Assay for insulin

B. V. Fisher and D. Smith, HPLC as a replacement for the animal response assays for insulin, J. Pharm. Biomed. Anal., 4 311... 

Lochner, N., Lobmaier, C., Wirth, M., Leitner, A., Pittner, F., and Gabor, F. 2003. Silver nanoparticle enhanced immunoassays One step real time kinetic assay for insulin in serum. Eur Pharm Biopharm 56(3) 469-477. 

Assays for insulin antibodies fall into three categories (1) quantitative radioimmunoelectrophoresis, which measures the binding of IgG antibody to radiolabeled insulin by rocket Immunoelectrophoresis into anti-IgG-containing agarose (2) RIAs with separation of bound and free insulin by precipitation with PEG or a second antibody and (3) solid phase immobilization of insulin to test tubes or Sepharose. These are discussed in more detail in Reeves. ... 

Rosenfeld RG, Gargosky SE. Assays for insulin-like growth factors and their binding proteins Practicalities and pitfalls. J Pediatr (United States) 1996 128 S52-S57. 

G12. Guyda, H. J., Corvol, M. T., Rappaport, R., and Posner, B. I., Radioreceptor assay for insulin-like peptides in human plasma Growth hormone dependence and correlation with sulfation activity by two bioassays. /. Clin. Endocrinol. Metab. 48, 739-747 (1979). 

Cells expressing an insulin-F36M fusion protein were exposed to AP21998 for three 1-h periods as indicated, and medium was collected every hour and assayed for insulin levels [55],... 

For the purpose of example the treatment of a quantal response assay by probit transformation using two doses of standard and two of the test preparation will be considered. The data used are taken from the assay for insulin in which the number of mice displaying hypoglycaemic convulsions is taken as the response. 

Butter, N.L., Hattersley, A.T., and Clark, RM. (2001) Development of a bloodspot assay for insulin. Clinica Chimica Acta, 310,141-150. 

The diabetic rats were treated with 18 IU of bovine insulin imbibed into polyacid resins b.i.d. orally using 1 cc syringes and gavage tubes. After 14 days of treatment the rats were sacrificed about 1.5 hours after the last dose. Blood samples were taken and assayed for immunoactive insulin activity (Amersham-Searie RIA kit) and serum glucose levels (glucose oxidase colorimetric assay, Sigma 510 Glucose Kit). 

Kato, K., Hamaguchi, Y., Fukui, H., and Ishikawa, E. (1975a) Enzyme-linked immunoassay. I. Novel method for synthesis of the insulin-b-D-galactosidase conjugate and its applicability for insulin assay. /. Biochem. (Tokyo) 78, 235. 

With this assay, basal insulin values of 5-15 pU/mL (30-90 pmol/L) are found in normal humans, with a peak rise to 60-90 U/mL (360-540 pmol/L) during meals. Similar assays for measuring all of the known hormones of the endocrine pancreas (including C-peptide and proinsulin) have been developed. 

Free fatty acids are elevated in the plasma of obese patients and are known to cause muscle and liver insulin resistance. The Wako HR series NEFA-HR(2) is an in vitro enzymatic colorimetric method assay for the quantitative determination of non-esterified fatty acids (NEFA) in serum. Perform the assay on serum collected from mice fasted for a period greater than 4 h, but less than 16 h. Perform the test on samples immediately after collection, without freezing. Also note that hemolysis in the serum samples may interfere with the assay. 

Several RIAs for insulin had been compared before aiming at 100% cross-reactivity with Apidra. The rat insulin RIA from Linco showed similar reactivity for Apidra and human insulin. Therefore, this assay was used with only slight modifications rat insulin standards were replaced by Apidra standards in an appropriate human serum matrix for calibration. 

CRITICAL ASSESSMENT OF THE METHOD The method described above could not differentiate Apidra from naturally occurring insulin/metabolites and precursors. This lack of specificity renders the characterisation of the pharmacokinetics difficult. Therefore, a specific assay for the determination of Apidra was required for its further development. 

Applications. The general feasibility of CE to separate immunocomplex from unbound reagent was first demonstrated by Grossman et al. [4] and Nielsen et al. [23], whose reports set the stage for further development of quantification techniques in CE-IA. The first quantitative noncompetitive CE assay of insulin was reported by Schultz and Kennedy [24], To increase the sensitivity of analysis, the authors used FITC-labeled insulin and LIF detection. Due to the separation power of CE, multiple products of fluorescence labeling of insulin did not hamper analysis (see Figure 6). Two types of calibration curves were con-... 

J. B., and Oprian, D.D., In vitro assay for fran -phosphorylation of rhodopsin by rhodopsin kinase. Biochemistry 36, 7064-7070, 1997 Cann, A.D., Bishop, S.M., Ablooglu, A.J., and Kobanski, R.A., Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation. Biochemistry 37,11289-11300,1998 Iwasaki, Y., Nishiyama, H., Suzuki,... 

Since Berson and Yalow first described the radioimmunoassay for insulin, similar assays have appeared in the literature for several hundred substances that differ markedly in chemical structure and biological activity. These represent only a small fraction of the biologically important molecules that could be analyzed by this technique. This overview is intended primarily for the investigator who wishes to develop a new radioimmunoassay for a specific substance. It directs his attention to papers that provide background information and necessary procedural details. 

Fig. 1. Dose-response curve for amount of dextran-coated charcoal added versus percentage free and bound, adsorbed. These are theoretical curves drawn from generalizations derived from several assays. —, Curve showing binding of free ligand —, curve showing binding of antibody bound ligand. The exact amounts of charcoal giving a particular bound value vary with each assay (e.g., growth hormone assay differs from insulin assay). For significance of arrows A and B, see the section on selection of charcoal dose.

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