人IgG Fc ELISA试剂盒

ABSbio™ 人IgG Fc ELISA试剂盒用于定量检测血清、血浆、细胞上清液和其他基质中的人IgG Fc和Fc融合蛋白。该检测对人IgG Fc具有特异性，与小鼠IgG Fc无交叉反应。

The kit uses a double‐antibody sandwich enzyme‐linked immunosorbent assay (ELISA) to analyze the level of human Fc in samples. A Monoclonal Antibody specific for Human IgG Fc has been pre-coated onto a 96-well microplate. Standards and unknown samples were loaded into the wells and incubated. After washing away any unbound substances, an enzyme-linked anti-human antibody specific for IgG Fc is added to the wells and incubated. Following a washing to remove any unbound antibody-enzyme reagent. Tetramethylbenzidine (TMB) is added to the microplate wells. TMB reacts with peroxide in the presence of HRP to form a colorimetric signal proportional to the amount of IgG Fc bound by the capture antibody. Color development is stopped by the addition of acid to the microplate wells. The Stop Solution changes the color from blue to yellow and the intensity of the color optical density (OD) is measured at 450 nm using a spectrophotometer. The minimum detectable dose of this kit typically less than 0.1 ng/mL, detection range is 0.05-10 ng/mL.

试剂盒组分

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

应用:Measurement of human IgG Fc in serum, plasma and culture media.

反应物种:Human, Mouse, Rat

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

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

检测时间:3-4 hrs

储存:2-8°C

保质期:6 months after receipt.

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. Human Fc Protein Standard: The vial contains 10 μl of the standard sufficient for a 96-well plate. Add 2 μl of the standard into 998 μl of Assay Diluent to make the high standard concentration of 10 ng /ml. A seven point standard curve is generated using 2-fold serial dilutions in Assay Diluent, vortex briefly for each of dilution step.

3. Detection Antibody: Immediately before use, add 50 μl of the antibody into 11 mL of Assay Diluent for one plate (for partial plate assay, adjust the volumes accordingly).

4. Sample: Sample dilution with Assay diluent may need to be optimized by customer.

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 100 μl to standard wells, diluted sample 100 μl to sample wells, assay diluent 100 μl only to blank wells.

2. 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 600-700 rpm on a micro-plate shaker.

3. Decant as much liquid as possible, fill the wells with 300 μl wash solution, shaking for 5 min, decant the wash solution and remove residual liquid with absorbent paper. Repeat wash four times.

4. Add 100 µl of 1x Detection antibody solution per well. Cover the plate with plate sealer and incubate the plate at room temperature for 1h, shaking the plate at 600-700 rpm on a micro-plate shaker.

5. Wash 5 times as outlined in step 3.

6. Add 100 µl of TMB Solution to each well and incubate at room temperature for 20~30 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.

7. Add 100 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.

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

9. 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 calculate 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).

10. If molecular weight of sample differs from Fc standard (MW 50kD) apply the following equation to the reading concentration to obtain the actual concentration = [MW Sample]/[MW Fc protein] x Sample reading (ng/mL)

分析特点

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

精确度:Intra-assay Precision (Precision within an assay) C.V. < 7.6%. Inter-assay Precision (Precision between assays) C.V. <8.0%.

回收率:The recovery of the assay was determined by adding various amounts Human Fc 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 IgG Fab, IgA, IgM undetectable; Mouse, Rat, Rabbit IgG undetectable.