YK161

Total GLP-１-HS ELISA Kit

For Measurement of Mouse, Rat & Human Total GLP-1
in Plasma(EDTA), Culture medium supernatant

FOR LABORATORY USE ONLYYANAIHARA INSTITUTE INC.
2480 - 1 AWAKURA, FUJINOMIYA-SHISHIZUOKA, JAPAN 418-0011

Contents
Ⅰ． Introduction

2

Ⅱ． Characteristics

3

Ⅲ． Composition

4

Ⅳ． Method

5-7

Ⅴ． Notes

7-8

Ⅵ． Performance Characteristics

8-12

Ⅶ． Stability and Storage
Ⅷ.

12

References

12-13

1

− Please read all the package insert carefully before beginning the assay −YK161 Total GLP-1-HS ELISA Kit
Ⅰ．Introduction
GLP-1 is a peptide hormone from the intestinal mucosa, which is produced from its precursor,
proglucagon by post transnational processing. The mammalian proglucagon 1) is synthesized in theneuroendocrine L-cell of the intestine and the alpha-cells of the pancreas. It contains within its
structure the sequences of glucagon and two glucagon-like peptides (GLP-1 and GLP-2) in tandem
flanked at their amino and carboxyl termini by dibasic residues. GLP-1 is a 37 amino acids peptide
and produced in the small intestine and in the pancreas in the human, in either C-terminal-amidated
on glycine-extended form2) 3).
GLP-1 (7-36) amide and its receptor are present in several brain regions and may play a role in thephysiological control of feeding4). Several reports have been presented as follows as to the biologicalactivities of GLP-1. GLP-1 (7-37) and (7-36) amide is known as one of the most potent insulin
secretagogues 5).
GLP-1 (7-36) amide was supposed to improved glycemic control in patients with type 2 diabetes
by increasing insulin secretion, by inhibiting glucagon secretion and by delaying gastric emptying
rather than by altering extrapancreatic glucose metabolism6). Intravenous GLP-1 (7-37) and (7-36)
amide could normalize fasting hyperglycaemia in type 2 diabetic patients7). Hyperglycaemia during
parenteral nutrition could be controlled by exogenous GLP-1, whereas the chronic therapy of type 2
diabetes required GLP-1 derivatives with longer duration of action8). Recombinant GLP-1 (7-36)
amide was recently shown to cause significant weight loss in type 2 diabetics when administered for
6 weeks as a continuous subcutaneous infusion, 5-day treatment of hereby obese human subjects with
GLP-1 at high doses by prandial subcutaneous infusion promptly slowed gastric emptying as aprobable mechanism of action of increased satiety, decreased hunger and reduced food intake with an
ensuing weight loss9).

2

YK161 Total GLP-1-HS ELISA Kit

Contents▼ The assay kit can measure GLP-1 within therange of 1.24 – 300 pM▼ The assay is completed within 18+1+0.5 and 0.5
hr.
▼ With one assay kit, 41 samples can be measured
in duplicate.
▼ The 96-wells plate in kit is consisted by 8-wells
strips, and the strips can be used separately.
▼ Stability and storage
Store all of the components at 2-8°C.
This kit is stable under the condition for
24 months from the date of manufacturing.
The expiry date is stated on the package

1)
2)
3)
4)
5)
6)
7)
8)

Antibody coated plate
GLP-1(7-36)amide standard
Labeled antibody solution
SA-HRP solution
Enzyme substrate solution (TMB)
Stopping solution
Buffer solution
Washing solution(Concentrated)
9) Adhesive foil

Ⅱ．Characteristics
This ELISA kit is used for quantitative determination of total GLP-1 in mouse, rat and humanplasma sample. The kit is characterized by sensitive quantification and high specificity. In addition, it
is not influenced by other components in plasma sample and needlessness of sample pre-treatment.
<Specificity>
This ELISA kit has high specificity to GLP-1(1-36) amide, GLP-1(7-36) amide and GLP-1(9-36)
amide and shows low crossreactivity to GLP-1(1-37) and GLP-1(7-37), and shows no crossreactivity
to rat and human GLP-2, rat Glicentin, human Glucagon, rat mouse and human GIP (1-42), rat andhuman GIP (3-42).
<Assay principle>
This kit for determination of total GLP-1 in mouse, rat and human plasma(EDTA) and culture
supernatant sample is based on the sandwich enzyme immunoassay. During first immune incubation,
GLP-1(7-36)amide in standards or in samples bind to the rabbit anti GLP-1(7-36)amide antibody,which is coated on the surface of the microtiter plate. After incubation and plate washing, labeled
antibody solution (biotinylated rabbit anti GLP-1(7-36)amide polyclonal antibody) is added to bindto the antibody-antigen complex. Then, HRP labeled streptoavidin (SA-HRP) is added to form
antibody-antigen-biotinylated antibody complex. Finally, HRP enzyme activity is determined by
3,3’,5,5’-Tetramethylbenzidine (TMB) and the concentration of total GLP-1 is calculated.

