Glucagon for IDF

Development and validation of a high-sensitivity enzyme-linked immunosorbent assay for specific measurement of glucagon
Mattias Gäreskog, Annika Carlsson, Maria Schwanbeck, Eleni Karamihos, Carolina Gäredal, Lisa Grufman, Hanna Ritzén, Magnus Simonsson,
Eva Ludvigsen and Robert Gunnarsson
Research and Development, Mercodia AB, Uppsala, Sweden.
CONCLUSION
The novel ELISA is sensitive and highly specific for glucagon, with no need for sample extraction.
AIM
The correlation between glucagon and glicentin in Sprague Dawley rats is weak.
Our aim was to develop a highly sensitive ELISA for specific measurement of glucagon.
Different methods of sample preservation and extraction were also investigated.
Specific measurement of glucagon assists in further understanding the physiological function of this peptide.
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT
Oxyntomodulin
HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA
Glicentin, human
RSLQDTEE KSRSFSAS QADPLSDP DQMNEDKR HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA
Glicentin, mouse
HALQDTEE NPRSFPAS QTEAHEDP DEMNEDKR HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA
1 = serum
2 = EDTA
3 = EDTA + aprotinin
4 = EDTA + protease + esterase + DPP-4 inhibitor (p800)
5 = EDTA + DPP-4 inhibitor (p700)
Proglucagon 1-61
GLP-1
IP-2
GLP-2
Glicentin
Oxyntomodulin
Proglucagon 1-61
POSTTRANSLATIONAL
PROCESSING
M5F9S
Y
L-intestinal cells
E6A11K
Y
L2B4G
Brain
IP-1
Glucagon levels
The glucagon concentration range was 5.1 – 26.9 pmol/L
in 10 non-fasted rat samples and 4.9 – 22.2 pmol/L in 10
fasted rat samples. For mouse samples, the glucagon range
was 5.5 – 23.4 pmol/L in 10 non-fasted mouse samples
and 3.3-13.1 pmol/L in 10 fasted mouse samples.
1st screening
Glucagon
Glicentin
(pmol/L)
5.2 ± 0.9
39 ± 5.1
5.1 ± 7.1
46 ± 5.5
4.4 ± 1.0
6.4 ± 1.2*
–
47 ± 5.6
56 ± 7.1*
–
2nd screening
Glucagon Glicentin
(pmol/L)
–
–
9.9 ± 2.1* 65 ± 14.8*
20 20 0 0 –
15 ± 3.2
15 ± 3.0
S-‐1 S-‐1 S-‐2 S-‐2 E-mail
[email protected]
[email protected]
S-‐4 S-‐4 8080 S-‐5 S-‐5 72 ± 16.7
73 ± 16.2
S-‐7 S-‐7 S-‐8 S-‐8 S-‐9 S-‐9 S-‐10 S-‐11 S-‐12 S-‐13 S-‐14 S-‐15 S-‐16 S-‐17 S-‐18 S-‐19 S-‐20 S-‐10 S-‐11 S-‐12 S-‐13 S-‐14 S-‐15 S-‐16 S-‐17 S-‐18 S-‐19 S-‐20 4040 S-‐1 S-‐2 S-‐3 S-‐4 S-‐5 S-‐6 S-‐7 S-‐8 S-‐9 S-‐10 S-‐11 S-‐12 S-‐13 S-‐14 S-‐15 S-‐16 S-‐17 S-‐18 S-‐19 S-‐20 Sample ID
Figure 1. Glicentin and glucagon in EDTA samples from Sprague Dawley rats.
140 140
p<0.05, ANOVA (n=8).
Extraction
Extraction by acetonitril may be used to concentrate samples, but
extraction is not needed for specific capture of glucagon in this assay.
S-‐6 S-‐6 00 *
p<0.05, ANOVA (n=10).
Glicen8n 60 2020 1st screening
p800 tubes were the most stable method of sample preservation.
2nd screening
p700 and p800 showed similar stability in sample preservation.
