AMH ELISA 试剂盒

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

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

1. picoAMH Calibrators B-F and picoAMH Controls I & II: Tap and reconstitute picoAMH Calibrator B-F and picoAMH Controls I & II each with 1 mL deionized water. Solubilize, mix well and use after reconstitution. Note: In case sensitivity below calibrator B level is desired, dilute reconstituted calibrator B as below.
2. (a) CAL B/2: Mix 150 μL of reconstituted Cal B with 150 μL of Cal A/Sample diluent. (b) CAL B/3: Mix 100 μL of reconstituted Cal B with 200 μL of Cal A/Sample diluent.
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.

• Serum and Lithium heparin plasma is the recommended sample type.
• 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 serum separation products.
• Within two hours after centrifugation, transfer at least 500 μL of cell free sample to a storage tube, vortex and tightly stopper the tube immediately.
• Samples may be stored at 4 °C if assayed within 7 days, 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.

Allow all specimens and reagents to reach room temperature and mix thoroughly by gentle inversion before use. Calibrators, controls, and unknowns should be assayed in duplicate. NOTE: All serum/plasma samples reading higher than the highest calibrator should be diluted as appropriate in the 0 pg/mL Calibrator A/Sample diluent prior to assay. In general, pediatric males (<3yrs) subject samples should be diluted 1 part serum/plasma in 20 parts of calibrator A (10 μL of sample + 190 μL of Cal-124A/Sample diluent) prior to assay in protocol-1 Protocol-1 (Female ≤ 40yrs) EDIIIIIIIT

1. Label the microtitration strips to be used.
2. Add 50 μL of the AMH/MIS Assay Buffer to each well using a repeater pipette.
3. Pipette 100 μL of the reconstituted Calibrator and Controls to the appropriate wells.
4. Pipette 10 μL of samples using precision pipette to the sample designated wells.
5. Pipette 90 μL of Cal-124A/Sample-diluent to the sample added wells.
6. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 3 hrs at room temperature (23 ± 2 °C).
7. Aspirate and wash each strip 5 times with Wash Solution (350 μL/per well) using an automatic microplate washer.
8. Add 100 μL of the Antibody-Biotin Conjugate RTU to each well using a repeater pipette.
9. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 1 hr at room temperature (23 ± 2 °C).
10. Aspirate and wash each strip 5 times with the Wash Solution (350 μL/per well) using an automatic microplate washer.
11. Add 100 μL of the Streptavidin-Enzyme Conjugate-RTU to each well using a repeater pipette.
12. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 30 mins at room temperature (23 ± 2 °C).
13. Aspirate and wash each strip 5 times with the Wash Solution (350 μL/per well) using an automatic microplate washer.
14. Add 100 μL of the TMB chromogen solution to each well using a repeater pipette. Avoid exposure to direct sunlight.
15. Incubate the wells, shaking at 600-800 rpm on an orbital microplate shaker, for 8-12 min at room temperature (23 ± 2 °C). NOTE: Visually monitor the color development to optimize the incubation time.
16. Add 100 μL of the Stopping solution to each well using a repeater pipette. Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to (1) 450 nm, (2) 405 nm and 630 nm (machine blank).
17. IMPORTANT: All diluted specimens should be multiplied by the appropriate dilution factor (i.e. 10) for the final concentration. Alternatively, multiply the calibrators by a factor of 10 and input the corrected calibrator values in the data reduction software of the spectrophotometer prior to reading the assay. NOTE: a) While reading the absorbance of the microtitration well, it is necessary to program the zero calibrator as a "Blank". b) Each lab should establish their extrapolation criteria 1) 450 nm-630 nm at the low end and 405 nm-630 nm at the high end of the curve if needed. c) All diluted samples reading lower than the limit of detection should be run following protocol-2. Protocol 2 (Female age > 40yrs) Females of all ages with diminished ovarian reserve Allow all specimens and reagents to reach room temperature and mix thoroughly by gentle inversion before use. Calibrators, controls, and unknowns should be assayed in duplicate. NOTE: All serum/plasma samples reading higher than the highest calibrator should be diluted as appropriate in the 0 pg/mL Calibrator A/Sample diluent prior to assay.
    1. Label the microtitration strips to be used.
    2. Add 50 μL of the AMH/MIS Assay Buffer to each well using a repeater pipette.
    3. Pipette 100 μL of the reconstituted Calibrator and Controls to the appropriate wells.
    4. Pipette 50 μL of samples using precision pipette to the sample designated wells.
    5. Pipette 50 μL of Cal-124A/Sample-diluent to the sample added wells.
    6. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 3 hrs at room temperature (23 ± 2 °C).
    7. Aspirate and wash each strip 5 times with Wash Solution (350 μL/per well) using an automatic microplate washer.
    8. Add 100 μL of the Antibody-Biotin Conjugate RTU to each well using a repeater pipette.
    9. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 1 hr. at room temperature (23 ± 2 °C).
    10. Aspirate and wash each strip 5 times with the Wash Solution (350 μL/per well) using an automatic microplate washer.
    11. Add 100 μL of the Streptavidin-Enzyme Conjugate-RTU to each well using a repeater pipette.
    12. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 30 mins at room temperature (23 ± 2 °C).
    13. Aspirate and wash each strip 5 times with the Wash Solution (350 μL/per well) using an automatic microplate washer.
    14. Add 100 μL of the TMB chromogen solution to each well using a repeater pipette. Avoid exposure to direct sunlight.
    15. Incubate the wells, shaking at 600-800 rpm on an orbital microplate shaker, for 8-12 min at room temperature (23 ± 2 °C). NOTE: Visually monitor the color development to optimize the incubation time.
    16. Add 100 μL of the Stopping solution to each well using a repeater pipette. Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to to (1) 450 nm, (2) 405 nm and 630nm (machine blank).
    17. IMPORTANT: All diluted specimens should be multiplied by the appropriate dilution factor (i.e. 2) for the final concentration. Alternatively, multiply the calibrators by a factor of 2 and input the corrected calibrator values in the data reduction software of the spectrophotometer prior to reading the assay. NOTE: a) While reading the absorbance of the microtitration well, it is necessary to program the zero calibrator as a "Blank". b) Each lab should establish their extrapolation criteria 1) 450 nm-630 nm at the low end and 405 nm-630 nm at the high end of the curve if needed. c) All diluted samples reading lower than the limit of detection can be run neat (100 μL sample in step-4 and skip step-5) in protocol-2. No calibration factor is required if run neat. Combination Protocol NOTE: Protocol-1 and Protocol-2 can also be performed simultaneously in the same run by marking the sample wells as per the protocol used. The sample results then should be processed as per the protocol by applying the protocol calibration factor.

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. 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 samples.
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 Controls and unknown sample from the calibration curve by matching their mean OD readings with the corresponding AMH concentrations.
5. All serum/plasma samples reading higher than the highest calibrator should be diluted as appropriate in the 0 pg/mL Calibrator A/Sample diluent and re-assayed.
6. Any sample reading lower than the analytical sensitivity should be reported as such.
7. Multiply the diluted sample value by the dilution factor, if required.