Human MIS/AMH ELISA Kit (HUFI00379)- High Sensitivity

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Human MIS / AMH ELISA Kit
Product Code:	HUFI00379
Size:	96 Assays
Alias:	AMH, MIS, muellerian-inhibiting factor, Mullerian inhibiting factor, Anti-Muellerian hormone, AMH, Muellerian-inhibiting substance, MIS
Detection Method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human AMH concentrations in serum plasma and other biological fluids.
Sensitivity:	28.125pg/ml
Range:	46.875-3000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Human AMH and the recovery rates were calculated by comparing the measured value to the expected amount of Human AMH in samples.


Matrix	Recovery Range (%)	Average (%)
serum(n=5)	88-105	94
EDTA plasma(n=5)	86-105	94
UFH plasma(n=5)	89-100	93

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human AMH and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.


Sample	1:2	1:4	1:8
serum(n=5)	88-104%	89-104%	87-102%
EDTA plasma(n=5)	87-101%	82-99%	Text
UFH plasma(n=5)	80-95%	90-98%	80-99%

CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate (SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Protein Information

Heading 1	Heading 2
Uniprot	P03971
NCBI GenInfo Identifier	313104218
NCBI Gene ID	268
Protein Family	Muellerian-inhibiting factor
UniProt Gene Name	AMH

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Sample Type

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

MIS/AMH

Mullerian inhibiting Substance (MIS) / Anti-Mullerian hormone (AMH)

MIS/AMH (Müllerian inhibiting substance/anti-Müllerian hormone) is a protein that plays a crucial role in the development of the reproductive system in both males and females. During fetal development, MIS/AMH is produced by the Sertoli cells in the testes of male embryos. It functions by suppressing the development of the Müllerian ducts, which are responsible for the formation of the uterus, fallopian tubes, and upper vagina in females. The presence of MIS/AMH causes regression of the Müllerian ducts in males, leading to the development of male reproductive organs. In females, MIS/AMH is produced by the granulosa cells of the ovarian follicles. Its expression is highest during the pre-antral and early antral stages of follicle development.

MIS/AMH Structure

AMH, also known as Müllerian-inhibiting substance (MIS), belongs to the TGF-β superfamily. It is a glycoprotein consisting of two identical subunits held together by disulfide bonds, forming a homodimeric structure. With a molecular weight of 140 kDa, AMH is a disulfide-linked protein. The gene encoding AMH is located on the short arm of chromosome 19 in humans, specifically on band 19p13·3. It spans 2750 base pairs and is divided into five exons. The bioactive region of AMH is encoded by the 3' portion of the fifth exon, which is notably GC rich. During development, AMH is highly expressed in Sertoli cells from testicular differentiation through puberty. It is also expressed to a lesser extent in granulosa cells from birth until menopause.

MIS/AMH Levels

AMH levels can vary depending on age. In women, AMH levels typically begin to increase during adolescence and reach their peak around the age of 25. Subsequently, it is natural for AMH levels to gradually decline. When measuring AMH levels, healthcare providers typically use nanograms per milliliter (ng/mL) as the unit of measurement. While there is ongoing debate among experts regarding the definition of typical AMH levels, there are some general ranges that can be considered. On average, AMH levels can fall within the range of 1.0 ng/mL to 3.0 ng/mL. Levels below 1.0 ng/mL are considered low, and a level of 0.4 ng/mL is classified as severely low. It is important to note that slight variations in results may occur between different laboratories due to the use of different equipment and testing methods.

MIS/ AMH ELISA Kits FAQs

What is the MIS/AMH ELISA Kit used for?

The MIS/AMH ELISA kit is used for the quantitative measurement of MIS/AMH levels in blood serum or plasma samples. It provides researchers, clinicians, and fertility specialists with a reliable and accurate tool for assessing ovarian reserve, predicting ovarian response to fertility treatments, and diagnosing reproductive disorders.

What are the advantages of using the MIS/AMH ELISA Kit?

The MIS/AMH ELISA Kit offers several advantages, including high sensitivity, accuracy, and reproducibility. It provides a user-friendly and reliable method to quantify MIS/AMH levels in biological specimens, allowing for precise measurements and robust data analysis.

What sample types are compatible with MIS/AMH ELISA Kit?

The MIS/AMH ELISA Kit is compatible with various sample types, including serum, plasma, cell lysates, and tissue homogenates. It provides flexibility in sample selection, allowing researchers to analyze MIS/AMH levels in different biological matrices.

What are the storage requirements with MIS/AMH ELISA Kit?

The MIS/AMH ELISA Kit components should be stored according to the instructions provided in the kit manual. Generally, it is recommended to store the kit components at the recommended temperature to ensure their stability and optimal performance.

What should I do if my assay results are not optimal?

If you encounter any issues or have suboptimal assay results, we recommend contacting our dedicated support team for assistance. They will be available to provide troubleshooting guidance, answer your questions, and ensure you achieve the best possible results with the MIS/AMH ELISA Kit.