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DHT Dihydrotestosterone ELISA Kit
Germany IBL imported, article number: DB52021
Germany IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
1 , the scope of application
This ELISA kit can be used for the quantitative detection of dihydrotestosterone in human serum. Note: For in vitro diagnostic use only.
2 , the principle of experiment
This ELISA kit uses a typical competition method. Standards, controls, and unlabeled antigens and enzyme-labeled antigens in patient samples compete for binding to a limited antibody binding site on the coated plate. The plate was washed to remove unbound material. After washing the plate, the enzyme substrate solution was added. Finally, a stop solution was added to terminate the enzyme reaction, and the OD value was read on a microplate reader. The color displayed by the solution in the coating is proportional to the amount of DHT in the sample. The standard used in the experiment can be used as a standard curve. We can read the amount of DHT in the sample and control directly from the standard curve.
3 , clinical application
5α-dihydrotestosterone (DHT) is a steroid hormone similar to testosterone and androstenedione, and belongs to the class of androgens. DHT is a 19-carbon steroid with androgenic activity. Androgen is mainly secreted by Leydig cells of the testes. Androgen circulating in the blood binds to some proteins, especially to SHBG (sex hormone binding protein) and albumin. Only a small fraction is present in the blood in unbound form and is called the free part. The affinity of DHT for SHBG is three times that of testosterone to SHBF. In men, 70% of DHT is converted from peripheral testosterone; in women, most DHT is converted from androstenedione. The liver is the main organ that inhibits androgen function. Therefore, steroid hormones undergo structural changes in the liver, and it is widely believed that these various structural changes are necessary for the loss of biological activity. This type of sterol forms some metabolites before reaching the kidney excretion, and some steroids return to the blood circulation. Therefore, steroid hormones in the human body are excreted through the urine. Clinical tendency: In Cranfield syndrome, the level of DHT is much lower than that of normal people. In patients with congenital hirsutism, more than 40% of patients have elevated DHT levels. In patients with polycystic ovaries, approximately 35% of patients have elevated DHT levels. The DHT level in adults is much higher than that in normal elderly people, so the secretion of androgen in adolescence will increase, so that it can promote the formation of male secondary sexual characteristics. It has been confirmed that the seminiferous tubules of human testes can produce DHT. Therefore, when the seminiferous tubules are damaged, the production of DHT is blocked, resulting in a decrease in plasma DHT levels (for example, germ cell dysplasia and sperm deficiencies). The level of plasma DHT in patients without testicular malformations is very low. It has been reported that in some prostate cancer patients (especially the fourth stage), the detection of DHT is useful for predicting the therapeutic effect of antiandrogen therapy.
4 , sample collection and storage
A double test is required to require approximately 0.2 ml of serum. Collect 4-5 ml of blood into the corresponding labeled tube and wait for it to coagulate and centrifuge to obtain serum. It can be stored stably for 24 hours at 4 °C. If the experiment is done in a few days, the sample can be stored frozen at -10 °C or lower.
5 , sample pretreatment
This detection system is a direct detection system that does not require any sample pretreatment.
6 , the equipment required for the experiment but the kit does not provide
1) pipette, 50; 100; 150 and 300 μl
2) Sampling tip
3) distilled or deionized water
4) Shaker
5) Microplate reader
7 , kit components
1) Rabbit anti-DHT antibody coated detachable coated plate, ready to use. Contains: anti-DHT antibody coated 96-well coated plate, sealed in a semi-package containing desiccant, stored: refrigerated at 2-8 ° C, stability: 12 months or the expiration date on the label .
2) HRP-labeled DHT enzyme conjugate, 50-fold concentrated type, containing: HRP-labeled DHT enzyme conjugate, protein buffer and mercury-free preservative, volume: 200 μl / support, storage: refrigerated at 2-8 ° C Environment, stability: 12 months or expiration date on the label, preparation: dilute 1:500 with detection buffer before use (eg 20 μl HRP enzyme conjugate 2 ml detection buffer. If the entire package is For plate use, 120 μl of HRP Enzyme Link can be diluted with 12 ml of assay buffer and the remaining reagents discarded.
