IGF-1 600 ELISA kit
1. Introduction
DRG's IGF-1 600 ELISA kit provides the required materials for the quantitative detection of serum IGF-1. This test kit is for diagnostic use only.
2. Clinical significance
Insulin-like growth factor-1 (IGF-1) is a polypeptide composed of 70 amino acid molecules (molecular weight of 7650 Daltons), which is one of many related insulin-like growth factors in the insulin circulation process. This molecule has about 50% homology with pre-insulin, and it has many biological functions similar to insulin. This polypeptide is highly growth hormone (GH) dependent, but it is also increasingly showing GH independent secretion. IGF-1 has many growth-promoting functions, including promoting cell division and cartilage sulfation. It can also show the rate of bone renewal. Almost 95% of circulating IGFP-1 binds to specific IGF-binding proteins (currently known to have six subspecies). BP3 is the main binding protein of IGF-1. It forms a ternary complex with a molecular weight of 140,000 Daltons together with IGF-1 and an acidic variable subunit.
3. Clinical application
The detection of serum IGF-1 is of great significance in the diagnosis and treatment of children with growth disorders and bronchial hypertrophy. The concentration of IGF-1 will change with the age, nutritional status, body structure and growth hormone secretion. The detection of IGF-1 is beneficial to the evaluation of short stature children and the nutritional research of patients with malignant diseases. In the diagnosis of bronchial hypertrophy, IGF-1 detection is more reliable than random GH detection.
4. Experimental principle
DRG's IGF-1 600 ELISA kit is a solid-phase enzyme-linked immunoassay, using the principle of competition. Before starting the experiment, acidify and neutralize the patient samples, standards and quality control products. The microwells of the coated plate are coated with monoclonal antibodies that are specific to a single antigenic site on the IGF-1 molecule. The pre-treated samples were incubated with enzyme conjugate (biotin-labeled IGF-1) at room temperature. After washing the plate, the enzyme complex (horseradish peroxidase-labeled streptavidin) is added and incubated. After adding the substrate solution, the color intensity of the solution is inversely proportional to the concentration of IGF-1 in the patient sample.
5. Kit components
1) 1M HCl, 1 bottle, 3ml, ready to use, used for acidification of samples.
2) 1M NaOH, 1 bottle, 3ml, ready to use for neutralization.
3) Coated plate, 12 × 8 (removable), 96 wells, coated with anti-IGF-1 antibody (monoclonal antibody)
4) Standard products (standard products 0-6), 7 bottles, 1ml, concentration: 0; 5; 10; 50; 150; 300; 600ng / ml. NIBSC number: 87/518, conversion factor: 1ng / ml × 0.13 = nmol / l. Contains 0.03% Proclin300 and 0.005% gentamicin sulfate as preservatives.
5) Enzyme conjugate, 1 bottle, 14ml, ready-to-use, biotin-labeled IGF-1, containing 0.01% methyl isothiazolone, 0.02% BND preservative.
6) Enzyme complex, 1 branch, 20ml, ready-to-use, HRP-labeled streptavidin complex.
7) Substrate solution, 1 bottle, 14ml, ready to use, TMB
8) Stop solution, 1 bottle, 14ml, ready to use, containing 0.5M H2SO4. Avoid contact with the stop fluid to avoid burning or irritating the skin.
9) Washing solution, 1 bottle, 30ml (40 times concentrated)
Note: The zero standard used for sample dilution should be as required.
6. Equipment needed for the experiment (but the kit does not provide)
1) Microplate reader
2) Pipette
3) Absorbent paper
4) Distilled water
5) 1.5ml reaction cup (for sample preparation)
6) General instruction paper
7. Storage and stability of the kit
Unopened reagents are stored at 2-8 ° C and can remain active during the validity period. Do not use expired reagents. Unopened reagents must be stored at 2-8 ° C. Microwell reaction plates must also be stored at 2-8 ° C. Once the bag is opened, it must be carefully sealed tightly. After unpacking, the immune activity of the coated microwell reaction plate can be stable for about 6 weeks, but it must be sealed in a bag containing desiccant.
8. Sample
Serum can be used for this experiment. Note: It is observed that the concentration of IGF-1 in plasma is significantly reduced. Do not use hemolytic, jaundice, or lipemia samples. Please note: Samples containing sodium azide cannot be used for this experiment.
