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HTRF Mouse Insulin Serum Detection Kit, 1,000 Assay Points
HTRF Mouse Insulin Serum Detection Kit, 1,000 Assay Points
Generic HTRF biomarker image
| Feature | Specification |
|---|---|
| Application | Protein Quantification |
| Sample Volume | 5 µL |
Product variants
Unit Size: 200 assay points
Part #:
62IN3PEF
Unit Size: 1,000 assay points
Part #:
62IN3PEB
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
HTRF Mouse Insulin Serum Detection Kit, 1,000 Assay Points
Generic HTRF biomarker image
HTRF Mouse Insulin Serum Detection Kit, 1,000 Assay Points
Product information
Overview
For years, researchers have sought the ideal insulin assay that would combine absolute specificity with sensitivity, accuracy, low sample consumption, and ease-of-use. The search has come to an end! This serum insulin assay offers superior specificity and sensitivity for a reliable quantification of insulin in serum and plasma samples. Built on a truly homogeneous protocol, the assay does not require any washing or complex extraction steps, leading to significant time savings over ELISA methods.
How it works
Assay principle
Insulin is measured using a sandwich immunoassay of two monoclonal antibodies, one labelled with Cryptate (donor) and the other with XL665 (acceptor). The intensity of the FRET signal obtained is proportional to the concentration of insulin in the sample.
Assay protocol
10 µL of cell supernatants are supplemented with 10 µL of acceptor-labelled antibodies and 5 µL of donor-labelled antibodies (or 15 µL of pre-mixed reagents for a single dispensing step). The mix is then incubated from 4h to overnight at RT and results are read on an HTRF compatible reader.
Assay details
Sensitivity
The detection limit* is calculated as two standard deviations above the Calibrator 0. The HTRF® Insulin assay was calibrated against the NIBSC international standard 66/304. The following equivalence was found: 1 ng Insulin HTRF® represent 1 ng Insulin NIBSC 66/304 (i.e.: 2.3x10-05 IU). Samples were incubated overnight at room temperature. The assay ranges from 0,24 to 8 ng/mL and the detection limit measured was 0,064 ng/mL.
Precision intra-assay
| Mouse serum sample | Mean [Insulin] (ng/mL) | CV |
|---|---|---|
| 1 | 0.25 | 7.6% |
| 2 | 0.73 | 3.7% |
| 3 | 0.9 | 5.4% |
| 4 | 2.84 | 4.6% |
| 5 | 5.17 | 4.0% |
| Mean CV | 5.1% |
Precision inter-assay
| Mouse serum sample | Mean [Insulin] (ng/mL) | CV |
|---|---|---|
| 1 | 0.37 | 14.6% |
| 2 | 0.67 | 9.5% |
| 3 | 0.9 | 4.0% |
| 4 | 1.1 | 8.1% |
| 5 | 6.9 | 9.2% |
| Mean CV | 9.1% |
Dilutional linearity
| Mouse serum sample | Dilution factor | [Insulin] expected (ng/mL) | [Insulin] measured (ng/mL) | Recovery |
|---|---|---|---|---|
| 1 | 1 | - | 0.97 | - |
| 2.5 | 0.388 | 0.368 | 95% | |
| 5 | 0.194 | 0.188 | 97% | |
| 2 | 1 | - | 4 | - |
| 2.5 | 1.6 | 1.57 | 98% | |
| 5 | 0.8 | 0.9 | 110% | |
| 3 | 1 | - | 7.1 | - |
| 2.5 | 2.84 | 3 | 105% | |
| 5 | 1.42 | 1.66 | 117% | |
| Mean Recovery | 103.7% |
Spike and recovery
| Mouse serum sample | [Insulin] added (ng/mL) | [Insulin] expected (ng/mL) | [Insulin] measured (ng/mL) | Recovery |
|---|---|---|---|---|
| 1 | 0 | - | 0.75 | - |
| 2.3 | 3.05 | 3.6 | 118% | |
| 4.8 | 5.55 | 6.4 | 115% | |
| 2 | 0 | - | 2.5 | - |
| 2.4 | 4.9 | 4.8 | 98% | |
| 4.8 | 7.3 | 8 | 110% | |
| 3 | 0 | - | 4.2 | - |
| 0.8 | 5 | 5.4 | 108% | |
| 1.6 | 5.8 | 6.2 | 107% | |
| Mean Recovery | 109.3% |
Correlation with leading ELISA
The HTRF Mouse serum insulin assay showed an excellent correlation to the leading absorbance and chemiluminescent ELISA, both on sera with low titers and high titers of insulin. With a slope of 1.1 and 0.9 respectively, the regression line was close to the ideal line, demonstrating the assay reliability.
Assay validation
Validation on normal mouse serum
C57BL/6J mice were randomized into two groups of 7 animals. One group was fasted overnight while the second one was normally fed. The mice were sacrificed and the blood was collected for serum preparation. The insulin concentration was determined in each sample using the HTRF Mouse Serum Insulin kit reagents.
Validation on diabetic mouse plasma
A group of five db/db diabetic male mice and a group of five db/+ control male mice were fasted overnight. The mice were sacrificed and the blood was collected through cardiac puncture for K3 EDTA-plasma preparation. The insulin concentration of each sample was determined using the HTRF Mouse Serum Insulin kit reagents.
Validation on obese mouse serum
A group of eight ob/ob obese mice and a group of eight ob/+ control mice were sacrificed and the blood was collected through cardiac puncture for serum preparation. The insulin concentration of each sample was determined using the HTRF Mouse Serum Insulin kit reagents.
Specifications
| Application |
Protein Quantification
|
|---|---|
| Brand |
HTRF
|
| Detection Modality |
HTRF
|
| Product Group |
Kit
|
| Sample Volume |
5 µL
|
| Shipping Conditions |
Shipped Ambient
|
| Target Class |
Biomarkers
|
| Target Species |
Mouse
|
| Technology |
TR-FRET
|
| Therapeutic Area |
Metabolism/Diabetes
|
| Unit Size |
1,000 assay points
|
Video gallery
HTRF Mouse Insulin Serum Detection Kit, 1,000 Assay Points
HTRF Mouse Insulin Serum Detection Kit, 1,000 Assay Points
Citations
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Flyer
Obesity, diabetes and metabolic disease reagents
This flyer details HTRF™ and AlphaLISA™ assays for investigating key biomarkers and signaling pathways in metabolic disease...
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