Glucagon 60
–
*
Specificity
Oxyntomodulin (human/rat/mouse) 4%
Glicentin (human)
0.8%
Mini-glucagon (a.a. 19-29)
<0.1%
GLP-1
<0.3%
GLP-2
<0.3%
GRPP
<0.0005%
S-‐3 S-‐3 100 100
S-17
EDTA +
aprotinin
p800
p700
40 40 S-16
A recovery between 80% and 120% is generally deemed
to be acceptable to confirm selectivity and linearity of a
method.
Sample
types
Serum
EDTA
120 120
S-15
Recovery
Upon addition 96-101% (Mean 98%)
Upon dilution 81-96% (Mean 86%)
Sample types
60 60 S-14
Detection limit
Detection limit is 1 pmol/L
Glucagon Glucagon
Glucagon Glicen8n Glicen8n Glicentin
140 140
S-13
RESULTS
80 80 S-12
Concentra)on Concentra)on (pmol/L) (pmol/L) 100 100 S-11
GLP-2
S-4
Proglucagon 1-61
GLICENTIN
Mercodia Rat Glicentin ELISA:
L2B4G Coated plate (Capture Ab)
RG-OC5 HRP-Conjugate (Detection Ab)
Mercodia120 Glucagon ELISA:
M5F9S 120 Coated plate (Capture Ab)
E6A11K HRP-Conjugate (Detection Ab)
S-3
Glucagon
GLP-1
S-2
GRPP
MPGF
Or visit:
www.mercodia.com
GLP-1(7-36)AMIDE
GLP-1(7-37)
S-1
GLP-2
Oxyntomodulin
Concentra)on (pmol/L) GLP-1
Glucagon IP-1
140 140 Concentration (pmol/L)
Pancreatic alpha cells
Glicentin
y=3.9268x + 7.0135
R2=0.36293
120 120
100 100
y = 3,9268x + 7,0135 R² = 0,36293 80 80
Glicen'n (pmol/L) Glucagon
Glucagon IP-1
GLUCAGON
RG-OC5
Glicentin (pmol/L)
GRPP
GRPP
5
Sample extraction using acetonitril was also evaluated.
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S-10
Glucagon IP-1
S-9
GRPP
S-8
Proglucagon
4
S-20
GLP-2
3
S-19
IP-2
2
S-18
GLP-1
S-7
Glucagon IP-1
S-6
GRPP
S-5
PS
HAPQDTEE NARSFPAS QTEPLEDP DQINEDKR HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA
Y
Preproglucagon
1
Y
Glicentin, rat
Glucagon and glicentin preservation was examined in five
different sample types:
Serum
Glucagon
p700
Monoclonal antibodies against mammalian glucagon were generated in mice. A solid phase two-site enzyme immunoassay was developed, using a N-terminal anti-glucagon clone for capture and a peroxidase-conjugated C-terminal
clone for detection. Calibrators were prepared from synthetic glucagon calibrated against WHO 1st International
reference preparation 69/194.
Specificity was evaluated against oxyntomodulin and glicentin. Selectivity and linearity were evaluated by recovery
studies.
Glucagon and glicentin, in samples from Sprague Dawley rats, were measured in the Mercodia Glucagon ELISA
and Mercodia Rat Glicentin ELISA (under development) that does not cross-react with glucagon.
p800
Until today, it has been difficult to specifically measure glucagon in rat and mouse
samples because of its shared sequence with the proglucagon-derived intestinal peptides glicentin and oxyntomodulin, found in blood. There is a need for a specific and
robust glucagon assay that does not require labor-intense procedures of extraction.
Our aim was to develop and validate a highly sensitive ELISA using a low sample volume, for specific measurement of glucagon. Different methods of sample preservation
and extraction were investigated.
EDTA + aprotinin
METHODS
EDTA
BACKGROUND
60 60
40 40
20 20
00 0 0
5 5
10 10
15 15
Glucagon (pmol/L) 20 20
Glucagon (pmol/L)
Figure 2. Correlation of glucagon and rat glicentin.
25 25
30 30

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