Estradiol (E2) ELISA Kit Instructions

Estradiol (E2) ELISA Kit Instructions

(Germany DRG : EIA2693 )

1. Description of the preface

E2 is a phenol A ring 18C Steroids. The molecular weight of this steroid hormone is 272.4 , which is the most effective estrogen in the human body. It is mainly produced by the gravre follicle and placenta of the female ovary, and a small amount is produced by the adrenal gland and the male testis. E2 (estradiol) is secreted into the bloodstream. In the blood, 98% of estradiol binds to sex hormone binding proteins, and some bind to other serum proteins (such as serum albumin). Some of them exist in free form. The activity of estrogen is affected by the estradiol receptor compliant, which can elicit a corresponding response at the target site and at the nucleic acid level. These targets include follicles, uterus, breasts, vagina, urethra, hypothalamus, pituitary gland and slightly less sensitive liver and skin. In non-pregnant women with normal menstrual cycles, estradiol secretion follows a cyclical pattern with two distinct peaks before ovulation. High concentrations of estradiol can exert positive feedback at the pituitary level, which affects the secretion of pituitary gonadotropins , such as follicle stimulating hormone and luteinizing hormone, the former is necessary for follicular maturation, and the latter is necessary for ovulation formation. . After ovulation, the level of estradiol decreased rapidly until the luteal cells were activated, resulting in a second peak in estradiol levels and a balanced assessment in the luteal phase. During pregnancy, the level of maternal serum estradiol is extremely high and exceeds the peak before ovulation and is maintained at a high level throughout pregnancy. The detection of serum estradiol is an effective indicator for evaluating various menstrual dysfunction, such as precocious and late maturing, primary, secondary amenorrhea and menopause. Serum estradiol levels are reported to be elevated in women with feminization syndrome, gynecomastia and testicular cancer. In infertility patients, the detection of serum estradiol is beneficial to monitor the induction of ovulation after drug treatment (such as clomiphene citrate, luteinizing hormone releasing hormone or exogenous gonadotropin). In patients with ovarian hyperstimulation syndrome due to in vitro fertilization, serum estradiol concentrations are monitored daily to provide optimal timing for hCG management and oocyte collection.

2 , detection principle :

   DRG 's estradiolase-free kit is a solid-phase enzyme-free adsorption assay ( ELISA ) based on the principles of competition. The microassay well on the reaction plate is coated with a monoclonal antibody that binds to a unique antigenic site on the estradiol molecule. A patient sample containing endogenous estradiol competes with horseradish peroxidase-labeled estradiol for binding to antibodies on the plate. After incubation, the unbound enzyme conjugate was washed away with washings. The amount of peroxidase bound to the coated antibody is inversely proportional to the concentration of estradiol in the sample. After the addition of the substrate solution, the intensity of the color displayed is inversely proportional to the concentration of estradiol in the patient sample.

3 , kit components :

1)        Coated plate, 12 × 8 (detachable), 96- well, microencapsulated rabbit monoclonal antibody against estradiol.

2)        Standard: 7 1.0ml / support. The content is: 0 ; 25 ; 100 ; 250 ; 500 ; 1000 ; 2000pg/ml . (1pg/ml=3.67pmol/L)

3)        Enzyme linkage: 1 branch, 25 ml , containing horseradish peroxidase-labeled estradiol.

4)        Substrate solution: 1 part , containing, 14ml , containing TMB

5)        Stop solution: 1 branch, 0.5M The H ₂ SO _4, 14ml, to avoid contact with stop solution, in order to avoid burning the skin and skin irritation.

6)        Washing solution (40×) , 1 stick, 30ml

Note: Zero standards for sample dilution are available on request.

4 , the experiment requires equipment (but the kit does not provide)

1)         Microplate reader (450 ± 10nm) .

2)         Pipette and the suction nozzle 25 μ l, 100 μ l, 200 μ l.

3)         Coordinate paper.

4)         Deionized water.

5 , reagent storage :

   Unopened reagents at 2 — 8 °C When stored under storage, its activity is stable during the period of validity. Do not use expired reagents. All unopened reagents must be stored in 2 -8 °C The microporous reaction plate must also be stored in 2 - 8 °C The environment. Once the bag is opened, it must be carefully sealed.

6 , reagent preparation

All reagents and the required number of slats were equilibrated to room temperature before the start of the experiment.

Washing solution: Add 30ml washings were concentrated in 1170ml of deionized water in the diluted, the diluted cleaning liquid at room temperature is stable for 2 weeks.

7. Sample collection and preparation

7.1 sample collection

Serum: Whole blood was collected by venipuncture, until it was coagulated, and serum was obtained by centrifugation at room temperature.

Plasma: Whole blood was collected into a centrifuge tube containing an anticoagulant and centrifuged immediately after collection.

7.2 Storage of samples

The sample should be covered with a lid before the start of the experiment, 2 -8 °C It can be stored stably for 5 days under the environment . If you want to store the sample for a longer period of time before the experiment, you should freeze the sample in -20 °C The environment. Thawed samples should be inverted several times before the experiment.

7.3 dilution of the sample

In the first experiment, if the concentration of testosterone in the serum sample is higher than the concentration of testosterone in the highest standard, the sample should be diluted 10 or 100 times with the zero standard and retested according to the experimental procedure. This dilution factor should be taken into account when calculating the concentration.

E.g:

a)   Dilution ratio of 10:: press 1 90μl zero standard (mixed) 10 l serum +

b)   Dilute in a 1 : 100 ratio: 10 μl diluted a ) +90 μl zero standard (fully mixed)

8 , experimental steps :

All standards, samples and controls are double tested to ensure all test conditions are the same.

1)        Secure the required number of slats to the pallet.

2)        Standard, control, and diluted samples were added to the corresponding microwells using a new sampling tip of 25 μL .

3)        200 μL of enzyme conjugate was added to each well .

4)        Mix well for 10 seconds and it is important to mix thoroughly at this step.

5)        Incubate for 120 minutes at room temperature.

6)        The wells were quickly discarded and the plates were washed 3 times with 400 μL of wash solution per well and patted dry on blotting paper. Note: The correct operation of the washing step will significantly affect the sensitivity and accuracy of the experiment.

7)        Add 100 μ l substrate solution in each microwell.

8)        Incubate for 15 minutes at room temperature.

9)         50 μ l stop solution was added to each test well to stop the enzymatic reaction.

10)      The OD value was measured at 450 ± 10 nm using a microplate reader within 10 minutes after the addition of the stop solution.

9 , the result calculation :

1)        Calculate the average absorbance value for each standard, control, and patient sample.

2)        Using the absorbance value as the ordinate ( y ) and the concentration value as the abscissa ( x ), the corresponding concentration values ​​of each standard are plotted to make a standard curve.

3)        The corresponding concentration was determined on the standard curve using the average absorbance value of each sample.

4)         Automatic calculation method: On the IFU , it automatically calculates the result using a four-parameter curve. Other inductive methods may yield slightly different results.

5)         The concentration of the sample can be read directly from the standard curve. Further dilution of the HPL concentration in the sample above the highest standard must be used. This dilution factor should be taken into account when calculating the sample concentration.

10 , reference value

We strongly recommend that each experimental city establish its own normal and outliers. In a study of a significant normal population, we used DRG 's estradiol ELISA kit to detect the following results:

This translation is for reference only, please refer to the original for details.

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