LH Elisa Kit Luteinizing Hormone

1. Intended use

Immunoassay for the in vitro quantitative determination of luteinizing hormone in human serum.

2. Summary

• LH (luteinizing hormone), together with FSH (follicle stimulating hormone), belongs to the gonadotropin family. LH and FSH regulate and stimulate the growth and function of the gonads (ovaries and testes) synergistically. Like FSH, TSH and hCG, LH is a glycoprotein consisting of two subunits (α- and β-chains). (1, 2, 3) This proteohormone, which consists of 121 amino acids and three sugar chains, has a molecular weight of 29500 daltons. In women, the gonadotropins act within the hypothalamus-pituitary-ovary regulating circuit to control the menstrual cycle.
• LH and FSH are released in pulses from the gonadotropic cells of the anterior pituitary and pass via the bloodstream to the ovaries. Here the gonadotropins stimulate the growth and maturation of the follicle and hence the biosynthesis of estrogens and progesterones. The highest LH-concentrations occur during the mid-cycle peak and induce ovulation and formation of the corpus luteum, the principal secretion product of which is progesterone. In the Leydig cells of the testes, LH stimulates the production of testosterone. Determination of the LH concentration is used in the elucidation of dysfunctions within the hypothalamus-pituitary-gonads system. The determination of LH in conjunction with FSH is utilized for the following indications: congenital diseases with chromosome aberrations (e.g. Turner’s syndrome), polycystic ovaries (PCO), clarifying the causes of amenorrhea, menopausal syndrome, and suspected Leydig cell insufficiency. (1, 4, 5)

3. Test principle

Sandwich principle. Total duration of assay: 80 minutes.

• Sample, Anti-LH coated microwells and enzyme labeled Anti-LH are combined.

• During the incubation, LH presents in the sample is allowed to react

simultaneously with the two antibodies, resulting in the LH molecules being

sandwiched between the solid phase and enzyme-linked antibodies.

• After washing, a complex is generated between the solid phase, the LH within the sample and enzyme-linked antibodies by immunological reactions.

• Substrate solution is then added and catalyzed by this complex, resulting in a chromogenic reaction. The resulting chromogenic reaction is measured as absorbance.

• The absorbance is proportional to the amount of LH in the sample.

Reagents

Materials provided

• Coated Microplate, 8 x 12 strips, 96 wells, pre-coated with mouse monoclonal

Anti-LH.

• Calibrators, 6 vials, 1 ml each, ready to use; Concentrations: 0(A), 5(B), 20(C), 50(D), 100(E) and 200(F) mIU/mL.

• Enzyme Conjugate, 1 vial, 11 mL of HRP (horseradish peroxidase) labeled mouse monoclonal Anti-LH in Tris-NaCl buffer containing BSA (bovine serum albumin). Contains 0.1% ProClin300 preservative.

• Substrate, 1 vial, 11ml, ready to use, (tetramethylbenzidine) TMB.

• Stop Solution, 1 vial, 6.0 ml of 1 mol/L sulfuric acid.

• Wash Solution Concentrate, 1 vial, 25 mL (40X concentrated), PBS-Tween

wash solution.

• IFU, 1 copy.

• Plate Lid: 1 piece.

Materials required (but not provided)

• Microplate reader with 450nm and 620nm wavelength absorbent capability.

• Microplate washer.

• Incubator

4. Test procedure

• Use only the number of wells required and format the microplates’ wells for

each calibrator and sample to be assayed.

• Add 25 μL of calibrators or samples to each well.

• Add 100 μL of enzyme conjugate to each well.

• Shake the microplate gently for 30 seconds to mix.

• Cover the plate with a plate lid and incubate at 37 °C for 60 minutes.

• Discard the contents of the micro plate by decantation or aspiration. If

decanting, tap and blot the plate dry with absorbent paper.

• Add 350 μL of wash solution, decant (tap and blot) or aspirate. Repeat 4 additional times for a total of 5 washes. An automated microplate strip washer can be used. At the end of washing, invert the plate and tap out any residual wash solution onto absorbent paper.

• Add 100 μL of substrate to each well.

• Incubate at ambient temperature (18-25℃)in the dark for reaction for 20 minutes. Do not shake the plate after substate addition.

• Add 50 μl of stop solution to each well.

• Shake for 15-20 seconds to mix the liquid within the wells. It is important to

ensure that the blue color changes to yellow completely.

• Read the absorbance of each well at 450 nm (using 620 to 630 nm as the

reference wavelength to minimize well imperfections) in a micro plate reader.