent changes in sinusoidal apposition.

Product Citations (1)

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Altered thrombopoiesis in spontaneously hypertensive rats

This publication cites Rat Thrombopoietin ELISA Kit (A80372).

Article Abstract

Platelet hyperreactivity is a risk factor for cardiovascular disease development and mortality in hypertensive patients. Although several studies have linked platelet hyperreactivity with increased bone marrow thrombopoiesis, extramedullary thrombopoiesis remains highly unexplored in hypertension and other cardiovascular diseases. Here, we investigated thrombopoiesis-related parameters in the bone marrow, spleen, and lungs of 5-week-old spontaneously hypertensive rats (SHR) and their normotensive counterparts, Wistar Kyoto rats by quantitative histological imaging, flow cytometry, and enzyme-linked immunoassays. Flow cytometry assays and quantitative histological imaging strongly suggest that thrombocytosis in SHR is caused solely by bone marrow thrombopoiesis. Moreover, analysis of the lungs showed a significant decrease in the density of megakaryocyte and CKIT^pos/CD41^pos cells, suggesting potential implications for lung megakaryopoiesis of SHR rats. Finally, while IL6 levels increased by twofold in the bone marrow, TPO levels decreased in both the bone marrow and serum of SHR rats. Overall, our results raise the question of whether pulmonary and bone marrow thrombopoiesis may regulate each other through TPO-based feedback, posing key questions about platelet biology under steady state and pathological conditions.

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