Chromogranin A ELISA Kit

Catalog No. ABIN2014346

Product Details Chromogranin A ELISA Kit

Target: Chromogranin A (CHGA)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.4-2500 ng/mL

Minimum Detection Limit: 1.4 ng/mL

Application: ELISA

Purpose: This ELISA (enzyme-linked immunosorbent assay) kit is intended forthe quantitative determination of human chromogranin A levels inEDTA-plasma and serum samples. This assay exclusively measureshuman chromogranin A without the high dose hook effect up to1,000,000 ng/ml. This test may be used as an aid for detectingpatients with pheochromocytoma and neuroendocrine tumors(carcinoids).

Brand: EDI™

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Components: 1. Anti-CgA Antibody Coated Microplate
Two vials each containing human chromogranin A in a lyophilized bovine serum-based matrix with a non-azide, non-mercury preservative. Refer to vials for exact concentration range for each control.
Both controls should be stored at 2-8 °C and are stable until the expiration date on the kit box.

Material not included: 1. Precision single channel pipettes capable of delivering 15 µL, 50 µL, 100 µL, and 1000 µL etc.
2. Repeating dispenser suitable for delivering 100 µL.
3. Disposable pipette tips suitable for above volume dispensing.
4. Disposable 12 x 75 mm or 13 x 100 glass or plastic tubes.
5. Disposable plastic 100 mL and 1000 mL bottle with caps.
6. Aluminum foil.
7. Deionized water.
8. Plastic microtiter well cover or polyethylene film.
9. ELISA multichannel wash bottle or automatic (semi-automatic) washing system.
10. Spectrophotometric microplate reader capable of reading absorbance at 450/650 nm or 450/620 nm.

Application Details

Sample Volume: 30 μL

Assay Time: 4 h

Plate: Pre-coated

Protocol: This ELISA is designed, developed and produced for the quantitativemeasurement of human chromogranin A in EDTA-plasma or serumsample. The assay utilizes the two-site sandwich technique withtwo selected antibodies that bind to different epitopes of humanchromogranin A.Assay standards, controls and patient samples are added directly towells of microplate that is coated with a polyclonal chromogranin Aantibody. After the first incubation period, the antibody on the wall ofmicrotiter well captures human chromogranin A in the sample andunbound protein in each microtiter well is washed away. Then ahorseradish peroxidase (HRP)-labeled monoclonal anti-humanchromogranin A antibody is added to each microtiter well and asandwich of monoclonal antibody - human chromogranin A polyclonal antibody is formed. The unbound monoclonal antibody isremoved in the subsequent washing step. For the detection of thisimmunocomplex, the well is incubated with a substrate solution in atimed reaction and then measured in a spectrophotometricmicroplate reader. The enzymatic activity of the immunocomplexbound to the chromogranin A on the wall of the microtiter well isdirectly proportional to the amount of chromogranin A in the sample.A standard curve is generated by plotting the absorbance versus therespective human chromogranin A concentration for each standardwith a 4 parameter curve fit. The concentration of humanchromogranin A in test samples is determined directly from thisstandard curve.

Reagent Preparation:

(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) ELISA Wash Concentrate must be diluted to working solution prior to use. Please see REAGENTS section for details.
(3) Reconstitute all assay standards and controls by adding 0.5 mL of deminerialized water to each vial. Allow the standards and controls to sit undisturbed for 10 minutes, and then mix well by inversions or gentle vortexing. Make sure that all solids are dissolved completely prior to use. These reconstituted standards and controls must be stored at -10 C or below. Do not exceed 3 freeze-thaw cycles.

Sample Collection: EDTA-plasma is the preferred sample for Chromoganin A measurement. Only 30 µL total (15 µL each) of human EDTA-plasma is required. No special preparation of individual is necessary prior to specimen collection. Whole blood should be collected with lavender-top Vacutainer. Separate the plasma from cells by centrifugation (850 ? 1500xg for 10 minutes). The plasma should be separated from the cells within one hour of blood collection and transferred to a clean test tube. Plasma samples should be stored at ? 15 C if the assay is not to be performed within 72 hours. Otherwise, the plasma samples should be stored at room temperature for up to 72 hours. It is important that the plasma samples must not be stored at 2 ?8 C for long term storage. Avoid more than three freeze-thaw cycles of specimen. Serum sample can also be used for chromogranin A measurement. Tests with paired EDTA-plasma and serum sample from same donor shows that serum gives almost the same chromogranin A level as EDTA-plasma by using this ELISA.

