Chromogranin A ELISA Kit

Produktnummer ABIN454721

Produktdetails Chromogranin A ELISA Kit

Target: Chromogranin A (CHGA)

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 78-5000 pg/mL

Untere Nachweisgrenze: 78 pg/mL

Applikation: ELISA

Verwendungszweck: This immunoassay kit allows for the specific measurement of human Chromogranin,CgA concentrations in cell culture supernates, serum, and plasma.

Proben: Cell Culture Supernatant, Serum, Plasma

Analytische Methode: Quantitative

Spezifität: This assay recognizes recombinant and natural human Chromogranin A.

Kreuzreaktivität (Details): No significant cross-reactivity or interference was observed.

Produktmerkmale: Homo sapiens,Human,Chromogranin-A,CgA,Pituitary secretory protein I,SP-I,CHGA

Bestandteile: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A 1×120µl Detection Reagent B 1×120µl Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

Anwendungsinformationen

Probenmenge: 100 μL

Plattentyp: Pre-coated

Protokoll: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Chromogranin A has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Chromogranin A present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for Chromogranin A is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Chromogranin A bound in the initial step. The color development is stopped and the intensity of the color is measured.

Aufbereitung der Reagenzien:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution 3 produces a stock solution of 500ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (500 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

Probennahme: Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.

Testdurchführung:

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:
1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming. 4
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

Ergebnisberechnung:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Chromogranin A concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Details zu Chromogranin A

Target: Chromogranin A (CHGA)

Andere Bezeichnung: CHGA (CHGA Produkte)

Synonyme: 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

Hintergrund: Chromogranin A (CgA) is a glycoprotein found in neuroendocrine cells and may be useful as a tumor marker for neuroendocrine tumors. Elevated circulating chromogranin A (CgA) levels are found in neuroendocrine tumors (NETs), but the diagnostic usefulness of this marker is still debatable. Research demonstrates that higher CgA levels associate with metastatic disease, confirming that CgA levels can provide a helpful practical biochemical marker for the clinical management of NETs, but with low sensitivity and specificity. Human chromogranin A is 439 amino acid protein with a molecular weight of 49 kilodalton. It is a secretory protein that is produced by the dense-core vesicles of neuroendocrine cells. The physiologic role of this protein has not been fully elucidated, but it has been hypothesized that it may serve as a precursor to other biologically active peptides or may serve a function in the intracellular production of hormones and neuropeptides. Chromogranin A has been used as an immunohistochemical marker for normal and neoplastic neuroendocrine tissues. Elevated plasma concentrations have been demonstrated in patients with tumors of endocrine origin and levels have been shown to correlate with tumor volume. Several studies suggest that measurement of plasma chromogranin A may be a valuable diagnostic in subjects with tumors such as pheochromocytoma, carcinoid tumor, neuroblastoma, and small cell lung carcinoma. Neuroendocrine tumors that secrete no other hormone markers, referred to as “nonfunctioning” tumors, can sometimes be detected serologically because they retain the ability to secrete chromogranin A. Chromogranin A is more stable than serotonin, making its measurement a useful alternative for the detection of carcinoid tumor. Elevated levels of chromogranin A in patients with prostate cancer have been shown to correlate with poor prognosis.

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