ABSBio™ Mouse/Rat Insulin ELISA kit is used for the quantification of insulin in mouse and rat sera.

The kit uses a double‐antibody sandwich enzyme‐linked immunosorbent assay (ELISA) to analyze the level of insulin in samples. The micro-plate is pre-coated with a monoclonal antibody against insulin. Standards and samples are added into the wells and co-incubated with a monoclonal antibody conjugated to horseradish peroxidase (HRP) enzyme. After wash step to remove any unbound substances, TMB substrate is added and color develops in proportion to the amount of insulin bound initially. The assay is stopped and the optical density of the wells determined using a micro-plate reader. Since the increases in absorbance are directly proportional to the amount of captured insulin, the unknown sample concentration can be interpolated from a reference curve included in each assay.

试剂盒组分

储存和处理: Store kit at 2-8°C, protected from light. Shelf life: 6 months after receipt. Warm Reagents to room temperature before use.

用途:Measurement of insulin in mouse or rat sera.

适用物种:Human, Mouse, Rat

样品类型:serum, plasma, cell and tissue culture supernatant, urine, fecal and homogenate cell and tissue samples

检测方法:Colorimetric sandwich ELISA assay (OD450 nm)

时间:3-4 hrs

储存:2-8°C

运输:Icepacks

所需其他材料:

Multi-channel pipette for washing

Cell culture incubator

Orbital shaker

micro plate reader

试剂准备

1. Wash Solution: 10x dilute Wash Solution with dH2O to prepare 1x Wash Solution.

2. Detection Antibody: Prepare 1×Detection antibody solution by dilution of the 100×Detection antibody solution in Sample Diluent, mix well. 100 µl of the 1×Detection antibody solution is required per well. Prepare only as much 1×Detection antibody solution as needed. Return the 100×Detection antibody solution to 2-8°C immediately after the necessary volume is removed.

程序指南

1. It is recommended that all standards and samples should be run in duplicate. Set standard wells, testing sample and blank wells on the assay plate/strip. Transfer diluted standard 5 μl to standard wells, diluted sample 5 μl to sample wells, sample diluent 5 μl.

2. Add 100 µl of 1x Detection antibody solution per well. Cover the plate with plate sealer and incubate the plate at room temperature for 2 hrs or at 37 °C for 1h, shaking the plate at 300 rpm on a micro-plate shaker.

3. Decant as much liquid as possible, fill the wells with 200 μl wash solution, shaking for 1 min, decant the wash solution and remove residual liquid with absorbent paper. Repeat wash five times.

4. Add 100 µl of TMB Solution to each well and incubate at room temperature for 10~20 minutes protect from light, or keep close monitoring on the developing process until desired developing blue color observed. Note: please be aware that the color may develop more quickly or more slowly that the recommended incubation time depending on the localized room temperature.

5. Add 50 µl of Stop Solution to each well to stop the reaction (the blue color change to yellow), gently tap the plate frame for a few seconds to ensure thorough mixing.

6. Read absorbance of the plate on a microplate reader at 450 nm within 5 min.

7. Average the duplicate readings for each standard and samples, subtract the average zero (blank) standard optical density. Construct standard curve (plotting the mean OD450 for each standard on the X‐axis against concentration on the Y‐axis, draw a best‐fit curve through the points) and lculate linear regression equation, then use corrected sample OD values and regression equation to calculate the corresponding sample concentration. It should be remembered that the sample has been diluted and its actual concentration should be justified by dilution factor (the measurement and calculation can be accomplished by software like SoftMax).

检测特点

灵敏度:The lowest insulin level that can be measured by this assay is 0.05 ng/ml.

精密度:Intra-assay Precision (Precision within an assay) C.V. < 10%. Inter-assay Precision (Precision between assays) C.V.

回收率:The recovery of the assay was determined by adding various amounts insulin to a sample. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 96-103 %.

特异性:Percent of cross reactivity Human insulin 100 %; Rat insulin 100 %.