EASYStep Human Anti-Mullerian Hormone (AMH) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDE-AMH-Hu
The EASYStep Human Anti-Mullerian Hormone (AMH) ELISA Kit (RDE-AMH-Hu) is an enzyme-linked immunosorbent assay for quantitative detection of AMH in Human samples. This kit uses the EASYStep ELISA format for colorimetric detection of natural AMH in samples including serum and plasma. The kit is easy to use and comes with a pre-coated microplate and all necessary reagents and buffers. The standard curve generated in the experiment is used to calculate the concentration of AMH in samples. For research use only.
Product Specifications
Product Name
EASYStep Human Anti-Mullerian Hormone (AMH) ELISA Kit
Catalog Number
RDE-AMH-Hu
Detection Range
0.31-20ng/mL
Sensitivity
0.19 ng/mL
Species Reactivity
Human
Species
Homo sapiens Human
Assay Length
2.5 hours
Experimental Method
Sandwich
Detection Method
Colorimetric
Sample Type
serum and plasma
Storage
2-8°C
Shelf Life
6 months
Product Details
Intended Use
This kit is a sandwich enzyme immunoassay for in vitro quantitative determination of human AMH concentrations in serum and plasma.
Test Principle
This kit employs Sandwich-ELISA principle. The micro-ELISA plate provided in this kit has been pre-coated with an antibody specific to AMH. Samples (or Standards) and biotinylated detection antibody specific for AMH are added to the micro-ELISA plate wells. AMH binds with the specific antibody. Then Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain AMH, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 ± 2 nm. The OD value is proportional to the concentration of AMH. You can calculate the concentration of AMH in the samples by comparing the OD of the samples to the standard curve.
Application Data
Reagents and Materials Provided
- Data Table: Reagents | Quantity Micro ELISA Plate (Detachable) | 96T: 8 wells ×12 strips 48T: 8 wells × 6 strips 24T: 8 wells × 3 strips Reference Standard | 96T: 2 vials 48T: 1 vial 24T: 1 vial Diluent Buffer | 1 × 20 mL Detection Solution | 1 × 6mL HRP Conjugate Diluent | 1 × 14 mL Wash Buffer (25×concentrate) | 1 × 30 mL HRP Conjugate | 96T: 1 × 120 μL 48T: 1 × 60 μL 24T: 1 × 60 μL Substrate Reagent | 1 × 10 mL Stop Solution | 1 × 10 mL Plate Sealer | 5 pieces Instruction manual | 1
Materials Required but not Supplied
- Microplate reader with 450 ± 10 nm filter.
- Precision single or multi-channel pipettes and disposable tips.
- Eppendorf Tubes.
- Incubator capable of maintaining 37°C.
- Deionized or distilled water.
- Absorbent paper.
- Container for Wash Buffer.
Storage of the kits
- For unopened kits: All the reagents can be stored at 2-8°C for 6 months.
- For opened kits: Once the kit is opened, the remaining wells and reagents should be stored according to the table below. Data Table: Item | Storage conditions Micro ELISA Plate (Detachable) | 2-8°C, 1 month Reference Standard | Discard any unused reconstituted standard and dilutions Diluent Buffer | 2-8°C Detection Solution | HRP Conjugate Diluent | Wash Buffer (25×concentrate) | HRP Conjugate | 2-8°C (Protect from light) Substrate Reagent | Stop Solution | 2-8°C
- For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this date.
- Return the unused wells to the foil pouch containing the desiccant pack and reseal along entire edge of zip-seal.
- It is highly recommended to use the remaining reagents within 1 month of opening.
Sample Collection and Storage
- Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2-8°C within 30 minutes of collection. Collect the supernatant for use in the assay. Hemolyzed samples are not suitable for use with this ELISA assay.
- Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and/or contamination.
- Noticeable hemolysis will affect antibody-antigen reactions. Samples with any sign of hemolysis are not suitable for this assay.
- When performing the assay, bring samples to room temperature.
Reagent Preparation
- Bring all kit components and samples to room temperature (18-25°C) before use.
- Wash Solution - Dilute 30 mL of Wash Buffer (25×) with 720 mL of deionized or distilled water to prepare 750 mL of Wash Solution (1×).
- Standard - Centrifuge the standard at 10,000 × g for 1 minute. Add 1.0 mL of Diluent Buffer, let it stand for 10 minutes and invert it gently several times (not to foam). The concentration of the standard in the stock solution is 20 ng/mL. Prepare 7 EP tubes containing 0.5 mL Diluent Buffer and use the diluted standard to produce a double dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Prepare a dilution series with 7 points, for example: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.62 ng/mL, 0.31 ng/mL, and the last EP tube with Diluent Buffer is the blank at 0 ng/mL.
- HRP Conjugate working solution - Calculate the required amount before the experiment (100 μL/well). Prepare slightly more than necessary for your experiment. Centrifuge the HRP Conjugate at 800 × g for 1 minute, then dilute to the working concentration with HRP Conjugate Diluent (1:100).
- Do not perform a serial dilution directly in the wells.
- Prepare standard within 15 minutes of performing the assay. Do not dissolve the reagents at 37°C directly.
- Carefully reconstitute Standards according to the instruction. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated.
- The reconstituted Standards can be used only once.
- If crystals have formed in the Wash Buffer (25×), warm to room temperature and mix gently until the crystals are completely dissolved.
- Any contaminated water or container used during reagent preparation will influence the detection result.
Assay Procedure Summary
- Add 50 µL standard or sample to each well, immediately add 50 µL Detection Solution to each well. Incubate for 90 minutes at 37°C.
- Aspirate and wash the plate for 3 times.
- Add 100 µL HRP conjugate working solution. Incubate for 30 minutes at 37°C.
- Aspirate and wash 5 times.
- Add 90 µL Substrate Solution. Incubate for 15 minutes at 37°C.
- Add 50 µL Stop Solution. Read at 450 nm immediately.
Calculation of Results
Average the duplicate readings for each standard, control and sample, then subtract the average zero standard optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with AMH concentration on the y-axis and absorbance on the x-axis. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Product Data
Sensitivity
- The minimum detectable dose of AMH is typically less than 0.19 ng/mL.
- The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
Specificity
- This assay has high sensitivity and excellent specificity for detection of human AMH.
- No significant cross-reactivity or interference between human AMH and analogs was observed.
- Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between AMH and all analogs, therefore, cross reactivity may still exist.
Precision
- Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level AMH were tested 20 times on one plate, respectively.
- Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level AMH were tested on 3 different plates, 8 replicates in each plate.
- CV (%) = SD/mean × 100
- Intra-Assay: CV<10%
- Inter-Assay: CV<12%
Stability
- The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage conditions.
- To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity, and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.