1

Human Anti-Müllerian Hormone ELISA
MATERIAL REQUIRED BUT NOT SUPPLIED

Cat. No.: BA1002
Enzyme Immunoassay for the quantitative determination of Anti-MüllerianHormone (AMH) in human serum and plasma.
Anti-Müllerian Hormone (AMH) is a glycoprotein belonging to thetransforming growth factors (TGF-P). In females, AMH is secreted bygranulosa cells of small follicles in the ovary.1 Serum AMH levels strongly
correlate with the number of growing follicles. Serum AMH levels are used
in individualized follicle-stimulating hormone dosing protocols and maypredict the risk of poor response or ovarian hyperstimulation syndrome.3
Serum concentrations of AMH gradually decrease and fall below detectablelevels in menopause. AMH is the best current available measure of ovarian
reserve for different clinical conditions.2
PRINCIPLE OF AMH ELISA

1.
2.
3.
4.
5.

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.

The microtiter plate is coated with the antibody specifically binding the AntiMüllerian Hormone. The human serum or plasma is incubated in the platewith the capture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith biotin-labelled detection antibody. Following another washing step,
Streptavidin-HRP conjugate is added into the well.
Unbound reagent is then washed out. Horseradish peroxidase (HRP) boundin the complex reacts with the chromogenic substrate (TMB) creating theblue colour. The reaction is stopped by addition of STOP solution (H2SO4).
The absorbance values are measured at 450 nm (optionally 450/630 nm)
and are proportional to the concentration of AMH in the specimen. The
concentration of AMH in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

Glassware and test tubes
Microtiter plate washer
Precision pipettes (various volumes) with tips
Orbital shaker
Microtiter plate reader capable of measuring absorbanceat 450 nm or 450/630 nm with software for data generation

6.
7.

For research use only
For professional laboratory use
The reagents with different lot numbers should not be mixed
To prevent cross sample contamination, use disposable labware
and pipette tips
To protect laboratory stuff, wear protective gloves andprotective clothing
The substrate solution should remain colourless, keep it
protected from light
The test should be performed at standard laboratory conditions(temperature 25oC ± 2oC).

STORAGE CONDITIONS
1.
2.

The kit must be stored at 2 – 8oC.
The opened components can be stored for one week at 2 – 8oC.

PREPARATION OF REAGENTS

Use new pipette tip for pipetting different reagents and samples
to prevent cross-contamination.
All reagents and samples should be allowed to reach thetemperature 25oC ± 2oC.

Preparation of Standards
Reconstitute lyophilized Human AMH Standard in Dilution Buffer, for thevolume information see the Certificate of Analysis. Let it rehydrate for
15 min and dilute 1:4 prior to use. The concentration of human AMH inMaster Standard is 3 ng/mL.
Prepare set of Standard solution as follows:
Use the Master Standard for serial dilution (as below). Mix each tube
thoroughly before the next transfer. The Dilution Buffer serves as Blank.
Volume of Standard

Kit Contents
Item
Antibody Coated Microtiter Plate
Biotin-labelled Antibody
Streptavidin-HRP Conjugate
Master Standard (lyophilized)
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution
v_02

Qty.
96 wells
13 mL
13 mL
1 vial1 vial1 vial
13 mL
50 mL
13 mL
13 mL

Std1

Standard 3 ng/mL (lyophilized)

Dilution
Buffer1000 µL

Concentration

Std2300 µL of Std1300 µL

1.5 ng/mL

Std3300 µL of Std2300 µL

0.75 ng/mL

Std4300 µL of Std3

300 µL

0.375 ng/mL

Std5300 µL of Std4

300 µL0.188 ng/mL

Std6300 µL of Std5300 µL

0.094 ng/mLBlank-300 µL0 ng/mL3 ng/mL

Preparation of Quality Control A and B
Reconstitute the lyophilized human serum Quality Controls withdeionized/distilled water, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:4 in Dilution
Buffer, prior to use, see Preparation of samples.

