Mouse GIP Active ELISA Assay Kit

$820.00

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Mouse GIP (Active) ELISA Assay kit:
For Research Use Only
Size:  1×96 wells
Dynamic Range:  7.8 – 500 pg/mL
Incubation Time:  3.5 hours
Sample Type:  Mouse plasma, cell culture supernatant
Sample Size: 20 μL

Intended Use
This Eagle Biosciences Mouse GIP (Active) ELISA Assay kit kit is used for quantitative determination of mouse GIP (1-42) active form in plasma and culture medium supernatant. The kit is characterized by its sensitive quantification and high specificity. In addition, it has no influence by other components in samples. GIP (1-42) standard is highly purified synthetic product.

Assay Background
The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagons-like peptide-1 (GLP-1), are a group of gastrointestinal hormones that cause an increase in the amount of insulin released from the beta cells of the islets of Langerhans after ingestion of food.

The intestinal peptide GIP was first isolated from porcine upper small intestine1). The sequences of porcine, bovine and Mouse GIP have been determined, each has 42 amino acids, and the sequences is highly conserved. The porcine and bovine peptides differ from the Mouse at two and three site, respectively. Takeda et al. have isolated a Mouse cDNA encoding the GIP precursor and confirming that GIP belongs to the vasoactive intestinal peptide (VIP)/Glucagon/secretin family. GIP is a gastrointestinal peptide hormone that is released from duodenal endocrine K cells after absorption of glucose or fat7). GIP is a potent releaser of insulin in experimental animals and in man provided that the blood glucose is above basal level.

Plasma level of GIP is elevated after an oral glucose load or a meal in normal man. This increase after a meal is below normal in newly diagnosed insulin–dependent diabetics). It is now being recognized that GIP receptor is also expressed in organs and cells such as duodenum, small intestine, pancreatic alpha-cell, adipocyte and osteoblast. These results demonstrate GIP may have a lot of physiological effect in addition to their glucoregulatory effects. GIP is rapidly inactivated by the enzyme dipeptidyl peptidase- 4 (DPP- 4) to GIP (3-42) with a blood half-life of only several minutes. DPP- 4 inhibitor can prolong the half-life of GIP, that expecting treatment of incretin effect. This Eagle Biosciences Mouse GIP (Active) ELISA kit has high specificity to mouse GIP (1-42) active form and shows no cross-reactivity to mouse GIP (3-42) inactive form.

Assay Principle
This Mouse GIP (Active) ELISA Assay kit for determination of mouse GIP (1-42) active form is based on a sandwich enzyme immunoassay. To the wells of plate coated with highly purified mouse monoclonal antibody against mouse GIP (1-42) active form, standards or samples are added for the 1st step immunoreaction. After the 1st step incubation and plate washing, HRP labeled antibody solution against mouse GIP (1-42) is added as the 2nd step to form antibody – antigen – labeled antibody complex on the surface of the wells. After the 2nd step incubation and rinsing out excess labeled antibody, Finally, HRP enzyme activity is determined by 3,3’,5,5’-Tetramethylbenzidine (TMB) and the concentration of mouse GIP (1-42) active form is calculated.

  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 into the wells and aspirate the washing solution in the wells. Repeat this washing procedure further twice (total 3 times). Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
  3. Add 25µL of buffer solution to the wells first, and then introduce 25µL of each of standard solutions (0, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 pg/mL) or samples to the wells.
  4. Cover the plate with adhesive foil and incubate it at room temperature for 2 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 4 times with 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 100µL of HRP labeled antibody solution to each of the wells.
  7. Cover the plate with adhesive foil and incubate it at room temperature 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 4 times with 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 µL of Enzyme substrate solution (TMB) to each of the wells, cover the plate with adhesive foil and keep it for 30 minutes at room temperature in a dark place for color reaction (keep still, plate shaker not need).
  10. Add 100 µL of stopping solution to each of the wells to stop color reaction.
  11. Read the optical absorbance of the solution in 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 calculated with any computer program having a 5 (or 4)-parameter logistic function. Otherwise calculate mean 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.