3

Ⅲ．Composition

Component

Form

Quantity

Main Ingredient1.

Antibody coated plate

microtiterplate

1 plate (96 wells)

Rabbit anti GLP-1 (7-36) amide
polyclonal antibody coated2.

Standardlyophilized

1 vial (300 fmol)

Synthetic GLP –1 (7-36)amide

3.

Labeled antibody
solution

liquid1 bottle (12 mL)

Biotinylated rabbit anti GLP -1(7-36)amide polyclonal antibody

4.

SA-HRP solution

liquid1 bottle (12 mL)

Horseradish peroxidase labeledstreptoavidin

5.

Enzyme substratesolution

liquid1 bottle (12 mL)

3,3’,5,5’-Tetramethylbenzidine
(TMB)

6.

Stopping solution

liquid1 bottle (12 mL)

1M H2SO4

7.

Buffer solution

liquid2 bottles(20 mL x 2)

Buffer containing a reaction
accelerator

8.

Washing solution(concentrated)liquid1 bottle (50 mL)

Concentrated saline

9.

Adhesive foil

4 pieces4

Ⅳ．Method<Equipment required>
1. Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
2. Photometer for microtiter plate（plate reader）, which can read the extinction 3.0 at 450 nm
3. Microtiter plate shaker
4. Glass test tubes for preparation of standard solution
5. Washing device for microtiter plate, dispenser with aspiration system
6. Graduated cylinder（1,000 mL）
7. Distilled water or deionized water<Preparatory work>
1. Preparation of the standards：
Reconstitute the GLP-1 (7-36) amide standard (lyophilized 300 fmol/vial) with 1 mL of buffer solution,
which affords 300 pM standard solution. The reconstituted standard solution (0.1 mL) is diluted
with 0.2 mL of buffer solution that yields 100 pM standard solution. Repeat the dilution
procedure to make each standard solution of 33.3, 11.1, 3.70 and 1.24 pM standard solutions.
Buffer solution itself is used as 0 pM standard solution.
2. Dilution of mouse plasma (x5 dilution) :Add 80 L of buffer solution into an appropriate glass test tube. Pipette 20 L of mouse plasma
into the tube and vortex to mix.
There is no need to dilute for rat and human plasma.
3. Preparation of the washing solution：
Dilute 50 mL of the washing solution (concentrated) to 1,000 mL with distilled or deionizedwater.
4. Other reagents are ready for use.

