Alternative Sequential Protocol for Increased Specificity in Mercodia Glucagon ELISA (10-1271-01)

The Mercodia Glucagon ELISA is validated with a simultaneous assay protocol. See specifications in the Directions for Use for the Mercodia Glucagon ELISA 10-1271-01.

Under certain conditions like after bariatric surgery, kidney disease and exogenous administration, the levels of Glicentin1, Proglucagon 1-612 and Oxyntomodulin can be elevated to levels that might interfere in the Glucagon ELISA. By using a modified sequential protocol described here, which allows for an extra wash step after sample application, the highest possible specificity is achieved. The modification of the protocol will improve specificity (Table 1) by washing away un-specifically bound material from the antibody-coated plate, without losing sensitivity (detection limit <1.0 pmol/L).

Sample correlation between the protocols is good (Figure 1) and the average recovery for Dilutional Linearity and Parallelism with the new protocol is accepted.

Please read the original Directions for Use for more details about the assay procedure before performing the assay. Observe that this protocol is for research use only and is not validated for CE/IVD use.

Please Observe: Extra Reagents Needed

This protocol requires one extra vial of Conjugate Buffer (art no 20-7055), which can be ordered by emailing info(at)bio-connectdiagnostics.nl.

References

1. Roberts, G. P. et al. Gastrectomy with Roux-en-Y reconstruction as a lean model of bariatric surgery. Surg. Obes. Relat. Dis. (2018). doi:10.1016/j.soard.2018.01.039
2. Wewer Albrechtsen, N. J. et al. Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production. Cell Rep. 21, 1452–1460 (2017)

Sequential Protocol For Mercodia Glucagon ELISA (10-1271-01)

Please read the original Directions for Use for the Mercodia Glucagon ELISA (10-1271-01) for more information before performing the assay.

Add Calibrators, Controls and samples
25 µL
Add Enzyme Conjugate Buffer (art no 20-7055) to all wells
200 µL
Incubate
O/N (18-22h)
at 4°C on a plate shaker
(700-900 rpm)
Wash plate with wash buffer 1X solution
6 times*
Add enzyme conjugate 1X solution to all wells
200 µL
Incubate
1 h
at 18-25°C on a plate shaker
(700-900 rpm)
Wash plate with wash buffer 1X solution
6 times*
Add Substrate TMB
200 µL
Incubate
30 minutes at 18-25°C on the bench
Add Stop Solution
50 µL
Shake for 5 seconds to ensure mixing
Measure A450
Evaluate results

* Wash 6 times with 700 μL wash buffer 1X solution per well, using an automatic plate washer with an overflow wash function. After the final wash, invert and tap the plate firmly against absorbent paper. Do not include a soak step in the washing procedure.
For manual washing, see Technical Note No: 34-0106 Instruction for manual washing procedure for microplates (available online).

Performance Characteristics

Detection Limit

Detection limit is defined as the Capability of Detection according to ISO11843-Part 1. Capability of Detection should be seen as part of a method validation, rather than the lowest concentration that can be measured.

The detection limit is 1 pmol/L as determined with the methodology described in ISO11843-Part 4. Concentrations of samples with absorbances below Calibrator 1 should not be calculated, but expressed as less than or equal to (≤) the concentration indicated on the vial for Calibrator 1.

Cross-reactivity

The sequential assay protocol minimizes cross-reactivity from all pro-glucagon derivatives achieving the highest possible specificity.

Table 1. Cross-reactivity using the sequential protocol. Values below detection limit will not be detected (noted n.d.).

Concentrations tested (pmol/L)
Cross-reactivity (%)
Glicentin
3-600n.d. 
Oxyntomodulin
3-40n.d.
60-3003.1-3.6
Proglucagon (1-61)
5-25n.d. 
Glucagon (3-29)
5-1000n.d.
n.d. = not detected

Recovery

Dilutional Linearity: The average recovery is 103 % (99-108 %) for EDTA plasma samples

Paralllelism: The average recovery is 106 % (98-120 %) for EDTA plasma samples

Sample correlation

Correlation between the two different assay protocols was good with an R2-value above 0.98.


Figure 1. Correlation plot including 29 samples (11 P800 plasma and 18 EDTA plasma samples).



Source:
Technical Note: Alternative Sequential Protocol for Increased Specificity in Mercodia Glucagon ELISA

Related to:
Brands: Mercodia
Product groups: Assays