Insulin ELISA kit instructions
Insulin ELISA kit instructions
(Germany DRG : EIA2935 )
1. Description of the preface
DRG 's Insulin Enzyme Free Kit can be used for the quantitative detection of insulin in human serum and plasma (with heparin or citric acid added). This test kit is for in vitro diagnostic use only.
Insulin is the main hormone regulating glucose metabolism. It is secreted by the β-cells of the Langerhans island and is present in the form of a precursor of the former insulin, which can be processed to form C- peptide and insulin. Both hormones are secreted into the portal circulation in equal amounts. The mature insulin molecule consists of two polypeptide chains, the A chain and the B chain ( 21 and 30 amino acids, respectively). These two chains are joined together by a disulfide bond within the bond. The secretion of insulin is mainly affected by the concentration of plasma glucose, which has a series of important metabolic functions. Its main function is to control the uptake and utilization of glucose in peripheral tissues through the transport of glucose. The effects of insulin and other hypoglycemic hormones (such as inhibition of hepatic glycogen xenobiotics and glycogenolysis) can be neutralized by the action of hyperglycemic hormones (glucagon, adrenaline, growth hormone and cortisol). In insulin-dependent diabetes ( IDDM ) and other diseases such as pituitary hypofunction , the concentration of insulin is significantly reduced. In patients with non-insulin-dependent diabetes, obese patients, insulinoma patients, and some patients with endocrine dysfunction (such as Cushing's syndrome and acromegaly), insulin levels rise.
2 , the principle of experiment
DRG 's insulinase-free assay is a solid-phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich method . The micro-detection wells on the reaction plate are coated with monoclonal antibodies that bind to unique antigenic sites on the insulin molecule. A patient sample containing endogenous insulin is incubated with the enzyme conjugate in a coating well, which is a biotinylated anti-insulin antibody. After incubation, the unbound enzyme conjugate was washed away with washings. At the second incubation, the streptavidin peroxidase complex binds to the biotinylated anti-insulin antibody and the amount of peroxidase complex bound is proportional to the concentration of insulin in the sample. After the addition of the substrate solution, the intensity of the color displayed is proportional to the concentration of insulin in the patient sample.
3 , kit components
1)       Microplate: 12 × 8 , 96 well, coated with anti-insulin monoclonal antibody.
2)       Zero standard : 1 branch, 3ml , ready to use, 0 μ IU/ml .
3)       Standards 1 - 5 : 5 sticks, 1 ml , ready to use, 6.25; 12.5; 25; 50; 100 μl U/ml .
4) Â Â Â Â Â Â Enzyme-linked enzyme: 1 branch, 5 ml , ready-to-use, biotinylated murine monoclonal antibody.
5) Â Â Â Â Â Â Enzyme complex: 1 branch, 7 ml , ready to use, horseradish peroxide - streptavidin complex.
6) Â Â Â Â Â Â TMB Substrate Solution: i.e. with a 14ml.
7) Â Â Â Â Â Â Stop Solution: 1 with 14ml i.e. contain
8) Â Â Â Â Â Â Washing solution: 1 bottle, 30ml , 40 times concentrated, see reagent preparation.
Note: Zero standards diluted with samples are subject to availability.
4 , the materials required for the experiment (but not provided in the kit)
1) Â Microplate reader
2) Â Standard pipette
3) Â Absorbent paper
4) Â Distilled water
5 , reagent storage and stability
All unopened reagents are in 2 -
6 , sample preparation
All reagents and the required strips were equilibrated to room temperature before use.
Washing solution: Dilute 30 ml of the concentrated washing solution with 1170 ml of distilled water to obtain a final volume of 1200 ml . The diluted washing solution can be stably stored for two weeks at room temperature.
7 , sample collection
This ELISA kit can use either serum or plasma (heparin or citrate anticoagulated plasma only) as a sample.
Samples with significant hemolysis, jaundice, and lipemia should not be used.
Serum: Whole blood was collected by venipuncture, and after coagulation, serum was separated by centrifugation at room temperature.
Plasma: Whole blood was collected into a centrifuge tube containing an anticoagulant and centrifuged immediately after collection.
8. Storage of samples:
After the sample is covered, it can be used before the experiment 2
9 , the dilution of the sample:
In the first experiment, if the concentration of the sample was higher than the highest standard, the sample was diluted 10 or 100 times as described in the experimental procedure . This dilution is multiplied when calculating the concentration.
example:
a)  By 1:10 dilution: 10 μ L of serum +90 μ L zero standard (fully shake).
b)  1: 100 diluted Release: 10 μ L of diluted by a) +90 μ L of serum zero standard (Shake)
10 , experimental steps
All standards, controls, and samples must be double tested to ensure that all microwells are tested under the same conditions.
1 ) Place the required number of slats on the pallet. .
2) The disposable nozzle ﹑ standards controls and samples were added to 25 μ L corresponding to the test wells.
3) 25 μ L per well of enzyme conjugate.
4 ) Mix well for 10 seconds, and it is very important to mix well in this step.
5 ) Incubate for 30 minutes at room temperature .
6) Rapid discarded contents of the test wells, each well was added 400 μ L of the plate was washed three times with washing, shoot dry on absorbent paper to remove residual liquid. Note: The correct operation of the washing step will significantly affect the sensitivity and accuracy of the experiment.
7) Add 50 μ L per well enzyme complex.
8 ) Incubate for 30 minutes at room temperature .
9) Quick discarded contents of the test wells, each well was added 400 μ L of the plate was washed three times with washing, shoot dry on absorbent paper to remove residual liquid.
10) substrate solution added to each well of 50 μ L.
11 ) Incubate for 15 minutes at room temperature .
12) Add 50 μ L per well of stop solution to stop the reaction.
13 ) Read the OD value at 450 ± 10 nm within 10 minutes after the addition of the stop solution .
11 , calculation results:
1) Â Â Â Â Calculate the average absorbance of standards, controls, and patient samples.
2) Â Â Â Â A standard curve is drawn by taking the absorbance as the Y- axis and the concentration as the X- axis, according to the average absorbance obtained from the standard and its corresponding concentration value.
3) Â Â Â Â Using the average absorbance of the sample, the corresponding concentration value is obtained on the standard curve.
4) Â Â Â Â Automatic method: The result is obtained using a computer program in computer cube mode, four parameter mode or double logarithmic function.
5) Â Â Â Â The concentration of the sample can be obtained directly from the standard curve. Further concentration is required if the concentration of the sample is higher than the concentration of the highest standard. When calculating the concentration, multiply by the dilution factor of the sample.
The following is a typical example of a standard curve for an insulin ELISA .
Standard | Absorbance ( 450nm ) |
Zero standard ( 0 μIU/ml )      | 0.03 |
Standard 1 ( 6.25 μIU/ml )              | 0.07 |
Standard 2 ( 12.5 μIU/ml ) | 0.14 |
Standard 3 ( 25 μIU/ml ) | 0.35 |
Standard 4 ( 50 μIU/ml ) | 0.88 |
Standard 5 ( 100 μIU/ml ) | 2.05 |
Reference:
It is recommended that each laboratory establish its own normal and abnormal values.
In a study of an apparently normal adult, the following values ​​were obtained using the DRG insulin ELISA kit: 2-25 μIU/ml
This translation is for reference only, please refer to the original for details.
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