AMH ELISA Assay Kit

Additional Information

Assay Background

Anti-Müllerian Hormone (AMH) or Müllerian-inhibiting hormone (MIH) is a glycoprotein hormone structurally related to inhibin and activin from the transforming growth factor beta superfamily, whose key roles are in growth differentiation and folliculogenesis. AMH expression is critical to sex differentiation at a specific time during fetal development, and appears to be tightly regulated by nuclear receptor SF1, transcription GATA factors, sex reversal gene DAX1, and follicle-stimulation hormone (FSH). AMH is activated by SOX9 in the Sertoli cells of the male fetus thereby arresting the development of fallopian tubes, uterus, and upper vagina. AMH is also a product of granulosa calls of the preantral and small antral follicles in women. As such, AMH is only present in the ovary until menopause. AMH can serve as a molecular biomarker for relative size of the ovarian reserve and can also be used as a marker for ovarian dysfunction, such as women with polycystic ovary syndrome (PCOS).

Assay Principle

The Eagle Biosciences Human AMH ELISA Assay Kit is designed, developed and produced for the quantitative measurement of human Anti-Müllerian Hormone in serum or heparin plasma samples. The assay utilizes the two-site “sandwich” technique with two selected antibodies that bind to different epitopes of human AMH.

Assay calibrators, controls and patient samples are added directly to wells of a microtiter plate that is coated with antibody to N-terminal AMH along with another AMH specific antibody labeled with horseradish peroxidase (HRP). After an initial incubation period, the plate is washed a sandwich of solid-phase antibody – human AMH – HRP-conjugated monoclonal antibody is formed. The unbound monoclonal antibodies and buffer matrix are removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is directly proportional to the amount of human calprotectin in the test sample. A standard curve is generated by plotting the absorbance verse the respective human calprotectin concentration for each standard on a Cubic or point-to-point curve fitting. The concentration of human AMH in test samples is determined directly from this calibration curve.

Assay Procedure

1. Add 50 μL of Calibrators, Controls and patient samples into the designated microwells
2. Add 50 μL of the diluted tracer antibody into the designated microwells
3. Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 4 hours ± 15 minutes at 400 to 450 rpm or large orbit radius at 180 rpm.
4. Remove the aluminum foil and plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 μL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used
5. Immediately add 100 μL of ELISA HRP Substrate to each well.
6. Cover with foil or other material to protect from light, and incubate static at room temperature for 20 ± 2 minutes
7. Remove the aluminum foil and add 100 μL of ELISA Stop Solution into each of the wells. Mix gently
8. Read the absorbance at 450 nm with reference filter at 620, 630 or 650 nm immediately.

Typical Standard Curve