5

<Procedure>
1. Before starting the assay, bring all the reagents and samples to room temperature (20-30ºC).
2. Fill 0.35 mL/well of washing solution to each of the wells and aspirate the washing solution in
the wells. Repeat this washing procedure further twice (total 3 times). Finally, invert the plate andtap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual
washing solution.
3. <Human and Rat plasma samples (culture medium supernatant), Mouse culture medium
supernatant>
Add 100 µL of buffer solution to the wells first, and then introduce 25 L each of standard
solution (0, 1.24, 3.70, 11.1, 33.3, 100, 300 pM) or samples to each of the wells.
<Mouse plasma samples>
Add 200 µL of buffer solution to the wells first, and then introduce 30 L each of standard
solution (0, 1.24, 3.70, 11.1, 33.3, 100, 300 pM) or diluted (x5) plasma samples to each of thewells.
4. Cover the plate with adhesive foil and incubate it at room temperature (20-30°C) for 18 hours.
During the incubation, the plate should be shaken with a plate shaker (approximately 100 rpm).
5. After incubation, take off the adhesive foil, aspirate and wash the wells 3 times with approximate
0.35 mL/well of washing solution. Finally, invert the plate and tap it onto an absorbent surface,such as paper toweling, to ensure blotting free of most residual washing solution.
6. Add 100 L of labeled antibody solution to each of the wells.
7. Cover the plate with adhesive foil and incubate it at room temperature (20-30°C) for 1 hour.
During the incubation, the plate should be shaken with a plate shaker (approximately 100 rpm).
8. Take off the adhesive foil, aspirate and wash the wells 3 times with approximate 0.35 mL/well of
washing solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper
toweling, to ensure blotting free of most residual washing solution.
9. Add 100 L of SA-HRP solution to each of the wells.
10. Cover the plate with adhesive foil and incubate the plate at room temperature (20-30°C) for 30
minutes. During the incubation, the plate should be shaken with microtiter plate shaker(approximately 100 rpm).
11. Take off the adhesive foil, aspirate and wash the wells 4 times with approximate 0.35 mL/well of
washing solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper
6toweling, to ensure blotting free of most residual washing solution.
12. Add 100 L of Enzyme substrate solution (TMB) to each of the wells, cover the plate withadhesive foil and keep it for 30 minutes at room temperature in a dark place for color reaction(keep still, plate shaker not need).
13. Add 100 L of the stopping solution to each of the wells to stop color reaction.
14. Read the optical absorbance of the wells at 450 nm. The dose-response curve of this assay fits
best to a 5 (or 4)-parameter logistic equation. The results of unknown samples can be calculatedwith any computer program having a 5 (or 4)-parameter logistic function. Otherwise calculatemean absorbance values of wells containing standards and plot a standard curve on double
logarithmic graph paper (abscissa: concentration of standard; ordinate: absorbance values). Use
the average absorbance of each sample to determine the corresponding value by simple
interpolation from this standard curve.
In the case of diluted mouse plasma samples, multiply concentrations of unknown samples by 5
to obtain the final concentration of total GLP-1.

Ⅴ．Notes
1. EDTA-2Na (1 mg/mL) additive blood collection tube is recommended for the plasma collection.
Plasma samples must be used as soon as possible after collection. If the samples are tested later,
they should be divided into test tubes in small amount and frozen at or below -30°C. Avoid
repeated freezing and thawing of samples. If same blood sample is to be prepared for measuring
GLP-1 active form only using another kit, DPP-4 inhibitor should be added immediately to theblood, yielding 100 M final concentration.
2. Standard solution should be prepared immediately before use. This kit can be used dividedly in
strips of the plate. In such case, the rest of reconstituted standard in glass vial or tube (300 pM)
should be stored at 4°C or below –30°C is stable for 4 weeks. Diluted standard solutions should
not be reused for another assay.
3. The total pipetting time of standard solutions and samples for a whole plate should not exceed 30
minutes.
4. During storage of washing solution (concentrated) at 2-8°C, precipitates may be observed,
however they will dissolve when diluted.
5. Pipetting operations may affect the precision of the assay, so that pipette standard solutions or
samples precisely into each well of plate. In addition, use clean test tubes or vessels in assay anduse new tip for each standard or sample to avoid cross contamination.

7

6. When sample concentration exceeds 300 pM, it needs to be diluted with buffer solution to proper
concentration.
7. During the incubation except the color reaction, the plate should be shaken gently by a plateshaker to promote immunoreaction (approximately 100 rpm).
8. Perform all the determination in duplicate.
9. Read plate optical absorbance of reaction solution in wells as soon as possible after stop color
reaction.
10. To quantitate accurately, always run a standard curve when testing samples.
11. Protect reagents from strong light (e.g. direct sunlight) during storage and assay.
12. Satisfactory performance of the test is guaranteed only when reagents are used from combination
pack with identical lot number.

