GDA ELISA Kit

Catalog No. ABIN5564623

Product Details GDA ELISA Kit

Target: GDA (Guanine Deaminase (GDA))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.25-80 ng/mL

Minimum Detection Limit: 1.25 ng/mL

Application: ELISA

Purpose: The AssayMax™ Human Guanase ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human guanase in plasma, serum, urine, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human guanase in approximately 4 hours. A polyclonal antibody specific for human guanase has been pre-coated onto a 96-well microplate with removable strips. Guanase in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human guanase, which is recognized by a streptavidin-peroxidase (SP) 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, Plasma, Serum, Urine

Analytical Method: Quantitative

Components: Human Guanase Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human guanase. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Guanase Standard: Human guanase in a buffered protein base (80 ng, lyophilized, 2 vials). Biotinylated Human Guanase Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against human guanase (120 l). MIX Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Standard Diluent (1x): A buffered protein base with stabilizer (2 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution 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 30 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. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIX Diluent Concentrate 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C. Human Guanase Standard: Reconstitute the Human Guanase Standard (80 ng) with 0.5 mL of Standard Diluent to generate a 160 ng/mL standard stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution (160 ng/mL) 2-fold with MIX Diluent to produce 80, 40, 20, 10, 5, 2.5, and 1.25 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Aliquot remaining stock solution to limit repeated freeze-thaw cycles. This solution should be stored at -20 °C and used within 48 hours. Standard Point Dilution [Guanase] (ng/mL) P1 1 part Standard (160 ng/mL) + 1 part MIX Diluent 80 P2 1 part P1 + 1 part MIX Diluent 40 P3 1 part P2 + 1 part MIX Diluent 20 P4 1 part P3 + 1 part MIX Diluent 10 P5 1 part P4 + 1 part MIX Diluent 5.0 P6 1 part P5 + 1 part MIX Diluent 2.5 P7 1 part P6 + 1 part MIX Diluent 1.25 P8 MIX Diluent 0.0 5 Biotinylated Human Guanase Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50-fold with MIX Diluent to produce a 1x solution. The undiluted antibody should be stored at -20 °C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent to produce a 1x solution. The undiluted conjugate should be stored at -20 °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 and collect plasma. A 2-fold sample dilution is suggested into MIX Diluent, 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 2-fold sample dilution is suggested into MIX Diluent, 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. Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes. The sample is suggested for use at 1x, 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 2-fold sample dilution is suggested into MIX Diluent, 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. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes at 4 °C to remove debris and collect supernatants. Samples can be stored at -20 °C or below. Avoid repeated freeze-thaw cycles.

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 Guanase Standard or sample to each 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 Guanase Antibody to each 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 1 hour. Wash the microplate as described above. Add 50 l of SP Conjugate to each 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 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 to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 30 minutes or until 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. 6 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. 2 The kit should not be used beyond the expiration date.

Storage: 4 °C,-20 °C

Storage Comment: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store Standard, SP Conjugate, and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Standard Diluent (1x), 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.

Target Details for GDA

Target: GDA (Guanine Deaminase (GDA))

Alternative Name: Guanase (GDA) (GDA Products)

Synonyms: CYPIN ELISA Kit, GUANASE ELISA Kit, NEDASIN ELISA Kit, AU015411 ELISA Kit, AW047581 ELISA Kit, Cypin ELISA Kit, MGC81835 ELISA Kit, zgc:112282 ELISA Kit, AGR_C_4212 ELISA Kit, DDBDRAFT_0169373 ELISA Kit, DDBDRAFT_0230211 ELISA Kit, DDB_0169373 ELISA Kit, DDB_0230211 ELISA Kit, guanine deaminase ELISA Kit, guanine deaminase L homeolog ELISA Kit, short-chain dehydrogenase/reductase sdr ELISA Kit, GDA ELISA Kit, Gda ELISA Kit, gda.L ELISA Kit, gda ELISA Kit, SPO2956 ELISA Kit, Gdia_3192 ELISA Kit, Mchl_2046 ELISA Kit, Mnod_4655 ELISA Kit, guaD ELISA Kit, Dd586_1771 ELISA Kit, Kvar_2512 ELISA Kit, Mrub_1462 ELISA Kit, BC1002_2263 ELISA Kit, Cseg_1079 ELISA Kit, Mesil_1795 ELISA Kit, Trad_2850 ELISA Kit, Mesop_0346 ELISA Kit, gDA ELISA Kit, ML5_2802 ELISA Kit

Background: Guanine deaminase (GDA), also known as guanase, guanine aminase, or guanine aminohydrolase, is a 51 kDa enzyme containing 454 amino acids. GDA catalyzes the hydrolytic deamination of guanine. By producing xanthine and ammonia, this reaction irreversibly eliminates the guanine base from further reutilization as a guanylate nucleotide. The product xanthine is a substrate for xanthine oxidase in the production of uric acid (1-3). GDA regulates the guanine nucleotide pool, promotes microtubule assembly, and is involved in neuronal dendritic branching (4). It may also influence synaptic development and plasticity by regulating postsynaptic protein sorting (5).

Gene ID: 9615

UniProt: Q9Y2T3