Ghrelin ELISA Kit

Catalog No. ABIN6974068

Product Details Ghrelin ELISA Kit

Target: Ghrelin (GHRL)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.625 pg/mL - 40 pg/mL

Minimum Detection Limit: 0.625 pg/mL

Application: ELISA

Purpose: For the quantitative determination of endogenic human ghrelin (GHRL) concentrations in serum, plasma, cell culture supernates, tissue homogenates.

Sample Type: Cell Culture Supernatant, Plasma, Serum, Tissue Homogenate

Analytical Method: Quantitative

Specificity: This assay has high sensitivity and excellent specificity for detection of human GHRL. No significant cross-reactivity or interference between human GHRL and analogues was observed. Note: Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between human GHRL and all the analogues, therefore, cross reaction may still exist.

Sensitivity: 0.156 pg/mL

Components:

• Assay plate
• Standard
• HRP-avidin (100 x concentrate)
• Biotin-antibody (100 x concentrate)
• Sample Diluent
• HRP-avidin Diluent
• Biotin-antibody Diluent
• Wash Buffer (25 x concentrate)
• TMB Substrate
• Stop Solution
• Adhesive Strip

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Sample Volume: 100 μL

Assay Time: 1 - 4.5 h

Plate: Pre-coated

Protocol:

1. Prepare reagents, samples and standards as instructed.
2. Add 100 µL standard or sample to each well. Incubate 2 hours at 37 °C.
3. Remove the liquid of each well, don't wash.
4. Add 100 µL Biotin-antibody (1x) to each well. Incubate 1 hour at 37 °C.
5. Aspirate and wash 3 times.
6. Add 100 µL HRP-avidin (1x) to each well. Incubate 1 hour at 37 °C
7. Aspirate and wash 5 times.
8. Add 90 μL of TMB Substrate to each well. Incubate for 15-30 minutes at 37 °C. Protect from light.
9. Add 50 µL Stop Solution to each well. Read at 450 nm within 5 minutes.

Reagent Preparation:

1. Biotin-antibody (1x) - Centrifuge the vial before opening. Biotin-antibody requires a 100-fold dilution. A suggested 100-fold dilution is 10 μL of Biotin-antibody + 990 μL of Biotin-antibody Diluent.
2. HRP-avidin (1x) - Centrifuge the vial before opening. HRP-avidin requires a 100-fold dilution. A suggested 100-fold dilution is 10 μL of HRP-avidin + 990 μL of HRP-avidin Diluent.
3. Wash Buffer (1x) - If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 mL of Wash Buffer (1 x).
4. Standard Centrifuge the standard vial at 6000-10000rpm for 30s. Reconstitute the Standard with 1.0 mL of Sample Diluent. Do not substitute other diluents. This reconstitution produces a stock solution of 40 pg/mL. Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Pipette 250 μL of Sample Diluent into each tube (S0-S6). Use the stock solution to produce a 2-fold dilution series (below). Mix each tube thoroughly before the next transfer. The undi luted Standard serves as the high standard (40 pg/mL). Sample Diluent serves as the zero standard (0 pg/mL).

Note:

• Kindly use graduated containers to prepare the reagent. Please don't prepare the reagent directly in the Diluent vials provided in the kit.
• Bring all reagents to room temperature (18-25 °C) before use for 30 min.
• Prepare fresh standard for each assay. Use within 4 hours and discard after use.
• Making serial dilution in the wells directly is not permitted.
• Please carefully reconstitute Standards according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10 μL for once pipetting.
• Distilled water is recommended to be used to make the preparation for reagents. Contaminated water or container for reagent preparation will influence the detection result.

Sample Preparation:

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturationmay occur in these samples, leading to false results. Samples should therefore be stored for a short periodat 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thawcycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged toremove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determinecompatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibilityof causing a deviation due to the introduced chemical substance.The recommended dilution factor is for reference only.
• Please estimate the concentration of the samples before performing the test. If the values are not in therange of the standard curve, the optimal sample dilution for the particular experiment has to be determined.Samples should then be diluted with PBS (pH =7.0-7.2).

Note:

Recommend to dilute the serum or plasma samples with Sample Diluent (1:200) before test. The suggested 200-fold dilution can be achieved by adding 5 μL sample to 45 μL of Sample Diluent. Complete the 200-fold dilution by adding15 μL of this solution to 285 μL of Sample Diluent. The recommended dilution factor is for reference only. The optimal dilution factor should be determined by users according to their particular experiments.

