Luteinizing Hormone (LH) ELISA Kit Instructions

Luteinizing Hormone ( LH ) ELISA Kit Instructions

  ( Germany DRG : EIA-1289)

1, Application range

DRG 's LH ELISA kit can be used for the quantitative detection of luteinizing hormone ( LH ) in serum. This test kit is for in vitro diagnostic use only.

2 , the preface

Under the influence of the luteinizing hormone releasing hormone ( LH-RH or Gn-RH ) released by the hypothalamus, LH (luteinizing hormone) can be produced in both the anterior pituitary of both men and women. In men, LH is also called stromal cytokines ( ICSH ), a glycoprotein with a molecular weight of approximately 30,000 Daltons. It is a complex formed by two different amino acid chains joined by non-covalent bonds, an alpha chain, and a beta chain. The alpha chain is similar to the alpha chain glycoprotein found in human thyroid stimulating hormone ( TSH ), follicle stimulating hormone ( FSH ) and human chorionic gonadotropin. The difference in these hormones is mainly due to the different amino acid composition of their beta chains, which also gives them different immunological functions. The secretion of LH in men is intermittent, and its basic function is to stimulate the secretion of testosterone by mesenchymal cells (Leidrich cells). Changes in LH concentrations in women are affected by the complex ovulation cycle of normal menstruation, which also depends on a series of hormonal activities along the gonad - hypothalamus - pituitary axis. The reduction in progesterone and estradiol levels before ovulation initiates each menstrual cycle. As the hormone levels decrease, the hypothalamus increases the secretion of gonadotropin-releasing hormone ( GnRF ), which in turn stimulates the pituitary to increase the production and secretion of FSH . Increased FSH levels stimulate the maturation of some follicles during the follicular phase, and one of the follicles matures into an egg-containing follicle. As follicles develop, estradiol begins to secrete, which is very slow at first, but it begins to increase rapidly after a normal cycle of 12 or 13 days. Because of the direct stimulation of the pituitary and increased levels of GnRF and FSH , estradiol begins to increase rapidly, and LH begins to release. These events just form the pre-ovulation period. After the highest level of LH , ovulation lasts approximately 12 to 18 hours. After ovulation, a corpus luteum that secretes progesterone and estrogen, which are two feedback regulators of LH , is formed. The luteal phase quickly followed the ovulation period, with the luteal phase showing high progesterone levels, estradiol second increase, and low LH and FSH levels. Low LH and FSH levels are the result of a negative feedback response of estradiol and progesterone along the hypothalamic - pituitary axis. After conception, the development of the embryo can produce hCG , which causes the corpus lute to continue to produce progesterone and estradiol. If not pregnant, the corpus luteum will degenerate, and the corresponding levels of progesterone and estradiol will decrease, leading to the formation of menstruation. With the formation of these hormones with low hormone levels, the hypothalamus will again begin to undergo a menstrual cycle in patients with hypogonadal decline, and the serum LH concentration will increase. The secretion of steroids in women is reduced due to immature ovarian development, primary ovarian failure, polycystic ovarian disease, or menopause, and in these cases, LH secretion is not regulated. In men, but testicular development is not normal or there is no testicular deformity, it also loses regulatory hormones. In patients with primary testicular failure and Klinefelter syndrome, although there is no need to increase the LH concentration in the case of continuous secretion of androgen, we can also find that there is a high concentration of LH in the body. In the case of renal failure, cirrhosis, hyperthyroidism, and severe starvation, the concentration of LH may also increase. Insufficient secretion of anterior pituitary hormone may also result in low LH levels. As predicted, low LH levels may lead to infertility in men and women. Although the low level of LH may also occur in the absence of GnTH stimulation in the anterior pituitary, the level of LH may be reduced due to a decrease in GnRH secretion in the hypothalamus. Therefore, low LH levels may indicate dysfunction in the pituitary or hypothalamus, but the root cause of the specific problem must be determined by other experiments. In the differential diagnosis of hypothalamic, pituitary or gonadal dysfunction, the detection of LH concentration is often detected together with FSH (so that the functions of the two are closely linked). In addition, hormone levels can be used to determine whether menopause, calculate ovulation time, and monitor endocrine therapy outcomes.

3 , the principle of experiment

   DRG 's LH enzyme-free assay is a solid-phase enzyme-free adsorption assay ( ELISA ) based on the sandwich method principle. The microassay well on the reaction plate is coated with a monoclonal antibody that binds to a unique antigenic site on the LH molecule. A patient sample containing endogenous LH was incubated with the enzyme conjugate in a coated well, which is an anti- LH antiserum linked to horseradish peroxidase. After incubation, the unbound enzyme conjugate was washed away with washings. The total amount of horseradish peroxidase compliant was positively correlated with the concentration of LH in the sample. After the addition of the substrate solution, the apparent color intensity is proportional to the concentration of LH in the patient sample.