Typical Standard Curve

Precision & Reproducibility                

Intra-assay CV(%)    5.8~6.0
Inter-assay CV(%)    1.6~3.3

Cross-Reactivity

Related peptides        Crossreactivity (%)
GIP (1-42) (Mouse)    100
GIP (3-42) (Mouse)    <0.1
Glucagon                   <0.1
Mouse GLP-2             <0.1
GLP-1 (7-36) NH2      <0.1
GLP-1 (9-36) NH2      <0.1

References

  • Brown,J.C., Mutt, V. and Pedersen,R.A. (1970) Further purification of a polypeptide demonstrating enterogastrone activity. J.Physiol. 209, 57-64
  • Jörnvall H, Carlquist M, Kwauk S, Otte SC, McIntosh CH, Brown JC, Mutt V. (1981) Amino acid sequence and heterogeneity of gastric inhibitory polypeptide (GIP). FEBS Lett. 123, 205-210.
  • Moody,A.J., Damm Jorgensen, K.and Thim, L.(1981)Diabetologia 21, 306, abstr.
  • Carlquist M, Maletti M, Jörnvall H, Mutt V. (1984) A novel form of gastric inhibitory polypeptide(GIP) isolated from bovine intestine using a radioreceptor assay. Fragmentation with staphylococcal protease results in GIP1-3 and GIP4-42, fragmentation with enterokinase in GIP1-16 and GIP17-42.Eur.J. Biochem. 145, 573-577
  • Moody, A. J., Thim, L. & Valverde, I. (1984) The isolation and sequencing of human gastric inhibitory peptide(GIP). FEBS Lett. 172, 142-148
  • Takeda J, Seino Y, Tanaka K, Fukumoto H, Kayano T, Takahashi H, Mitani T, Kurono M, SuzukiT, Tobe T, et al.(1987) Sequence of an intestinal cDNA encoding human gastric inhibitory polypeptide precursor. Proc Natl Acad Sci U S A. 84(20):7005-8.
  • Pederson, R.A. (1994) in Gut Peptides: Biochemistry and Physiology, eds, Walsh, J.H.& Dockray, G.J. (Raven, New York), pp,217-260
  • Rabinovitch, A. and Dupre, J (1974)Effect of the gastric inhibitory polypeptide present in impure pancreozymin-cholecystokinin of plasma insulin and glucagons in the rat. Endocrinology 94, 1139-1144
  • Dupre,J., Ross, S.A.,Watson, D. and Brown, J.C. (1973) Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J. Clin. Endocrinol. Metab. 37, 826-828
  • Elahi, D., Andersen, D.K., Brown, J.C.,Debas, H.T., Hershcopf, R.J., Raizes, G.S., Tobin, J.D.and Andres, R.(1979) Pancreatic alpha-and-beta-cell response to GIP infusion in normal man. Am.J.Physiol. 237, E185-E191
  • Krarup T, Madsbad S, Moody AJ, Regeur L, Faber OK, Holst JJ, Sestoft L.(1983) Diminished immunoreactive gastric inhibitory polypeptide response to a meal in newly diagnosed type I (insulin-dependent) diabetics. J Clin Endocrinol Metab. 56, 1306-12.
  • Naitoh R, Miyawaki K, Harada N, Mizunoya W, Toyoda K, Fushiki T, Yamada Y, Seino Y, Inagaki N.(2008) Inhibition of GIP signaling modulates adiponectin levels under high-fat diet in mice. Biochem Biophys Res Commun. 376, 21-5.
  • Miyawaki K, Yamada Y, Yano H, Niwa H, Ban N, Ihara Y, Kubota A, Fujimoto S, Kajikawa M, Kuroe A, Tsuda K, Hashimoto H, Yamashita T, Jomori T, Tashiro F, Miyazaki J, Seino Y. (1999) Glucose intolerance caused by a defect in the entero-insular axis: a study in gastric inhibitory polypeptide receptor knockout mice. Proc Natl Acad Sci U S A. 96, 14843-7.
  • Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H, Fujimoto S, Oku A, Tsuda K, Toyokuni S, Hiai H, Mizunoya W, Fushiki T, Holst JJ, Makino M, Tashita A, Kobara Y, Tsubamoto Y, Jinnouchi T, Jomori T, Seino Y.(2002) Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat Med. 8, 738-42.
  • Tsukiyama K, Yamada Y, Yamada C, Harada N, Kawasaki Y, Ogura M, Bessho K, Li M, Amizuka N, Sato M, Udagawa N, Takahashi N, Tanaka K, Oiso Y, Seino Y. (2006) Gastric inhibitorypolypeptide as an endogenous factor promoting new bone formation after food ingestion. Mol Endocrinol. 20, 1644-51.