3) DHT standard, ready to use. 6 containing, DHT, human serum albumin and mercury-free preservative, prepared with buffer and a certain amount of DHT. The table below shows the approximate concentration of the standard. For the specific concentration, please refer to the reagent bottle label:
Standard
concentration
Volume/support
Standard A
0 pg/ml
2.0 ml
Standard B
25 pg/ml
0.6 ml
Standard C
100 pg/ml
0.6 ml
Standard D
500 pg/ml
0.6 ml
Standard E
1000 pg/ml
0.6 ml
Standard F
2500 pg/ml
0.6 ml
Store at 2-8 ° C, stability: 12 months or the expiration date of the label. Once opened, all standards must be used up within 14 days or frozen to avoid repeated freezing and thawing.
4) Quality control, ready-to-use, 1 containing: DHT, human serum albumin and mercury-free preservative, prepared with buffer and a certain amount of DHT. For specific concentration and acceptable range, please refer to the label of the reagent bottle. . Volume: 0.6ml / support, storage: refrigerated at 2-8 ° C environment, stability: 12 months or the expiration date on the label, once opened, all quality control products must be used within 14 days, must be repeated Freezing and thawing.
5) Concentrated sputum wash solution, 10 times concentrated type, 1 stick, containing: buffer, non-ionic detergent and mercury-free preservative, volume: 50ml / support, storage: refrigerated at 2-8 ° C environment Validity period: 12 months or the expiration date on the label. Preparation: Diluted with distilled water or deionized water at a ratio of 1:10 before use. If the whole coated board is used, 50 ml of concentrated sputum washing solution can be diluted with 450 ml of distilled water.
6) Detection buffer, ready-to-use, 1 containing: protein buffer and mercury-free preservative, volume: 15ml / support, stability: 12 months or the expiration date on the label.
7) TMB substrate solution, ready-to-use, 1 containing: TMB, hydrogen peroxide and non-DMF or DMSO, volume: 16ml / support, storage: refrigerated in 2-8 ° C environment, stability: 12 The expiration date on the month or label.
8) Stop solution, ready to use, 1 stick, containing: 1M sulfuric acid, volume: 6ml / support, storage: refrigerated at 2-8 ° C environment, stability: 12 months or the expiration date on the label.
8 , experimental steps
Sample pretreatment: none
All reagents must be equilibrated to room temperature before use. Standards, controls, and samples must be tested in duplicate. Once the experiment is performed, all steps must be completed.
1) Prepare a ready-to-use HRP enzyme conjugate and assay buffer. If urine is used as an experiment, the urine should be diluted.
2) Remove the required number of slats, reseal the unused slats in the package and refrigerate.
3) Add 50 μl of standard, control and sample (serum or diluted urine) to the corresponding microwells (double detection)
4) Add 100 μl of enzyme conjugate to each well (recommended for multi-channel pipettes).
5) Incubate for 1 hour on a coated plate shaker (200 rpm) at room temperature.
6) Wash the plate 3 times with 300 μl of washing solution per well and pat dry on absorbent paper.
7) 150 μl of TMB substrate solution was added to each well at the same time interval.
8) Incubate for 15-20 minutes on a coated plate shaker at room temperature.
9) Add 50 μl of stop solution to each well.
10) Read the OD value at 450 nm within 20 minutes after the addition of the stop solution.
* If the OD value exceeds the detection limit of the 450nm filter, we can replace it with 405n or 415nm, and the OD value may be lower, but it does not affect the experimental results of the sample and the control.
9 , the result of calculation
1) Calculate the average of the OD values ​​of the two standards.
2) On the semi-logarithmic coordinate paper, the calibration curve is made by taking the average OD value as the Y-axis and the concentration of the standard as the X-axis. If using immunoassay software, we can make a four-parameter curve.
3) Calculate the average value of the OD value of each unknown sample.
4) Read the concentration of the sample directly from the standard curve.
5) Multiply the obtained sample concentration value by the dilution factor. It is then multiplied by the volume of urine for 24 hours so that its flash unit can be changed to pg/24 hours. Finally, pg/24 hours is divided by 10 6 and the corresponding unit becomes μg/24 hours.
6) If the serum sample concentration is higher than 2500pg/ml, we can dilute it with Standard A (but the dilution ratio is not higher than 1:8). The result should be multiplied by the corresponding dilution factor.
This translation is for reference only, please refer to the original for details.
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