Ⅵ．Performance Characteristics

Typical standard curve
10.00

O.D. (450nm)

1.00

0.10

0.01
1.00

10.00

100.00

GLP-1(7-36)amide (pM)

81000.00

<Analytical recovery><Human Plasma A>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
3.41
8.43
23.06
106.36

Expected(pM)

Recovery
(%)

8.41
23.41
103.41

100.2498.51
102.85<Human Plasma B>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
4.30
9.15
23.01
98.45

Expected(pM)

Recovery
(%)

9.30
24.30
104.30

98.39
94.69
94.39

<Human Plasma C>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
7.99
13.33
28.99
116.98

Expected(pM)

Recovery
(%)

12.99
27.99
107.99102.62103.57108.33<Human Plasma D>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
21.03
26.32
39.00
114.36

Expected(pM)

Recovery
(%)

26.03
41.03
121.03101.11
95.05
94.49<Rat Plasma A>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
11.31
14.87
26.47
82.86

Expected(pM)

Recovery
(%)

16.31
31.31
111.31

91.17
84.54
74.44

<Rat Plasma B>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
7.91
11.81
22.82
81.87

Expected(pM)

Recovery
(%)

12.91
27.91
107.91

91.48
81.76
75.87

9

<Mouse plasma A>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
2.85
7.43
20.08
82.36

Expected(pM)

Recovery
(%)

7.85
22.85
102.85

94.65
87.88
80.08

<Mouse plasma B>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100

Observed
(pM)
2.93
7.21
20.53
83.53

Expected(pM)

Recovery
(%)

7.93
22.93
102.93

90.92
89.53
81.15

<Mouse plasma C>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
4.18
8.78
21.72
80.73

Expected(pM)

Recovery
(%)

9.18
24.18
104.18

95.64
89.83
77.49

<Mouse plasma D>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
2.97
7.39
19.71
78.44

Expected(pM)

Recovery
(%)

7.97
22.97
102.97

92.72
85.81
76.18

<Mouse plasma E>
Added GLP-1(7-36)amide
(pM)
0.00
5.00
20.00
100.00Observed
(pM)
4.10
8.78
21.83
87.80

Expected(pM)

Recovery
(%)

9.10
24.10
104.10

96.48
90.58
84.34

10

<Dilution test>
Dilution ratio
Human Plasma 1

Human Plasma 2

Human Plasma 3

Human Plasma 4

ｘ 1.0
ｘ 2.0
ｘ 4.0
ｘ 8.0
ｘ 1.0
ｘ 2.0
ｘ 4.0
ｘ 8.0
ｘ 1.0
ｘ 2.0
ｘ 4.0
ｘ 8.0
ｘ 1.0
ｘ 2.0
ｘ 4.0
ｘ 8.0
Dilution ratio

Rat Plasma 1

Rat Plasma 2

ｘ 1.0
ｘ 2.0
ｘ 4.0
ｘ 8.0
ｘ 1.0
ｘ 2.0
ｘ 4.0
ｘ 8.0

Observed
(pM)
27.62
15.73
7.14
3.08
37.22
20.65
10.66
5.24
32.07
15.78
7.14
3.18
15.31
8.52
3.96
1.41

Estimated(pM)

Recovery
(%)

31.46
28.56
24.64

113.90103.40
89.21

41.30
42.64
41.92

110.96114.56112.63

31.56
28.56
25.44

98.41
89.06
79.33

17.04
15.84
11.28

111.30103.4673.68

Observed
(pM)
14.87
6.17
2.89
1.32
10.30
4.93
2.32
1.03

Estimated(pM)

Recovery
(%)

12.34
11.56
10.56

82.99
77.74
71.02

9.86
9.28
8.24

95.73
90.10
80.00

<Precision and reproducibility>
Intra-assay: Human plasma CV (%) 1.98 – 5.43
Intra-assay: Mouse plasma CV (%) 4.87 – 4.98
Inter-assay: Human plasma CV (%) 2.21 – 3.84
Inter-assay: Mouse plasma CV (%) 4.86 – 9.76

11

<Crossreactivity>
GLP-1 fragments
GLP-1 (7-36) amide
GLP-1 (9-36) amide
GLP-1 (1-36) amide
GLP-1 (1-37)
GLP-1 (7-37)
GLP-1 Related peptides
Rat GLP-2
Human GLP-2
Rat Glicentin
Human Glucagon
Rat GIP (1-42)
Mouse GIP (1-42)
Human GIP (1-42)
Rat GIP (3-42)
Human GIP (3-42)

Crossreactivity(%)
100
100
100
.9.1
.9.4
Crossreactivity(%)
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1

Ⅶ．Stability and Storage<Storage>

Store all of the components at 2-8°C.

<Shelf life>

This kit is stable under the condition for 24 months from the date of
manufacturing.
The expiry date is stated on the package.

