Insulin is a peptide hormone exclusively produced in pancreatic beta-cells. It consists of A chain and B chain, which are linked by two disulfide bridges.
Insulin is the primary hormone responsible for glucose metabolism. Impaired insulin secretion and insulin resistance are key causes of type 2 diabetes (T2D).

This assay is a two-site ELISA. The micro-plate is pre-coated with a monoclonal antibody against insulin. Standards and samples are added into the wells and coincubated with a monoclonal antibody conjugated to horseradish peroxidase (HRP) enzyme. After wash step to remove any unbound substances, TMB substrate is added and colour develops in proportion to the amount of insulin bound initially. The assay is stopped and the optical density of the wells determined using a micro-plate reader. Since the increases in absorbance are directly proportional to the amount of captured insulin, the unknown sample concentration can be interpolated from a reference curve included in each assay.

A.Typical representation of standard curve

The following standard curve is provided for demonstration only. A standard curve should be generated for each assay.

 

Insulin (ng/mL)	Absorbance (450 nm)	Blanked Absorbance
0	0.061	0
0.025	0.07	0.009
0.075	0.096	0.035
0.2	0.223	0.162
0.5	0.77	0.709
1.5	2.852	2.791

 

B.Sensitivity

The lowest insulin level that can be measured by this assay is 0.025 ng/mL.

 

C. Precision

Intra-assay Precision (Precision within an assay) C.V. < 2.9%.

Inter-assay Precision (Precision between assays) C.V. <4.9%.

 

D. Recovery

The recovery of the assay was determined by adding various amounts insulin to a sample. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 92%.

 

E. Specificity

Percent of cross reactivity

Human insulin: 100%

Rat insulin: 100%

1. Shu L, Hoo RL, Wu X, Pan Y, Lee IP, Cheong LY, Bornstein SR, Rong X, Guo J, Xu A. A-FABP mediates adaptive thermogenesis by promoting intracellular activation of thyroid hormones in brown adipocytes. Nature communications. 2017 Jan 27;8(1):1-6.
2. Fan W, Zhang R, Han D, Jiang Z, Li S, Zhang J, Li Y, Wang Y, Cao F. Reduced Sirtuin1 signalling exacerbates diabetic mice hindlimb ischaemia injury and inhibits the protective effect of a liver X receptor agonist. Journal of cellular and molecular medicine. 2020 May;24(10):5476-90.
3. Wang B, Lin H, Li X, Lu W, Kim JB, Xu A, Cheng KK. The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic β-cells. Proceedings of the National Academy of Sciences. 2020 Nov 10;117(45):28307-15.
4. Zhang J, Ni Y, Qian L, Fang Q, Zheng T, Zhang M, Gao Q, Zhang Y, Ni J, Hou X, Bao Y. Decreased Abundance of Akkermansia muciniphila Leads to the Impairment of Insulin Secretion and Glucose Homeostasis in Lean Type 2 Diabetes. Advanced Science. 2021 Jun 4:2100536.
5. So WY, Liu WN, Teo AK, Rutter GA, Han W. Paired box 6 programs essential exocytotic genes in the regulation of glucose-stimulated insulin secretion and glucose homeostasis. Science Translational Medicine. 2021 Jun 30;13(600):eabb1038.
6. Liu D, Gu J, Shao W, Pang J, Qian X, Jin T. Comparison of metabolic beneficial effects of Liraglutide and Semaglutide in male C57BL/6J mice. Canadian Journal of Diabetes. 2021 Sep 8.
7. Xiao Y, Shu L, Wu X, Liu Y, Cheong LY, Liao B, Xiao X, Hoo RLC, Zhou Z, Xu A. Fatty acid binding protein 4 promotes autoimmune diabetes by recruitment and activation of pancreatic islet macrophages. JCI Insight. 2021 Mar 9, 6(7):e141814.
8. Wong THT, Wan JMF, Tse IMY, Sit WH, Louie JCY. Consumption of decaffeinated coffee with milk and sugar added before a high-glycemic-index meal lowers postprandial glucose surge when compared with consuming it after the meal. Human Nutrition & Metabolism. 2021 Jun, 200124.
9. Wu KKL, Long K, Lin H, Siu PMF, Hoo RLC, Ye D, Xu A, Cheng KKY. The APPL1-Rab5 axis restricts NLRP3 inflammasome activation through early endosomal-dependent mitophagy in macrophages. Nat Commun. 2021 Nov 17;12(1):6637.
10. Bai N, Lu X, Jin L, Alimujiang M, Ma J, Hu F, Xu Y, Sun J, Xu J, Zhang R, Han J, Hu C, Yang Y. CLSTN3 gene variant associates with obesity risk and contributes to dysfunction in white adipose tissue. Mol Metab. 2022 Sep;63:101531.
11. Ni Y, Qian L, Siliceo SL, Long X, Nychas E, Liu Y, Ismaiah MJ, Leung H, Zhang L, Gao Q, Wu Q, Zhang Y, Jia X, Liu S, Yuan R, Zhou L, Wang X, Li Q, Zhao Y, El-Nezami H, Xu A, Xu G, Li H, Panagiotou G, Jia W. Resistant starch decreases intrahepatic triglycerides in patients with NAFLD via gut microbiome alterations. Cell Metabolism. 2023 Sep 5;35(9):1530-1547.e8.

Insulin ELISA Kit	Assay Range (ng/mL)	Sample Volume Required Per Well (uL)	Assay Length (h)
Ultra sensitive (Rat Cat.: 33380/Mouse Cat.: 32380)	0.025-1.5	25	2.5
High sensitive (Rat Cat.:33270/Mouse Cat.:32270)	0.2-7	10	2
High sensitive Plus(Mouse only Cat.: 32393)	0.2-7	5	2.5
Wide range (Rat Cat.: 33100/Mouse Cat.:32100)	1-50	5	2