Could I use a plasma/serum insulin ELISA assay to dose intracellular insulin content?
Nicola! You can do it. I use Insulin ELISA to quantify total insulin content in islets. First I break the cells with an Alcoholic-acid solution (75ml EtOH+1.5ml HCL 37%+23.5ml water) and sonication (2 pulses of 30 seconds). Then you just need to know the right dilution so that it falls within the range of your ELISA kit.
I would like to express my opinion onto the insulin assay. Please see the original paper of RIA method (Yalow RS, Berson SA: Immunoassay of endogenous plasma insulin in man. J Clin Invest 39: 1157-1175, 1960. (file Yalow insulin)); i.e., RIA and/or ELISA using non-linear calibration curves might not be the true determination methods.
Then, HPLC determination method using the linear calibration line has been reported previously (Hayakawa K, Tanaka H: High-performance liquid chromatographic determination of bovine and porcine insulins in a commercial injection. J Chromatogr 312: 476-481, 1984; file Insulin RP-HPLC). Insulin molecule is rich in sulphur atoms, and protection of thiol residues by pyridylethylation method may be required (Friedman M, Krull LH, Cavins JF: The chromatographic determination of cystine and cysteine residues in proteins as S-β-(4-pyridylethyl)cysteine. J Biol Chem 245: 3868-3871, 1970. (file PEC synthesis). Automated method is also available; i.e., automated PEC synthesis by Nokihara K, Morita N, Yamaguchi M and Watanabe T (Sequence analysis of cysteine and cystine containing peptides and proteins by a gas-phase sequencer with isocratic separation of PTH-amino acids. Anal Letters 25: 513-533, 1992). In this case, PE-derivatized insulin chains of alpha and beta should be independently or simultaneously determined with RP-HPLC.
Human serum biocytinase (dimer-form of biotinidase) shows Km of 0.48 μM (0.48 nmol/mL). Then, 0.12 μg/mL of free biotin should be present in the human serum estimated from this Km value (see Oizumi J, Hayakawa K. Biocytin-specific 110 kDa biotinidase from the human serum. Clin Chim Acta 215: 63-71, 1993). Thus, we surely have found that 0.12 μg/mL of free biotin is present in the adult human serum (see attached file; Feed by median). Therefore, Km value is a good indicator of physiological concentration of substrate/product molecules.
The Km of dimer insulinase to insulin may be 6.2 μM (6.2 nmol/mL) of serum, then 18.6 μg/mL of serum insulin-chain may be present in serum. Therefore, this level of proteins may be determinative by using such high-recovery RP-HPLC method (see file; Insulin RP-HPLC).
Therefore, I would like to recommend the RP-HPLC method using some improvement for application onto your biological specimens.
Thank you all! Joan, I'm performing my experiments on INS-1E cells. Is sonication necessary? I would simply use a non-denaturing lysis buffer (0.1% TRITON-X-100 and EDTA). Could it work?
Nicola, I have never done it with INS-1E cells. You could do a trial with and without sonication and see if there is any difference.
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