Shanghai Korain Biotech Co., Ltd
Shanghai Korain Biotech Co Ltd
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96 Wells Customized Human Corticotropin Releasing Hormone CRH ELISA Kit
Cat.No E1000Hu
Standard Curve Range: 1ng/ml - 40ng/ml
Sensitivity: 0.53ng/ml
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.
Assay Principle
This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with human CRH antibody. CRH present in the sample is added and binds to antibodies coated on the wells. And then biotinylated human CRH Antibody is added and binds to CRH in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated CRH 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 human CRH. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.
Reagent Provided
| Components | Quantity |
| Standard Solution (40ng/ml) | 0.5ml x1 |
| Pre-coated ELISA Plate | 12 * 8 well strips x1 |
| Standard Diluent | 3ml x1 |
| Streptavidin-HRP | 6ml x1 |
| Stop Solution | 6ml x1 |
| Substrate Solution A | 6ml x1 |
| Substrate Solution B | 6ml x1 |
| Wash Buffer Concentrate (30x) | 20ml x1 |
| Biotinylated human CRH Antibody | 1ml x1 |
| User Instruction | 1 |
| Plate Sealer | 2 pics |
| Zipper bag | 1 pic |
Reagent Preparation
All reagents should be brought to room temperature before use.Standard Reconstitute the 120μl of the standard (40ng/ml) with 120μl of standard diluent to generate a 20ng/ml 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 (20ng/ml) 1:2 with standard diluent to produce 10ng/ml, 5ng/ml, 2.5ng/ml and 1.25ng/ml solutions. Standard diluent serves as the zero standard(0 ng/ml). Any remaining solution should be frozen at -20℃ and used within one month. Dilution of standard solutions suggested are as follows:
| 20ng/ml | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
| 10ng/ml | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
| 5ng/ml | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
| 2.5ng/ml | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
| 1.25ng/ml | Standard No.1 | 120μl Standard No.2 + 120μl Standard Diluent |
| Standard Concentration | Standard No.5 | Standard No.4 | Standard No.3 | Standard No.2 | Standard No.1 |
| 40ng/ml | 20ng/ml | 10ng/ml | 5ng/ml | 2.5ng/ml | 1.25ng/ml |
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.
Calculation of Result
Construct a standard curve by plotting the average OD for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis and draw a best fit curve through the points on the graph. These calculations can be best performed with computer-based curve-fitting software and the best fit line can be determined by regression analysis.