AMH ELISA Kit

Catalog No. ABIN3073426

Human AMH ELISA Kit Colorimetric assay for quantification of Human AMH.

Quick Overview for AMH ELISA Kit (ABIN3073426)

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

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.11-13 ng/mL

Application: ELISA

Sample Type: Dried Blood Spot (DBS)

Product Details AMH ELISA Kit

Minimum Detection Limit: 0.11 ng/mL

Purpose: The Anti-Müllerian hormone (DBS AMH) enzyme linked immunosorbent assay (ELISA) kit provides materials for the quantitative measurement of AMH in human dried blood spot. This kit is intended for laboratory research use only and is not for use in diagnostic or therapeutic procedures.

Analytical Method: Quantitative

Sensitivity: 0.035 ng/mL

Components:

• AMH Calibrator A
• picoAMH Calibrators B thru F (Lyophilized)
• picoAMH Controls I & II (Lyophilized)
• AMH/MIS Coated Microtitration strips
• AMH Extraction Buffer/sample diluent
• picoAMH Biotin Conjugate Ready-To-Use (RTU)
• picoAMH Streptavidin-Enzyme Conjugate-Ready-to-Use (RTU)
• TMB Chromogen Solution
• Stopping Solution
• Wash Concentrate A
• DBS AMH Calibration Card

Material not included:

1. Microplate reader capable of absorbance measurement at 450 nm, 405nm and 630 nm.
2. Microplate orbital shaker.
3. Microplate washer.
4. Semi-automated/manual precision pipette to deliver 2-250 μL.
5. Vortex mixer.
6. Deionized water.
7. Disposable 12 x 75 mm culture tubes.
8. Tight fitting 12 x 75 mm tube racks.
9. DBS 5/16" (7.937) round puncher. For manual punching Punchline catalog number 53700 from McGill incorporated can be used.

Application Details

Sample Volume: 50 μL

Assay Time: 3.5 h

Plate: Pre-coated

Reagent Preparation:

1. Intact IGFBP-4 calibrators B-F and Intact IGFBP-4 Controls I & II: Tap and reconstitute IGFBP-4 Calibrator B-F and IGFBP-4 Controls I & II each with 0.5 mL deionized water. Solubilize, mix well and use after reconstitution.
2. 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.
3. 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.
4. Intact IGFBP-4 Antibody-Biotin Conjugate Solution: The IGFBP-4 Antibody-Biotin Conjugate Concentrate should be diluted at a ratio of 1 part conjugate to 50 parts of IGFBP-4 Assay Buffer, according to the number of wells used. If an entire plate is to be used pipet exactly 220 μL of the Concentrate into 11 mL of the buffer.

Sample Collection: Dried blood spot is the recommended sample type.

• Use with capillary blood samples collected and dried on filter paper (Ahlstrom 226 or Whatman 903 (Protein Saver Card) according to the standard procedures established for blood collection on filter paper.
• Wipe away the first blood drop and apply surface of the first filter pape circle to the next large drop of blood, allowing the blood to fill and completely saturate the circle. Note: Alternatively, the filter paper can be spotted by adding 60 μL of whole blood from Li-Heparin or K2- EDTA tubes.
• Never use the front as well as back of the paper to fill the circle.
• Fill at least two circles and if possible all circles with blood.
• After collection, dry the blood impregnated filter papers for 1- 2 hours in a horizontal position at room temperature.
• The dried filter paper blood spots should be stored in a low permeability re- sealable pouch at 2-8 °C with a desiccant for up to 1 week or frozen at -20 °C or lower for up to 3 months.

Assay Procedure:

Allow all specimens and reagents to reach room temperature (23 ± 2 °C) and mix thoroughly by gentle inversion before use. Calibrators, controls, and unknowns should be assayed in duplicate. NOTE: All extracted samples reading higher than the highest calibrator should be diluted in the DBS AMH Extraction buffer prior to assay.

1. Label the microtitration strips to be used.
2. Pipette 100 μL of the reconstituted Calibrators and Controls to the appropriate wells and add 50 μL of the DBS AMH Extraction Buffer to calibrators and controls wells using a repeater pipette.
3. Pipette 150 μL of the extracted DBS samples (see DBS extraction procedure) to the appropriate wells. Note: Do not add DBS AMH Extraction Buffer to the extracted sample wells.
4. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 3 hrs at room temperature.
5. Aspirate and wash each strip 5 times with Wash Solution using an automatic microplate washer.
6. Add 100 μL of the 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 1 hr at room temperature.
8. Aspirate and wash each strip 5 times with the Wash Solution using an automatic microplate washer.
9. Add 100 μL of the 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.
11. Aspirate and wash each strip 5 times with the Wash Solution using an automatic microplate washer.
12. Add 100 μL of the TMB chromogen solution to each well using a precision pipette. Avoid exposure to direct sunlight.
13. Incubate the wells, shaking at 600-800 rpm on an orbital microplate shaker, for 8-12 min at room temperature. NOTE: Visually monitor the color development to optimize the incubation time.
14. Add 100 μL of the stopping solution to each well using a precision pipette. Read the absorbance of the solution in the wells within 20 minutes, using a microplate reader set to 450 nm.

Calculation of Results:

NOTE: The results in this package insert were calculated by plotting the data on a log vs. log scale using a cubic regression curve-fit. 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
3. Plot the log of the mean OD readings for each of the Calibrators along the y-axis versus log of the sample AMH concentrations in ng/mL along the x- axis, using a cubic regression curve-fit.
4. Determine the AMH concentrations of the Controls and unknowns from the calibration curve by matching their mean OD readings with the corresponding AMH calibrator concentrations.
5. Any sample reading higher than the highest Calibrator should be appropriately diluted with DBS AMH extraction buffer 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.

Restrictions: For Research Use only

Handling

Precaution of Use: For Research Use Only. Not for use in diagnostic procedures. 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 human source material (e.g. serum) 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

Pathways: Negative Regulation of Hormone Secretion