4 , the kit consists of :

1)       A microplate, 1 piece (96 wells ) , was coated with a monoclonal antibody against LH .

2)       Standard series : 6 pieces, lyophilized, 1ml , concentration : 0,10,20,40,100,200mIU/ml

3)       Enzyme conjugate, 1 branch, 11 ml , horseradish peroxidase-labeled anti- LH antiserum, 0.3% Proclin as a preservative.  

4)       Substrate solution, 14ml (TMB)

5)       Stop solution : 1 branch, 0.5MH ₂ SO ₄ , 14ml , avoid contact with stop solution to avoid irritation or burning skin.

Zero standards for sample dilution should be determined as needed.

5 , the equipment required for the experiment (but not provided by the kit)

1) Microplate reader

2) Precision pipette, 25 ; 50 ; 100 μl.

3) distilled water

4) Absorbent paper

5) Timer

6) Semi-logarithmic paper

6 , storage conditions

   Unopened reagent at 2 - 8 ℃ stored, may remain active within the validity period. Do not use the expired reagent, enzyme conjugate, a substrate solution, zero standard and standards must be 2 - stored at 8 ℃.

   Microplates must be 2 - stored at 8 ℃. Once the bag is opened, it must be carefully sealed. If the package of the coated board is opened, its immunological activity is stable for 6 weeks, provided that it is sealed in a bag containing desiccant.

7 , reagent preparation

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

Standard: Reconstitute the standard by adding 1.0 ml of distilled water to each standard of lyophilized powder. Note: The prepared standard can be stored stably for 2 months at 2-8 °C. If it is stored for a longer period of time, it should be stored at -20 °C.

8 , sample

Serum will be used in this experiment. Please do not use samples that are easy to hemolyze, jaundice, and lipemia. Note: Samples containing sodium azide should not be used in this experiment.

8.1 Sample collection

Serum: Whole blood was collected by venipuncture, and it was allowed to coagulate, and serum was separated by centrifugation at room temperature. Do not centrifuge before incomplete coagulation. Patients who have received anticoagulant therapy may require longer clotting times.

8.2 Sample storage

The sample was sealed with a lid before the start of the experiment and stored for 48 hours at 2-8 °C. If the sample was frozen at -20 ℃ the environment can be stored longer (freeze thaw only once). Thawed samples must be turned upside down several times before the experiment.

8.3 Sample dilution:

In the first experiment, samples with a sample concentration higher than the highest standard should be diluted with zero buffer before the experiment. This dilution factor should be taken into account when calculating the concentration. example:

a) Dilute 1 : 10 : 10 μL of serum + 90 μL of zero buffer (fully mixed).

b) Dilute 1 : 100 : 10 ul of diluted a ) +90 μL of zero buffer (fully mixed).

9 , experimental steps

All standards, samples, and controls must be double tested to ensure they are tested under the same conditions. There must be a standard curve for each test.

1)       Place the required number of slats on the pallet.

2)       The standards, controls, and serum samples were added 25 μ l to the respective wells, each had the new sampling tip.

3)       Add 100 μ l of anti-LH enzyme conjugate to each well.

4)       Mixing for 10 seconds , it is important to mix thoroughly in this step.

5)       Incubate at room temperature (22 °C ) for 30 minutes.

6)       Discard the reaction solution in the well. The plate was washed 5 times with distilled water ( 400 ul per well), and the plate was patted dry on absorbent paper to remove residual liquid. Note: The correctness of the washing step will significantly affect the sensitivity and precision of the experiment.

7)       By the original loading sequence, each well was added 100 μ l substrate solution.

8)       Incubate for 10 minutes at room temperature (22 °C ) .

9)       By sequential addition of substrate solution, 50 μ l per well of stop solution to each well.

10)   The absorbance was read at a wavelength of 450 nm within 10 minutes after the addition of the stop solution.

10 , the result of 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 value ( mIU/ml ) of each standard is 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: The results on the manual are calculated using four parameters, and other induction methods may give slightly different results.

5)       The concentration of the sample can be read directly from the standard curve. If the LH concentration in the sample is higher than the highest standard, further dilution must be performed. Any diluted sample must be calculated with the corresponding dilution factor.

11 , normal value

It is strongly recommended that each laboratory determine its own range of normal values ​​and ranges of abnormal values.

A study of the apparently healthy population using DRG 's LH ELISA kit yielded the following results:

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