Assay Precision: Intra-assay Precision (Precision within an assay): CV%<8% Three samples of known concentration were tested twenty times on one plate to assess.
Inter-assay Precision (Precision between assays): CV%<10% Three samples of known concentration were tested in twenty assays to assess.

Restrictions: For Research Use only

Handling

Storage: 4 °C,-20 °C

Storage Comment: Unopened kit Store at 2 - 8°C. Do not use the kit beyond the expiration date. May be stored for up to 1 month at 2 - 8°C. Coated assay Try to keep it in a sealed aluminum foil bag, plate and avoid the damp. Standard May be stored for up to 1 month at 2 - 8° C. If Biotin-antibody don't make recent use, better keep it store at HRP-avidin -20°C. Biotin-antibody Diluent Opened kit HRP-avidin Diluent Sample May be stored for up to 1 month at 2 - 8°C. Diluent Wash Buffer TMB Substrate Stop Solution *Provided this is within the expiration date of the kit.

Expiry Date: 6 months

References for Ghrelin ELISA Kit (ABIN6974068)

Casajoana, Pujol, Garcia, Elvira, Virgili, de Oca, Duran, Fernández-Veledo, Vendrell, Vilarrasa: "Predictive Value of Gut Peptides in T2D Remission: Randomized Controlled Trial Comparing Metabolic Gastric Bypass, Sleeve Gastrectomy and Greater Curvature Plication." in: Obesity surgery, Vol. 27, Issue 9, pp. 2235-2245, (2018) (PubMed).

Yannakoulia, Anastasiou, Zachari, Sidiropoulou, Katsaounou, Tenta: "Acute effect of smoking and smoking abstinence on energy intake and appetite-related hormones blood concentrations." in: Physiology & behavior, Vol. 184, pp. 78-82, (2018) (PubMed).

Vilarrasa, de Gordejuela, Casajoana, Duran, Toro, Espinet, Galvao, Vendrell, López-Urdiales, Pérez, Pujol: "Endobarrier® in Grade I Obese Patients with Long-Standing Type 2 Diabetes: Role of Gastrointestinal Hormones in Glucose Metabolism." in: Obesity surgery, Vol. 27, Issue 3, pp. 569-577, (2017) (PubMed).

Toru, Ayada, Genç, ?ahin, Ar?k, Acat, Bulut, Çetinkaya: "Visfatin and ghrelin: can they be forthcoming biomarkers or new drug targets for asthma?" in: International journal of clinical and experimental medicine, Vol. 8, Issue 4, pp. 6257-61, (2015) (PubMed).

Zhang, Xing, Cui, Wang, Xie, Zhang, Zhang, He, Lyu, Qiao, Chen, Jiang: "Mitochondria apoptosis pathway synergistically activated by hierarchical targeted nanoparticles co-delivering siRNA and lonidamine." in: Biomaterials, Vol. 61, pp. 178-89, (2015) (PubMed).

Emami, Nazem, Hedayati: "Is association between thyroid hormones and gut peptides, ghrelin and obestatin, able to suggest new regulatory relation between the HPT axis and gut?" in: Regulatory peptides, Vol. 189, pp. 17-21, (2014) (PubMed).

Target Details for Ghrelin

Target: Ghrelin (GHRL)

Alternative Name: ghrelin/obestatin prepropeptide (GHRL Products)

Synonyms: 2210006E23Rik ELISA Kit, Ghr ELISA Kit, MTLRP ELISA Kit, MTLRPAP ELISA Kit, m46 ELISA Kit, Gh1-1 ELISA Kit, GRHL ELISA Kit, MTLRP-AP ELISA Kit, GHR ELISA Kit, ghrelin ELISA Kit, ghrelin and obestatin prepropeptide ELISA Kit, ghrelin/obestatin prepropeptide ELISA Kit, Ghrl ELISA Kit, GHRL ELISA Kit, ghrl ELISA Kit

Background:

Abbreviation: GHRL

Alias: MTLRP, obestatin, OTTHUMP00000207794|OTTHUMP00000207795|OTTHUMP00000207796|OTTHUMP00000207798|ghrelin, growth hormone secretagogue receptor ligand|ghrelin/obestatin|ghrelin/obestatin preprohormone|m

UniProt: Q9UBU3

Pathways: Positive Regulation of Peptide Hormone Secretion, Hormone Transport, Peptide Hormone Metabolism, Negative Regulation of Hormone Secretion, Synaptic Membrane, Feeding Behaviour