8.1 Collection of samples
Serum: collect whole blood by venipuncture. After coagulation, centrifuge at room temperature to obtain serum. Do not centrifuge before the sample has completely coagulated. Samples of patients who have received anticoagulant therapy may require longer clotting times.
8.2 Storage of samples:
Samples should be tested immediately. If you want to store the sample for a longer time before the experiment, you should freeze the sample at -20 ℃. Thawed samples should be inverted several times before the experiment.
8.3 Dilution of samples:
During the first experiment, if the concentration of IGF-1 in the serum sample is higher than the concentration of IGF-1 in the highest standard, the sample should be diluted with zero standard and retested according to the experimental procedure. This dilution factor should be taken into account when calculating the concentration.
E.g:
a) Dilute at a ratio of 1:10: 10μl serum + 90μl zero standard (fully mixed)
b) Dilute at a ratio of 1: 100: 10μl diluted a) + 90μl zero standard (fully mixed)
9. Acidification and neutralization of patient samples and standards
1) Add 200μl of sample, standard and quality control well to the 1.5ml reaction Caps. Please note: Following the steps described below, the standard must be acidified and neutralized.
2) After adding 20μl of 1M HCl
3) Mix and incubate for 15min
4) Add 20 μl of 1M NaOH to all Caps for neutralization and mix the solution.
5) The pH value after neutralization should be between 7 and 8. Please check the pH value with a general indicator paper.
10. Experimental steps:
Each test will have a standard curve.
1) Fix the required number of slats to the rack.
2) Add 50μL of the standard, quality control and diluted samples to the corresponding microwells with new sampling tips.
3) Add 100 μL of enzyme conjugate to each microwell.
Mix thoroughly for 10 seconds. It is important to mix thoroughly at this step.
4) Incubate at room temperature for 120 minutes.
5) Quickly discard the reactants in the wells, wash the plate 3 times with 400 μL of distilled water in each well, and pat dry on absorbent paper. Note: The correct operation of the plate washing step will obviously affect the sensitivity and accuracy of the experiment.
6) Add 150 μl of enzyme-linked complex to each microwell.
7) Incubate for 60 minutes at room temperature.
8) Quickly discard the reactants in the wells, wash the plate 3 times with 400 μL of distilled water in each well, pat dry on absorbent paper.
9) Add 100μl of substrate solution to each well
10) Incubate at room temperature for 30 minutes.
11) Add 100μl of stop solution to each well to stop the enzyme reaction
12) Within 10 minutes after adding the stop solution, measure the OD value at 450 ± 10nm with a microplate reader
11. Result calculation
1) Calculate the average absorbance value of each standard, quality control and patient sample.
2) Using the absorbance value as the ordinate (y) and the concentration value as the abscissa (x), plot the corresponding concentration value with the absorbance value of each standard to make a standard curve.
3) Determine the corresponding concentration on the standard curve using the average absorbance value of each sample.
4) Automatic calculation method: On the IFU, it has automatically calculated the results using a four-parameter curve. Other induction methods may produce slightly different results.
5) The concentration of the sample can be read directly from the standard curve. Samples with HPL concentrations higher than the highest standard must be used for further dilution. This dilution factor should be taken into account when calculating the sample concentration.
12. Expected value
It is strongly recommended that each laboratory establish its own range of normal values.
Pre-pubertal period (3 to 8 years old): 20-250ng / ml
The level of IGF-1 in children with lower body weight may be lower than 50ng / ml.
Adolescent children (11-16 years old): 130-600ng / ml
Adults after puberty: 150-350ng / ml
Note: The level of IGF-1 may decrease slightly with age.
This translation is for reference only, please refer to the original text for details.
Shanghai Yifeng Biotechnology Co., Ltd. acts as an agent for ELISA kits of different brands and price levels. Tens of thousands of antibody products, etc., with many varieties, good quality, high sensitivity, affordable, and also provide free testing services
"Puzzle 3D Eraser Bulk,Puzzle 3D Eraser,Building Blocks Eraser,Toppers Cute Erasers "
Yiwu Shengshang Stationery Co., Ltd. , https://www.ss-stationery.com