AMH ELISA Kit

Catalog No. ABIN3073420

Product Details AMH ELISA Kit

Target: AMH (Anti-Mullerian Hormone (AMH))

Reactivity: Cow

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 37.5-2400 pg/mL

Minimum Detection Limit: 37.5 pg/mL

Application: ELISA

Purpose: The Bovine Anti-Müllerian hormone (AMH) enzyme-linked immunosorbent assay (ELISA) kit provides materials for the quantitative measurement of AMH in bovine EDTA plasma and other biological fluids.

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Sensitivity: 11 pg/mL

Components:

• AMH/MIS Calibrators A / Sample Diluent
• Bovine AMH Calibrator H (Lyophilized)
• Bovine AMH Controls I & II (Lyophilized)
• AMH Coated Microtitration strips
• Assay Buffer
• AMH Biotin Conjugate Ready-To-Use (RTU)
• AMH Streptavidin-Enzyme Conjugate-Ready-to-Use (RTU)
• TMB Chromogen Solution
• Stopping Solution
• Wash Concentrate A

Material not included:

1. Microtitration plate reader capable of absorbance measurement at 450 nm, 405nm and 630 nm.
2. Microplate shaker.
3. Microplate washer.
4. Semi-automated/manual precision pipette to deliver 10-250 μL.
5. Vortex mixer.
6. Deionized water.

Application Details

Sample Volume: 50 μL

Assay Time: 3.5 h

Plate: Pre-coated

Reagent Preparation:

1. Bovine AMH Calibrator H : a) Tap and reconstitute Bovine AMH Calibrator H with 2 mL deionized water. Solubilize for ten minutes, mix well before use. b) Prepare seven polystyrene tubes and label them as Cal A, Cal B, Cal C, Cal D, Cal E, Cal F and Cal G. c) Add 500 μL of AMH Calibrator A/Sample Diluent to each polystyrene tube labeled Cal A-G. d) Add 500 μL of reconstituted AMH Calibrator H (from step a) to the tube labeled Cal G. Vortex and mix the content in the tube thoroughly before the next dilution transfer. e) Add 500 μL of Cal G (from step d) to the tube labeled Cal F. Vortex and mix the content in the tube thoroughly before the next dilution transfer. f) Add 500 μL of Cal F (from step e) to the tube labeled Cal E. Vortex and mix the content in the tube thoroughly before the next dilution transfer. g) Add 500 μL of Cal E (from step f) to the tube labeled Cal D. Vortex and mix the content in the tube thoroughly before use. h) Add 500 μL of Cal D (from step g) to the tube labeled Cal C. Vortex and mix the content in the tube thoroughly before use. i) Add 500 μL of Cal C (from step h) to the tube labeled Cal B. Vortex and mix the content in the tube thoroughly before use. Note: If more sensitivity is desired, Cal B can be further diluted to B/2 and B/4 using Cal A. j) The tube labeled Cal A contains 500 μL AMH Calibrator A/Sample Diluent and has 0 AMH concentrations and should be used as Blank. k) The Calibrators A-H for instance should read as 0.0 pg/mL, 78.1 pg/mL, 156.3 pg/mL, 312.5 pg/mL, 625 pg/mL, 1250 pg/mL, 2500 pg/mL and 5000 pg/mL. Aliquot and freeze the AMH Cal H Stock immediately for multiple uses. Avoid repeated freeze thaws. Frozen aliquots at -20 °C are good for one year. l) The AMH concentration in the Bovine AMH calibrators H is traceable to the manufacturer's working calibrators. Values assigned by other methodologies may be different. Such differences, if present, may be caused by inter-method bias.
2. Bovine AMH Controls I and II: Tap and reconstitute Bovine AMH Controls I and II with 1 mL deionized water. Solubilize for ten minutes, mix well before use.
3. Wash Solution: Dilute wash concentrate 25-fold with deionized water. The wash solution is stable for one month at room temperature when stored in a tightly sealed bottle.
4. Microtitration Wells: Select the number of coated wells required for the assay. The remaining unused wells should be placed in the resealable pouch with a desiccant. The pouch must be resealed to protect from moisture.

Sample Collection:

• EDTA Plasma and serum are the recommended sample types. Other biological fluids may produce different results.
• Sample handling, processing, and storage requirements depend on the brand of blood collection tube that you use. Please reference the manufacturer's instructions for guidance. Each laboratory should determine the acceptability of its own blood collection tubes and EDTA plasma separation products.
• Samples may be stored at 4 °C if assayed within 24 hours, otherwise samples must be stored at -20 °C or -80 °C to avoid loss of bioactivity and contamination.
• Avoid assaying lipemic, hemolyzed or icteric samples.
• Avoid repeated freezing and thawing of samples. Thaw samples no more than 3 times.
• For shipping, place specimens in leak proof containers in biohazard specimen bags with appropriate specimen identification and test requisition information in the outside pocket of the biohazard specimen bag. Follow DOT and IATA requirements when shipping specimens.

