Insulin Mouse Serum Assay Kit

The specificity, sensitivity and accuracy of an ELISA. A fraction of the time and sample volume.

For years researchers have sought the ideal insulin assay which would combine absolute specificity with sensitivity, accuracy, low sample consumption and ease of use. The search has come to an end.

Cisbio’s 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 a significant time saving over Enzyme-linked immune-sorbent assay (ELISA) methods.

 

Cisbio offers 3 kits for the quantification of insulin
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Download Insulin guide

Assay principle

Insulin is measured using a sandwich immunoassay of two monoclonal antibodies, one labelled with Lumi4-Tb Cryptate (Donor) and one labeled with XL665 (Acceptor). The intensity of the FRET signal obtained is proportional to the concentration of insuline in the sample.

Designed for ease of use, the assay does not require lengthy extraction procedures or sample pre-treatment as may be the case for other technologies. Simply add the detection reagents and read. Both detection reagents may be pre-mixed for a single dispensing step.

Built for specificity, the assay is compatible with mouse plasma & serum samples.

Law sample volume protocols:

Product Perfomance

The mouse serum insulin assay is designed for in vivo experiments on mouse models secreting low, medium or high insulin levels: normal mice (e.g. C57BL/6J or N), diabetic mice (e.g. db/db vs. controls db/+), obese mice (e.g. ob/ob vs. controls ob/+).

C57BL/6J mice were randomized into two groups of 7 animals. One group was fasted overnight while the second one was normally fed. 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.

One group of five db/db diabetic male mice and one group of five db/+ control male mice were fasted overnight. 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.

One group of eight ob/ob obese mice and one 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.

Correlation with leading ELISA

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.

Analytical performance

Sensitivity

Assay range 0.24 - 8 ng/mL
Limit of Detection (LoD)* 0.064 ng/mL
Incubation time Overnight at room temperature

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: 1ng Insulin HTRF® ↔ 1ng Insulin NIBSC 66/304 (i.e.: 2.3x10-05 IU).

Precision

5 % CV obtained for the intra-assay and 9% CV for the inter-assay show the high robustness and reproducibility of the Mouse Insulin assay.

Intra-assay (n=24)

Mouse serum
sample
Mean [Insulin]
(ng/mL)
CV
1 0.25 7.6%
2 0.73 3.7%
3 0.90 5.4%
4 2.84 4.6%
5 5.17 4.0%
  Mean CV 5.1%

Each of the 5 mouse serum samples is measured 24 times, and % CV is calculated for each sample.

Inter-assay (n=4)

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%

5 different mouse serum samples are measured in 4 different experiments. and % CV is calculated for each sample.

Accuracy

Dilutional linearity

Mouse serum sample

Dilution
factor

[Insulin]
expected (ng/mL)
[Insulin]
measured (ng/mL)
Recovery
1

1
2.5
5

-
0.388
0.194

0.97
0.368
0.188

-
95%
97%

2

1
2.5
5

-
1.60
0.8

4.0
1.57
0.9

-
98%
110%

3

1
2.5
5

-
2.84
1.42

7.1
3.0
1.66

-
105%
117%

      Mean Recovery 103.7%

3 mouse serum samples were diluded 5- and 2.5-fold, the excellent % of recovery obtained from this experiment shows the good linearity of the assay

 

Spike and recovery

Mouse serum sample

[Insulin] added
(ng/mL)

[Insulin]
expected (ng/mL)
[Insulin]
measured (ng/mL)
Recovery
1

0
2.3
4.8

-
3.05
5.55

0.75
3.6
6.4

-
118%
115%

2

0
2.4
4.8

-
4.9
7.3

2.5
4.8
8.0

-
98%
110%

3

0
0.8
1.6

-
5.0
5.8

4.2
5.4
6.2

-
108%
107%

      Mean Recovery 109.3%

Two amounts of insulin (2.3 and 4.8 ng/mL) were added to 3 different mouse serum sample, the set of response obtained from a standard curve was compared to the expected values calculated. The 100%of recovery observed for the validates the sample matrix used for this assay

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Insulin Mouse Serum kit - 5 x 200 tests62IN3PEB
-
Insulin Mouse Serum kit - 200 tests62IN3PEF
-

Companion products

DescriptionCat. noProduct insertMSDS
Insulin kit standard62INSCDA
-

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