Kit Components：

Specificity：

This assay has high sensitivity and excellent specificity for detection of C4a ?. No significant cross-reactivity or interference between C4a ?and analogues was observed.

Note: Limited by current skills and knowledge, it is difficult for us to complete the cross-reactivity detection between C4a ?and all the analogues, therefore, crossr eaction may still exist.

Recovery：

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

Linearity：

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

Precision：?

Intra-Assay: CV<8%

Inter-Assay: CV<10%

Stability：

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 10% within the expiration date under appropriate storage condition.

Principle of the Assay：

Operation Procedure：

This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Capture ?antibody was pre-coated onto 96- well plates. And the biotin conjugated antibody was used as detection antibodies. The standards, test samples and biotin conjugated detection antibody were added to the wells subsequently, and washed with wash buffer. HRP-Streptavidin was added and unbound conjugates were washed away with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that ?changed into yellow after adding acidic stop solution. The density of yellow is proportional to the target amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of targetcan be calculated.

Precautions：

1.To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using standards and a small number of samples isrecommended.

2.After opening and before using, keep platedry.

3.Beforeusingthekit,spintubesandbringdownallcomponentstothebottomoftubes.

4.Storage TMB reagents avoidlight.

5.Washingprocessisveryimportant,notfullywasheasilycauseafalsepositiveandhighbackground.

6.Duplicate well assay is recommended for both standard and sampletesting.

7.Don’tletmicroplatedryattheassay,fordryplatewillinactivateactivecomponentsonplate.

8.Don’treusetipsandtubestoavoidcrosscontamination.

9.Avoid using the reagents from different batchestogether.

Material Required but Not Supplied：

1.Microplate reader(wavelength:450nm)。

2.37°Cincubator。

3.Automated platewasher。

4.Precision single and multi-channelpipette and disposable tips。

5.Clean tubes and Eppendorftubes。

6.Deionized or distilledwater。

Washing：

Manual: Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other absorbent material. Fill each well completely with 350ul wash buffer and soak for1to 2minutes,then aspirate contents from theplate,andclaptheplateonabsorbentfilterpapersorotherabsorbentmaterial.

Automatic: Aspirate all wells, and then wash plate with 350ul wash buffer. After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer shall be set for soaking1 minute.(Note:settheheightoftheneedles;besurethefluidcanbesippedupcompletely)。