Rat GHRL / Ghrelin ELISA Kit , High Specificity ELISA Sandwich Assay Kit

96wells Rat Ghrelin GHRL ELISA Kit Specificity and sensitivity

Cat.No E0896Ra

Standard Curve Range: 40ng/L - 12000ng/L

Sensitivity: 20.56ng/L

​Size: 96 wells

Storage: Store the reagents at 2-8°C. For over 6-month storage refer to the expiration date keep it at -20°C. Avoid repeated thaw cycles. If individual reagents are opened it is recommended that the kit be used within 1 month.

* This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.

Precision

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.

CV(%) = SD/mean x 100

Intra-Assay: CV<8%

Inter-Assay: CV<10%

Intended Use

This sandwich kit is for the accurate quantitative detection of Rat Ghrelin (also known as GHRL) in serum, plasma, cell culture supernates, cell lysates, tissue homogenates.

Assay Principle

This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Rat GHRL antibody. GHRL present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Rat GHRL Antibody is added and binds to GHRL in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated GHRL antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Rat GHRL. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.

Specimen Collection

Serum Allow serum to clot for 10-20 minutes at room temperature. Centrifuge at 2000-3000 RPM for 20 minutes.

Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C within 30 minutes of collection.

Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for approximately 20 minutes. When collecting pleuroperitoneal fluid and cerebrospinal fluid, please follow the procedures above-mentioned.

Cell Culture Supernatant Collect by sterile tubes when examining secrete components. Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect the supernatants carefully. When examining the components within the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge at 2000-3000 RPM for approximately 20 minutes.

Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly and weigh before homogenization. Mince tissues and homogenize them in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20 minutes.

Reagent Preparation

All reagents should be brought to room temperature before use.

Standard Reconstitute the 120μl of the standard (12800ng/L) with 120μl of standard diluent to generate a 6400ng/L standard stock solution. Allow the standard to sit for 15 mins with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the standard stock solution (6400ng/L) 1:2 with standard diluent to produce 3200ng/L, 1600ng/L, 800ng/L and 400ng/L solutions. Standard diluent serves as the zero standard(0 ng/L). Any remaining solution should be frozen at -20°C and used within one month. Dilution of standard solutions suggested are as follows:

Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or distilled water to yield 500 ml of 1x Wash Buffer. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved.

Summary

1. Prepare all reagents, samples and standards.
2. Add sample and ELISA reagent into each well. Incubate for 1 hour at 37°C.

3. Wash the plate 5 times.

4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.

5. Add stop solution and color develops.

6. Read the OD value within 10 minutes.

References

Wei W., Wang G., Qi X., Englander E.W., Greeley G.H. Jr
Endocrinology 146:1611-1625(2005)