2

Preparation of Wash Buffer 1x3.

Prepare a working solution of Wash Buffer by adding 50 mL of Wash Buffer
15x conc. to 700 mL of deionized/ distilled water (dH2O). Mix well. Store at4oC for two weeks or at -20oC for long term storage.
Preparation of samples
Human serum or plasma may be used with this assay. For long-term storage
the samples should be frozen at minimum -70oC. Lipemic or haemolytic
samples may cause false results.
Recommended dilution of samples is 1:4, i.e., for singlets 40 µL of sample +120 µL of Dilution Buffer, for duplicates 80 µL of samples + 240 µL of Dilution
Buffer, respectively.

Accuracy

Dilution linearity
SampleDilution

Measuredconcentration(ng/mL)
1.9
1.1
0.5
0.3
3.4
1.8
0.9
0.5

Expected
concentration(ng/mL)
0.96
0.5
0.2
1.7
0.9
0.4

Yield(%)

Measuredconcentration(ng/mL)
1.2
4.0
2.7
1.9

Expected
concentration(ng/mL)
4.23
2.75
1.98

Yield (%)

12x4x8x22x4x8x110
104
109
108
108
113

Do not store the diluted samples.
Spiking Recovery

ASSAY PROCEDURE
1.
2.

Prepare the reagents as described in the previous chapter.
Pipette 100 µL of set of Standards, Quality Controls, diluted
Samples and Dilution Buffer = Blank into each well. Incubate for
1 hour at 25oC ±2oC, shaking at 300 rpm.
3. Wash the wells 3-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
4. Pipette 100 µL of Biotin-labelled Antibody into each well.
Incubate for 1 hour at 25oC ±2oC, shaking at 300 rpm.
5. Wash the wells as described in point 3.
6. Pipette 100 µL of Streptavidin-HRP into each well. Incubate for
30 min at 25oC ±2oC, shaking at 300 rpm.
7. Wash the wells as described in point 3.
8. Pipette 100 µL Substrate solution, incubate for 10 min,at 25oC ±2oC. Avoid exposure to the light during this step.
9. Pipette 100 µL of STOP solution.
10. Read the signal at 450 or 450/630 nm within 15 min.

SampleSpike(ng/ml)

1

3.0
1.5
0.8

9596
94

Typical standard curve
The standard curve needs to be measured in every test. Most of themicroplate reader can automatically calculate the analyte concentration
using 4-parameter algorithm or alternative functions to fit the standard
points properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:4 as recommended and assayed.
The results are multiplied by the dilution factor.
1.

Sensitivity

The limit of detection, defined as a concentration of human AMH giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 0.024 ng/mL of sample.
2.

Precision

Intra-assay
Sample12

Mean (ng/mL)
1.9
3.4

SD
0.1
0.2

CV (%)
75SD
0.1
0.2

CV (%)
46Inter-assay (Run – to – run)
Sample12Mean (ng/mL)
2,0
3.7

RESOURCES
1 Zec I, Tislaric-Medenjak D, Megla ZB, Kucak I. Anti-Müllerian hormone: a uniquebiochemical marker of gonadal development and fertility in humans. Biochem Med(Zagreb). 2011;21(3):219-30. doi: 10.11613/bm.2011.031. PMID: 22420235.
2 Broer SL, Broekmans FJ, Laven JS, Fauser BC. Anti-Müllerian hormone: ovarian reserve

testing and its potential clinical implications. Hum Reprod Update. 2014 SepOct;20(5):688-701. doi: 10.1093/humupd/dmu020. Epub 2014 May 12. PMID:24821925.
3 Moolhuijsen LME, Visser JA. Anti-Müllerian Hormone and Ovarian Reserve: Updateon Assessing Ovarian Function. J Clin Endocrinol Metab. 2020 Nov 1;105(11):3361–73.
doi: 10.1210/clinem/dgaa513. PMID: 32770239; PMCID: PMC7486884.

v_02