Assay Procedure:

Allow all specimens and reagents to reach room temperature and mix thoroughly by gentle inversion before use. Calibrators and unknowns should be assayed in duplicate. NOTE: a) All male samples should be diluted 1:20 in Calibrator A/Sample diluent prior to assay. b) All samples reading higher than the highest calibrator should be mixed and diluted in the 0 pg/mL Calibrator A/Sample diluent prior to assay.

1. Label the microtitration strips to be used.
2. Pipette 50 μL of the Calibrators (Cal A-G, B/2 & B/4), Controls and Unknown sample to the appropriate wells.
3. Add 50 μL of the AMH Assay Buffer to each well using a repeater pipette.
4. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 120 minutes at room temperature (23 ± 2 °C).
5. Aspirate and wash each strip 5 times with Wash Solution (350 μL/per well) using an automatic microplate washer.
6. Add 100 μL of the AMH Antibody-Biotin Conjugate RTU to each well using a repeater pipette.
7. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 60 minutes at room temperature (23 ± 2 °C).
8. Aspirate and wash each strip 5 times with the Wash Solution (350 μL/per well) using an automatic microplate washer.
9. Add 100 μL of the AMH Streptavidin-Enzyme Conjugate-RTU to each well using a repeater pipette.
10. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 30 minutes at room temperature (23 ± 2 °C).
11. Aspirate and wash each strip 5 times with the Wash Solution (350 μL/per well) using an automatic microplate washer.
12. Add 100 μL of the TMB chromogen solution to each well using a repeater pipette. Avoid exposure to direct sunlight.
13. Incubate the wells, shaking at 600-800 rpm on an orbital microplate shaker, for 10-12 min at room temperature (23 ± 2 °C). NOTE: Visually monitor the color development to optimize the incubation time.
14. Add 100 μL of the stopping solution to each well using a repeater pipette. Read the absorbance of the solution in the wells within 20 minutes, using a microplate reader set to 450 nm. NOTE: Zero calibrator should be programmed as "Blank" while reading the optical density. If instrument has a wavelength correction, set the instrument to dual wavelength measurement at 450 nm with background wavelength correction at 630 nm.

Calculation of Results:

NOTE: The results in this package insert were calculated by plotting the log optical density (OD) data on the y-axis and log AMH concentration on X-axis using a cubic regression curve-fit. Alternatively, log vs. log quadratic regression curve-fit can be used. Other data reduction methods may give slightly different results.

1. Optimum results can be obtained at incubation temperature of 23 ± 2 °C.
2. Calculate the mean optical density (OD) for each calibrator, Control, or Unknown.
3. Plot the log of the mean OD readings for each of the Calibrators along the y-axis versus log of the AMH concentrations in pg/mL along the x-axis, using a cubic regression curve-fit.
4. Determine the AMH concentrations of the unknowns from the calibration curve by matching their mean OD readings with the corresponding AMH concentrations.
5. Any sample reading higher than the highest Calibrator should be appropriately diluted with the 0 pg/mL (CAL A / Sample Diluent) and re- assayed.
6. Any sample reading lower than the analytical sensitivity should be reported as such.
7. Multiply the value by a dilution factor, if required.

Assay Precision: Three control samples were assayed in 24 replicates to determine intra-assay precision. At concentrations between 611 and 1259ng/mL, the CV's ranged between 2.5 and 2.9%.

Restrictions: For Research Use only

Handling

Precaution of Use: For in-vitro research use. The following precautions should be observed: a) Follow good laboratory practice. b) Use personal protective equipment. Wear lab coats and disposable gloves when handling immunoassay materials. c) Handle and dispose of all reagents and material in compliance with applicable regulations WARNING: Potential Biohazardous Material This reagent may contain some animal and/or and human source material (e.g. serum or plasma) or materials used in conjunction with human source materials. Handle all reagents and patient samples at a Biosafety Level 2, as recommended for any potentially infectious human material in the Centers for Disease Control/National Institutes of Health manual "Biosafety in Microbiological and Biomedical Laboratories," 5th Edition, 2007. WARNING: Potential Chemical Hazard Some reagents in this kit contain Pro-Clean 400 and Sodium azide as a preservative. Pro-Clean 400 and Sodium azide in concentrated amounts are irritants to skin and mucous membranes. For further information regarding hazardous substances in the kit, please refer to the MSDS.

Storage: 4 °C

Target Details for AMH

Target: AMH (Anti-Mullerian Hormone (AMH))

Alternative Name: AMH (AMH Products)

Synonyms: AMH ELISA Kit, amh ELISA Kit, MIF ELISA Kit, MIS ELISA Kit, anti-Mullerian hormone ELISA Kit, amh ELISA Kit, AMH ELISA Kit, Amh ELISA Kit

Pathways: Negative Regulation of Hormone Secretion