SERPINA3 ELISA Kit

Catalog No. ABIN5564609

Product Details SERPINA3 ELISA Kit

Target: SERPINA3 (serpin Peptidase Inhibitor, Clade A (Alpha-1 Antiproteinase, Antitrypsin), Member 3 (SERPINA3))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.125-72 ng/mL

Minimum Detection Limit: 1.125 ng/mL

Application: ELISA

Purpose: The AssayMax™ Human alpha-1-Antichymotrypsin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of AACT in human plasma, serum, urine, milk, saliva, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures AACT in approximately 4 hours. A polyclonal antibody specific for AACT has been pre-coated onto a 96-well microplate with removable strips. Human AACT in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for AACT, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Brand: AssayMax™

Sample Type: Cell Culture Cells, Cerebrospinal Fluid, Milk, Plasma, Saliva, Serum, Urine

Analytical Method: Quantitative

Components: Human alpha-1-Antichymotrypsin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human AACT. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human alpha-1-Antichymotrypsin Standard: Human AACT in a buffered protein base (288 ng, lyophilized). Biotinylated Human alpha-1-Antichymotrypsin Antibody (50x): A 50- fold biotinylated polyclonal antibody against human AACT (120 l). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Material not included: Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)

Application Details

Assay Time: 4 h

Plate: Pre-coated

Protocol:

• Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 1 hour.
• Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 12 minutes.
• Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8 °C.

Sample Collection: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes. A 20000-fold sample dilution is suggested into EIA Diluent or within the range of 5000x - 100000x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes, and remove serum. A 20000-fold sample dilution is suggested into EIA Diluent or within the range of 5000x - 100000x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris and collect supernatants. Store the remaining samples at -20 °C or below. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 5-fold sample dilution is suggested into EIA Diluent or within the range of 2x - 50x, however, user should determine optimal dilution factor depending on application needs. Undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze- thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 1000-fold sample dilution is suggested into EIA Diluent or within the range of 100x - 10000x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 20-fold sample dilution is suggested into EIA Diluent or within the range of 2x - 100x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. CSF: Collect cerebrospinal fluid (CSF) using sample pot. Centrifuge samples at 3000 x g for 10 minutes. A 100-fold sample dilution is suggested into EIA Diluent or within the range of 10x - 1000x, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -80 °C for up to 3 months. Avoid repeated freeze-thaw cycles. 4 Refer to Sample Dilution Guidelines below for further instruction. Guidelines for Dilutions of 100-fold or Greater (for reference only, please follow the insert for specific dilution suggested) 100x 10000x A) 4 μL sample: 396 μL buffer (100x) = 100-fold dilution Assuming the needed volume is less than or equal to 400 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) = 10000-fold dilution Assuming the needed volume is less than or equal to 400 μL. 1000x 100000x A) 4 μL sample : 396 μL buffer (100x) B) 24 μL of A : 216 μL buffer (10x) = 1000-fold dilution Assuming the needed volume is less than or equal to 240 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) C) 24 μL of B : 216 μL buffer (10x) = 100000-fold dilution Assuming the needed volume is less than or equal to 240 μL.

Assay Procedure:

Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human alpha-1-Antichymotrypsin Standard or sample per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human alpha-1-Antichymotrypsin Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 1 hour. Wash the microplate as described above. 6 Add 50 l of Streptavidin-Peroxidase Conjugate per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 l of Chromogen Substrate per well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 12 minutes or till the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

Calculation of Results:

• Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
• To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
• Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this insert. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date. 2

Storage: 4 °C,-20 °C

Storage Comment: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Target Details for SERPINA3

Target: SERPINA3 (serpin Peptidase Inhibitor, Clade A (Alpha-1 Antiproteinase, Antitrypsin), Member 3 (SERPINA3))

Alternative Name: Alpha-1-Antichymotrypsin (AACT) (SERPINA3 Products)

Synonyms: SERPINA3-1 ELISA Kit, Pi2 ELISA Kit, AACT ELISA Kit, ACT ELISA Kit, GIG25 ELISA Kit, A1AT ELISA Kit, PI ELISA Kit, alpha-1-PI ELISA Kit, AAT ELISA Kit, Pi ELISA Kit, Spi1 ELISA Kit, SERPINA3-5 ELISA Kit, serpina3 ELISA Kit, serpin A3-5 ELISA Kit, serpin family A member 3 ELISA Kit, alpha-1-antiproteinase F precursor ELISA Kit, serpin family A member 1 ELISA Kit, serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5 ELISA Kit, serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 ELISA Kit, serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 L homeolog ELISA Kit, LOC396684 ELISA Kit, SERPINA3 ELISA Kit, LOC100008973 ELISA Kit, SERPINA1 ELISA Kit, Serpina1 ELISA Kit, SPIA5 ELISA Kit, serpina3.L ELISA Kit

Background: Alpha-1-antichymotrypsin (AACT, ACT, Serpin A3, Cell growth-inhibiting gene 24/25 protein) is a member of the serine proteinase inhibitor (serpin) family. It acts as an inhibitor by cleaving proteinases such as cathepsin G and mast cell chymases. In this way, AACT protects some tissues, such as the lower respiratory tract, from damage by proteolytic enzymes (1).

Gene ID: 12

UniProt: P01011