Assay Procedure:

(1) Place a sufficient number of antibody-coated microwell strips in a holder to run human chromogranin A standards, controls and unknown samples in duplicate.
(2) Test Configuration (1) Add 200 µL of assay buffer to each well.
(2) Add 15 µL of standards, controls and patient samples into the designated microwells.
(3) Cover the plate with one plate sealer and also with aluminum foil, and incubate plate on an ELISA plate shaker with a shaking rate at 350 rpm to 450 rpm at room temperature for 90 min.
(4) Remove aluminum foil and plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(5) Add 100 µL of Chromogranin A Tracer Antibody to each of the wells.
(6) Cover the plate with the plate sealer and also with aluminum foil, and incubate plate on an ELISA plate shaker with a shaking rate at 350 rpm to 450 rpm at room temperature for 30 min.
(7) Remove aluminum foil and plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(8) Add 100 µL of ELISA HRP Substrate into each of the wells.
(9) Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light. 10) Incubate plate at room temperature for 20 minutes.
(11) Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
(12) Read the absorbance at dual wavelength at 450/650 nm or 450/620 nm within 10 minutes in a microplate reader.

Calculation of Results:

The human chromogranin A concentrations for the controls and patient samples are read directly from the standard curve using their respective corrected absorbance.

Assay Precision: The intra-assay precision is validated by measuring two controls samples in a single assay with 12 replicate determinations. The inter-assay precision is validated by measuring two control samples in duplicate in 16 individual assays.

Restrictions: For Research Use only

Handling

Precaution of Use: The reagents must be used in a professional laboratory environment and are for research use. The source material for reagents containing bovine serum was derived in the contiguous 48 United States. It was obtained only from healthy donor animals maintained under veterinary supervision and found free of contagious diseases. Wear gloves while performing this assay and handle these reagents as if they were potentially infectious. Avoid contact with reagents containing TMB, hydrogen peroxide, or sulfuric acid. TMB may cause irritation to skin and mucous membranes and cause an allergic skin reaction. TMB is a suspected carcinogen. Sulfuric acid may cause severe irritation on contact with skin. Do not get in eyes, on skin, or on clothing. Do not ingest or inhale fumes. On contact, flush with copious amounts of water for at least 15 minutes. Use Good Laboratory Practices.

Storage: 4 °C

Target Details for Chromogranin A

Target: Chromogranin A (CHGA)

Alternative Name: Chromogranin A (CHGA Products)

Synonyms: CGA ELISA Kit, CG-ALPHA ELISA Kit, FSHA ELISA Kit, GPHA1 ELISA Kit, GPHa ELISA Kit, HCG ELISA Kit, LHA ELISA Kit, TSHA ELISA Kit, ChrA ELISA Kit, fj05f09 ELISA Kit, si:dkey-177p2.2 ELISA Kit, wu:fj05f09 ELISA Kit, zgc:101749 ELISA Kit, zgc:56075 ELISA Kit, CHGA ELISA Kit, cgA ELISA Kit, chga ELISA Kit, chromogranin A ELISA Kit, glycoprotein hormones, alpha polypeptide ELISA Kit, uncharacterized CHGA ELISA Kit, chromogranin A S homeolog ELISA Kit, CHGA ELISA Kit, CGA ELISA Kit, Chga ELISA Kit, chga ELISA Kit, chga.S ELISA Kit

Background: Chromogranin A is a 49 kDa acidic protein that consists of 439amino acids encoded on chromosome 14. Chromogranin A has beenidentified in a number of normal and neopastic endocrine tissues. Itis demonstrated that an elevated level of circulating chromogranin Ais a marker for tumors of neuroendocrine origin. However, the mostsignificant clinical use of chromogranin A is related to the diagnosticprocedure in patients with pheochromocytoma. The following is ashort summary of the potential usages of chromogranin A.1. A very sensitive (83 % ) and highly specific (96 %) marker in theevaluation of actual or suspected pheochromocytoma. Drugscommonly employed in the diagnosis or treatment ofpheochromocytoma have little effect on the plasma chromogranin Alevel, which is a great advantage of measuring chromogranin A overcatecholamines.2. To ascertain the source of a tumor. A high chromogranin A levelindicates that the tumor arises from neuroendocrine tissues.3. Endocrine tumors that do not produce their specific hormones, forexample, calcitonin negative but chromogranin A positive C-cellcarcinoma, zero-cell carcinoma, beta-cell carcinoma, parathyroidcarcinoma.

Pathways: Negative Regulation of Hormone Secretion, cAMP Metabolic Process, Regulation of G-Protein Coupled Receptor Protein Signaling