PAPPA ELISA Kit

Catalog No. ABIN3073448

Product Details PAPPA ELISA Kit

Target: PAPPA (Pregnancy-Associated Plasma Protein A, Pappalysin 1 (PAPPA))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 51-4100 ng/mL

Minimum Detection Limit: 51 ng/mL

Application: ELISA

Purpose: The PAPP-A enzyme linked immunosorbent assay (ELISA) kit provides materials for the quantitative measurement of PAPP-A in serum and other biological fluids. This kit is intended for laboratory research use only and is not for use in diagnostic or therapeutic procedures.

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Sensitivity: 10.10 ng/mL

Components:

• PAPP-A Calibrators A-F (Lyophilized)
• PAPP-A Controls (Lyophilized)
• Anti-PAPP-A Antibody Coated Microtitration Strips
• PAPP-A Assay Buffer
• PAPP-A Conjugate Diluent
• PAPP-A Antibody-Enzyme Conjugate Concentrate
• TMB Chromogen Solution
• Wash Concentrate A
• Stopping Solution

Material not included:

1. Microtitration plate reader capable of absorbance measurement at 450 nm, 405nm and 630 nm.
2. Microtitration orbital plate shaker.
3. Microtitration plate washer.
4. Semi-automated/manual precision pipette to deliver 10-250 μL.
5. Vortex mixer.
6. Deionized water.
7. Disposable 12 x 75 mm culture tubes.
8. Tight fitting 12 x 75 mm tube racks.

Application Details

Sample Volume: 50 μL

Assay Time: 3 h

Plate: Pre-coated

Reagent Preparation:

1. PAPP-A Calibrators A-F and PAPP-A Controls I & II: Tap and reconstitute PAPP-A Calibrator A-F and PAPP-A Controls I & II each with 1 mL deionized water. Solubilize, mix well and use after reconstitution.
2. Sample Diluent: Wash Concentrate A should be diluted at a ratio of 1 part into 24 parts of the deionized water, according to the number of wells used. For an entire plate, pipet exactly 10 mL of the Wash Concentrate A into 240 mL of the deionized water, mix well and label as 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. PAPP-A Antibody-Enzyme Conjugate Solution: The PAPP-A Antibody-Enzyme Conjugate Concentrate should be diluted at a ratio of 1 part into 50 parts of the PAPP-A conjugate diluent, according to the number of wells used. For an entire plate, pipet exactly 220 μL of the Antibody-Enzyme Conjugate Concentrate into 11 mL of the PAPP-A Conjugate Diluent. NOTE: The antibody-enzyme conjugate concentrate should be freshly diluted 10-15 minutes prior to use.
5. 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:

• Serum 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.
• 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 shippig, 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 samples and reagents to reach room temperature. Mix reagents thoroughly by gentle inversion before use. After reconstitution of reagents, mix thoroughly, avoiding foam. Calibrators, controls and samples should be assayed in duplicate.

1. Reconstitute PAPP-A Calibrator A-F and PAPP-A Controls I & II each with 1 mL deionized water. Solubilize for 10 minutes, Mix well.
2. Mark the microtitration strips to be used.
3. Pipet 50 μL of the calibrators, controls and unknown diluted samples (as described in the Sample Preparation section) to the appropriate wells.
4. Add 50 μL of the PAPP-A Assay Buffer to each well using a repeater pipette.
5. Incubate the wells, shaking at 600-800 rpm on an orbital microplate shaker, for 2 hours at room temperature (23 ± 2 °C).
6. Prepare the enzyme conjugate solution by diluting the antibody- enzyme conjugate concentrate with the PAPP-A conjugate diluent as described under the "Preparation of Reagents" section of this package insert.
7. Aspirate and wash each well 5 times with the wash solution (350 μL/per well) using an automatic microplate washer.
8. Add 100 μL of the antibody-enzyme conjugate solution to each well using a repeater pipette.
9. Incubate the wells, shaking at 600-800 rpm on an orbital microplate shaker, for 1 hour at room temperature (23 ± 2 °C).
10. Aspirate and wash each well 5 times with the wash solution (350 μL/per well) using an automatic microplate washer.
11. Add 100 μL of the TMB chromogen solution to each well using a repeater pipette. Avoid direct exposure to heat and sunlight.
12. Incubate the wells, shaking at 600-800 rpm on an orbital microplate shaker, for 8-10 min at room temperature (23 ± 2 °C). NOTE: Visually monitor the color development to optimize the incubation time.
13. Add 100 μL of the stopping solution to each well using a repeater pipette.
14. 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 PAPP-A 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 OD for each calibrator, control or diluted sample.
3. Plot the log of the mean OD readings for each of the Calibrators along the y-axis versus log of the PAPP-A concentrations in ng/mL on the x-axis, using a cubic regression curve fit. For reporting the PAPP-A concentrations in mIU/mL use the following conversion factor: 1 ng/mL (native htPAPP-A) = 0.00256 mIU/mL
4. Determine the PAPP-A concentrations of the controls and samples from the calibration curve by matching their mean OD readings with the corresponding PAPP-A concentrations.
5. Any sample reading higher than the highest calibrator should be appropriately diluted using sample diluent and re-assayed.
6. Any sample reading lower than the analytical sensitivity should be reported as such.

Assay Precision: Reproducibility of the PAPP-A ELISA assay was determined using three serum pools for a total of 12 assays. At concentrations between 369.8 and 1184.1 ng/mL, the CV's ranged between 2.5 and 3.4%.

Restrictions: For Research Use only

Handling

Precaution of Use: For Research Use Only. 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 as a preservative. Pro-Clean 400 and peroxide 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 PAPPA

Target: PAPPA (Pregnancy-Associated Plasma Protein A, Pappalysin 1 (PAPPA))

Alternative Name: PAPP-A (PAPPA Products)

Synonyms: 8430414N03Rik ELISA Kit, IGFBP-4ase ELISA Kit, PAG1 ELISA Kit, PAPP-A ELISA Kit, ASBABP2 ELISA Kit, DIPLA1 ELISA Kit, PAPA ELISA Kit, PAPPA1 ELISA Kit, Pappa ELISA Kit, pappalysin 1 ELISA Kit, pregnancy-associated plasma protein A ELISA Kit, PAPPA ELISA Kit, Pappa ELISA Kit, Pappa1 ELISA Kit