Insulin assay experiment

Summary

Radioimmunoassay of insulin can be used for (1) immunolabeling detection techniques using radioisotopes as tracers, and (2) competitive binding of isotope-labeled antigens and unlabeled antigens to be measured in samples to fixed amounts of specific antibodies, or competitive concordant reactions to determine the amount of antigen to be measured.

Operation method

radioimmunoassay

Principle

Insulin has a molecular weight of 5700 and consists of two amino acid peptide chains: the A chain has 21 amino acids and the B chain has 30 amino acids, with two disulfide bonds between the A and B chains. The islet B cells have a reserve of about 200 U of insulin and secrete about 40 U of insulin per day.During fasting, the plasma insulin concentration is 5-15 μU/mL.After a meal, plasma insulin levels can increase by 5 to 10 times.The rate of biosynthesis of insulin is about 1,000 μM. The rate of insulin biosynthesis is influenced by plasma glucose concentration. when blood glucose concentration is elevated, the insulinogen content of B cells increases and insulin synthesis is accelerated. Insulin is secreted into the bloodstream in equal molecules with C-peptide.

Materials and Instruments

Insulin Standard 125 I-Insulin
Antisera Secondary antibody PBS Normal rabbit serum
Counter Test tubes Sampler Centrifuge

Move

1. Proceed according to the table.

2. Measurements and calculations After the reaction was completed, the tubes were centrifuged at 3 000 r/ min for 30 min at room temperature, and the supernatant was carefully aspirated and collected in a storage container of radioactive waste liquid. The intensity of radioactivity (cpm) of the precipitate (B) in each tube was measured separately with a -counter and expressed as the average value of cpm in both tubes. Then the binding rate (B%, i.e., B/Bo×100%) was calculated by taking the cpm value of the 1st and 2nd tubes as Bo, respectively, and the standard curve was plotted by using B% as the vertical coordinate and different concentrations of insulin standards as the horizontal coordinate. Then, according to the B% of the tubes to be tested, the corresponding content of insulin can be found from the standard curve.

Caveat

The presence of insulin antibodies in the serum of patients treated clinically with insulin interferes with the determination of blood insulin levels by radioimmunoassay, in which case plasma C-peptide levels can be determined to understand the state of endogenous insulin secretion.

Common Problems

1. Normal value is 5-15mU/L in fasting, insulin-dependent type is below the lower limit of normal or not measurable, non-insulin-dependent type is in the normal range or higher than normal.
2. Insulin release test: no peak appears in insulin-dependent type, showing a low-flat curve; the peak of non-insulin-dependent type is lower than normal, or the peak is delayed.

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