<Package>

For 96 tests per one kit.

Ⅷ．References

1. Heinrich, G. (1984) Glucagon gene sequence. Four of six exons encode separate functional
domains of rat pre-proglucagon. J. Biol. Chem., 259, 14082-14087
2. Orskov, C. (1994) Tissue and plasma concentrations of amidated and glycine-extended
glucagon-like peptide I in humans. Diabetes., 43, 535-539
3. Yanaihara N. (1990) Recent advances in gastrointestinal hormones (article in Japanese).
Hum. Cell., 3, 1-8
4. Asarian, L. (1998) Intracerebroventricular glucagons-like peptide-1 (7-36)amide inhibits
sham feeding in rats without eliciting satiety., Physiol. Behav., 64, 367-372
5. Mojsov, S. (1990) Both amidated and nonamidated forms of glucagon-like peptide I are
synthesized in the rat intestine and the pancreas. J. Biol. Chem., 265, 8001-8008
6. Vella, A. (2000) Effect of glucagon-like peptide1 (7-36)amide on glucose effectiveness andinsulin action in people with type 2 diabetes. Diabetes., 49, 611-617
12

7. Nauck, MA. (1998) Normalization of fasting glycaemia by intravenous GLP-1 ([7-36
amide] or [7-37]) in type 2 diabetic patients., Diabet. Med., 15, 937-945
8. Nauck, MA. (2004) Blood glucose control in healthy subject and patients receiving
intravenous glucose infusion or total parenteral nutrition using glucagon-like peptide 1.
Regul. Pept., 118, 89-97
9. Naslund, E. (2004) Prandial subcutaneous injections of glucagon-like peptide-1 cause
weight loss in obese human subjects., Br. J. Nutr., 91, 439-446
10. Holst, JJ. (2002) Therapy of type 2 diabetes mellitus based on the actions of glucagon-like
peptide-1., Diabetes Metab. Res. Rev., 18, 430-441
11. Jaakko Makinen, Jarna C. Hannukainen, Anna Karmi, Heidi M. Immonen, Minna Soinio,
Lassi Nelimarkka, Nina Savisto, Mika Helmio, Jari Ovaska, Paulina Salminen, Patricia
Iozzo, Pirjo Nuutila (2015) Obesity-associated intestinal insulin resistance isameliorated after bariatric surgery., Diabetologia., 58, 1055-1062.
12. N Higuchi, T Hira, N Yamada, H Hara (2013) Oral administration of corn zeinhydrolysate stimulates GLP-1 and GIP secretion and improves glucose tolerance in malenormal rats and Goto-Kakizaki rats., Endocrinology, 154, 3089-98.
13. Shibata E, Aoki K, Tajima K, Taguri M, Terauchi Y.( 2016)
Comparison of efficacy and safety of taking miglitol dissolved in water during
a meal and taking a miglitol tablet just before a meal in patients with type 2 diabetes.
Expert Opin Pharmacother., 17,889-94.
14. Smith K, Karimian Azari E, LaMoia TE, Hussain T, Vargova V, Karolyi K, Veldhuis
PP, Arnoletti JP, de la Fuente SG, Pratley RE, Osborne TF, Kyriazis GA.(2018) T1R2
receptor-mediated glucose sensing in the upper intestine potentiates glucose absorption
through activation of local regulatory pathways. Mol Metab., 17, 98-111.
15. Nakagawa T, Nagai Y, Yamamoto Y, Miyachi A, Hamajima H, Mieno E, Takahashi M,
Inoue E, Tanaka Y. (2019) Effects of anagliptin on plasma glucagon levels and gastric
emptying in patients with type 2 diabetes: An exploratory randomized controlled trial versus
metformin., Diabetes Res Clin Pract.,158, 107892.
16. A Sato , Y Ohtsuka , Y Yamanaka (2019) Morning Mastication Enhances Postprandial
Glucose Metabolism in Healthy Young Subjects., Tohoku J Exp Med. ,249, 193-201.

<Manufacturer>
Yanaihara Institute Inc.
2480-1 Awakura, Fujinomiya-shi
Shizuoka, Japan 418-0011
TEL: +81-544-22-2771 FAX: +81-544-22-2770
Website: http://www.yanaihara.co.jp E-mail: ask@yanaihara.co.jp
Update at Jul. 10, 2023

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