C4a Des Arg

This ELISA kit applies to the in vitro quantitative determination of C4a Des Arg concentrations in serum, plasma and other biological fluids.

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to C4a Des Arg. Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for C4a Des Arg and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain C4a Des Arg, biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of C4a Des Arg. You can calculate the concentration of C4a Des Arg in the samples by comparing the OD of the samples to the standard curve.

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Intra-assay Precision (Precision within an assay):
3 samples with low, middle,high concentration of C4a Des Arg. And the sample of C4a Des Arg were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of C4a Des Arg. And the sample of C4a Des Arg were tested on 3 different plates, 30 replicates in each plate.

Matrices listed below were spiked with certain level of C4a Des Arg and the recovery rates were calculated by comparing the measured value to the expected amount of C4a Des Arg in samples.

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of C4a Des Arg and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

The stability of C4a Des Arg is determined by the loss rate of activity. The loss rate of C4a Des Arg is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Microplate reader with 450 nm wavelength filter
High-precision transfer pipette, EP tubes and disposable pipette tips
Incubator capable of maintaining 37℃
Deionized or distilled water
Absorbent paper
Loading slot for Wash Buffer

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.