NPPA ELISA Kit

Catalog No. ABIN612767

Product Details NPPA ELISA Kit

Target: NPPA (Natriuretic Peptide A (NPPA))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Minimum Detection Limit: 100 pg/mL

Application: ELISA

Purpose: The AssayMax rANP ELISA kit is designed for detection of rat ANP in plasma, serum, tissue extract and cell culture supernatants

Brand: AssayMax

Sample Type: Plasma, Cell Culture Supernatant

Analytical Method: Quantitative

Components: rANP Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against rANP. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. rANP Standard: Rat ANP in a buffered protein base (20 ng, lyophilized). Biotinylated rANP Antibody: A 100-fold biotinylated polyclonal antibody against rANP (80µl). MIx Diluent Concentrate (10x): A 10-fold buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). 1 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 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water.

Application Details

Sample Volume: 50 μL

Assay Time: 4 h

Plate: Pre-coated

Protocol: This assay employs a quantitative sandwich enzyme immunoassay technique that measures rANP in 4 hours. A polyclonal antibody specific for rANP has been pre-coated onto a microplate. The rANP in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for rANP, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute the MIx Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. rANP Standard: Reconstitute the 20 ng of rat ANP Standard with 5 ml of MIx Diluent to generate a standard solution of 4 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the Standard (4 ng/ml) twofold with MIx Diluent to generate 2, 1, 0.5 and 0.25 ng/ml. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [rANP] (ng/ml) P1 1 part Standard (4 ng/ml) 4.000 P2 1 part P1 + 1 part MIx Diluent 2.000 P3 1 part P2 + 1 part MIx Diluent 1.000 P4 1 part P3 + 1 part MIx Diluent 0.500 P5 1 part P4 + 1 part MIx Diluent 0.250 P6 MIx Diluent 0.000 Biotinylated rANP Antibody (100x): Dilute the antibody 1:100 with MIx Diluent. Spin down the Biotinylated Antibody briefly and only dilute the desired amount of the antibody. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C.

Sample Collection: Plasma: Collect plasma using a final concentration of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000x g for 10 minutes and assay undiluted plasma for medium and high level of ANP. Samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. For low level of ANP, please use ANP extraction protocol below. Low Level ANP Extraction Protocol Buffer A: 1% trifluoroacetic acid (TFA, HPLC Grade) in H2O Buffer B: 60% acetonitrile (HPLC Grade) in 1% TFA 1. Acidify the sample with equal amount of Buffer A (1 ml sample: 1 ml Buffer A). Mix and centrifuge samples at 6000 x g for 20 minutes at 4 C. 2. Pack an extraction column using 200 mg of C18 resin. Pre-equilibrate the column with 1 ml of Buffer B once and then with 3 ml of Buffer A three times. 3. Load the acidified plasma solution onto the pre-treated C18 column. 4. Slowly wash the column with 3 ml of Buffer A twice. 5. Elute the peptide slowly with 3 ml of Buffer B once and collect the eluant. 6. Evaporate and dry the eluant in a freeze dryer or use a suitable substitute method. 7. Keep the dried extract at -20 C and perform the assay as early as possible. Reconstitute the dried extract with 200 µl of MIx Diluent before the assay. Check sample pH with pH papers. If sample pH is below 6.5, neutralize the sample with 20 µl of 1M NaH2PO4. If the peptide value exceeds or does not fall in the range of detection, dilute or concentrate the sample accordingly. Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and perform the assay for medium and high level of ANP. Samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. For low level of ANP, please use the extraction protocol as above. Tissue: Extract tissue samples with 0.1 M phosphate-buffered saline (pH7.4) containing 1% Triton x-100 and centrifuge at 14000 x g for 20 min. Collect the supernatant and measure the protein concentration. Dilute 1:20 into MIx diluent and assay. Freeze remaining extract at -20°C or below. 2 Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze- thaw cycles. Dilute samples 1:4 with MIx diluent and assay.

Assay Procedure:

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash four times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and blot it on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated rANP Antibody to each well and incubate for two hours. Wash four times with 200 µL of Wash Buffer. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash four times with 200 µL of Wash Buffer. Add 50 µL of Chromogen Substrate per well and incubate for approximately 10 minutes or till the optimal color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately.

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 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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 4.8 % and 7.1% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: The kit should not be used beyond the expiration date.

Storage: 4 °C/-20 °C

Storage Comment: Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.

References for NPPA ELISA Kit (ABIN612767)

Otsuki, Nakamura, Harada, Yamamoto, Ogino, Morikawa, Ninomiya, Miyagawa, Sawa, Hisatome, Nishimura: "Adipose stem cell sheets improved cardiac function in the rat myocardial infarction, but did not alter cardiac contractile responses to β-adrenergic stimulation." in: Biomedical research (Tokyo, Japan), Vol. 36, Issue 1, pp. 11-9, (2015) (PubMed).

Target Details for NPPA

Target: NPPA (Natriuretic Peptide A (NPPA))

Alternative Name: ANP (NPPA Products)

Synonyms: anp ELISA Kit, ANF ELISA Kit, ANP ELISA Kit, Pnd ELISA Kit, RATANF ELISA Kit, ATFB6 ELISA Kit, CDD-ANF ELISA Kit, PND ELISA Kit, NPPA ELISA Kit, Anf ELISA Kit, atrial natriuretic peptide ELISA Kit, natriuretic peptide A ELISA Kit, natriuretic peptide type A ELISA Kit, anp ELISA Kit, nppa ELISA Kit, NPPA ELISA Kit, Nppa ELISA Kit

Background: Atrial natriuretic peptide (ANP), a 28 amino acids polypeptide, is mainly secreted from the atrium of the heart where it is stored in secretory granules as a 136 amino acids pro-hormone. Upon its secretion, induced by increases in atrial pressure and stretch, the pro-hormone is processed by a serine protease to the active 28 amino acids peptide. The peptide binds with high affinity to the membrane receptor guanylate cyclase GC-A, leading to increased intracellular cGMP levels. Increased ANP plasma level has been identified as predictors of cardiac dysfunction and prognosis in congestive heart failure and ischemic heart disease (3-5). Lower plasma levels of ANP will lead to sodium retention, and an increase in plasma volume, resulting in an increase blood pressure.