Leptin ELISA Kit

Catalog No. ABIN6973331

Product Details Leptin ELISA Kit

Target: Leptin (LEP)

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.068 ng/mL - 50 ng/mL

Minimum Detection Limit: 0.068 ng/mL

Application: ELISA

Purpose: For the quantitative determination of rat leptin (LEP) concentrations in serum, plasma, tissue homogenates.

Sample Type: Plasma, Serum, Tissue Homogenate

Analytical Method: Quantitative

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

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 50 ng/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 200 μL of Sample Diluent into each tube (S0-S6). Use the stock solution to produce a 3-fold dilution series (below). Mix each tube thoroughly before the next transfer. The undiluted Standard serves as the high standard (50 ng/mL). Sample Diluent serves as the zero standard (0 ng/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).

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 Leptin ELISA Kit (ABIN6973331)

Shen, Wei, Li, Qiao, Li: "Curcumin reverses the depressive-like behavior and insulin resistance induced by chronic mild stress." in: Metabolic brain disease, Vol. 32, Issue 4, pp. 1163-1172, (2018) (PubMed).

Zhao, Zhang, Ma, Tian, Shen, Zhou: "A combination of quercetin and resveratrol reduces obesity in high-fat diet-fed rats by modulation of gut microbiota." in: Food & function, Vol. 8, Issue 12, pp. 4644-4656, (2018) (PubMed).

Li, Li, Liu, Wang, Zhang, Li, Yang, Song, Yin, Gao, Cheng, Cai, Tian: "Telmisartan Ameliorates Nephropathy in Metabolic Syndrome by Reducing Leptin Release From Perirenal Adipose Tissue." in: Hypertension (Dallas, Tex. : 1979), Vol. 68, Issue 2, pp. 478-90, (2017) (PubMed).

Haghshenas, Jafari, Ravasi, Kordi, Gilani, Shariatzadeh, Hedayati, Rahimi: "The effect of eight weeks endurance training and high-fat diet on appetite-regulating hormones in rat plasma." in: Iranian journal of basic medical sciences, Vol. 17, Issue 4, pp. 237-43, (2014) (PubMed).

Sun, Song, Tamashiro, Moran, Yan: "Large litter rearing improves leptin sensitivity and hypothalamic appetite markers in offspring of rat dams fed high-fat diet during pregnancy and lactation." in: Endocrinology, Vol. 155, Issue 9, pp. 3421-33, (2014) (PubMed).

Kumar, Kaur: "Intermittent fasting dietary restriction regimen negatively influences reproduction in young rats: a study of hypothalamo-hypophysial-gonadal axis." in: PLoS ONE, Vol. 8, Issue 1, pp. e52416, (2013) (PubMed).

Vinoth, Patel, Lih, Seow, Cao, Meikle: "Appliance-induced osteopenia of dentoalveolar bone in the rat: effect of reduced bone strains on serum bone markers and the multifunctional hormone leptin." in: European journal of oral sciences, Vol. 121, Issue 6, pp. 517-24, (2013) (PubMed).

Tarry-Adkins, Chen, Jones, Smith, Ozanne et al.: "Poor maternal nutrition leads to alterations in oxidative stress, antioxidant defense capacity, and markers of fibrosis in rat islets: potential underlying mechanisms for development of the diabetic ..." in: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 24, Issue 8, pp. 2762-71, (2010) (PubMed).

Target Details for Leptin

Target: Leptin (LEP)

Alternative Name: leptin (LEP Products)

Synonyms: ob ELISA Kit, obese ELISA Kit, LEPD ELISA Kit, OB ELISA Kit, OBS ELISA Kit, leptin ELISA Kit, Lep ELISA Kit, LEP ELISA Kit, lep ELISA Kit

Background:

Abbreviation: LEP

Alias: FLJ94114, OB, OBS, leptin (murine obesity homolog)|leptin (obesity homolog, mouse)|obese, mouse, homolog of|obesity factor

UniProt: P50596

Pathways: JAK-STAT Signaling, AMPK Signaling, Hormone Transport, Peptide Hormone Metabolism, Hormone Activity, Negative Regulation of Hormone Secretion, Regulation of Carbohydrate Metabolic Process, Feeding Behaviour, Monocarboxylic Acid Catabolic Process