Standard Curve
Example of Human Complement C3a standard curve in Assay Diluent A1. Plotted are the background-subtracted data.
This standard curve is provided for demonstration only.
Human Complement C3a OneStep ELISA Kit
Human Complement C3a OneStep ELISA Kit
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Product Details
Product Details
Product Specification
| Antigen | Complement C3a; C3a |
| Immunogen | Recombinant Protein |
| Antibody Type | Recombinant mAb |
| Reactivity | Hu |
| Purification | Protein A |
| Stability & Storage | 12 months from date of receipt / reconstitution, 2 to 8°C as supplied. |
Kit
| Precision | Intra-assay: 4.6%； Inter-assay: 6.5% |
| Sample type | Serum; EDTA Plasma, Heparin Plasma, Citrate Plasma; Cell Culture Supernatant |
| Assay type | Sandwich (quantitative) |
| Sensitivity | 1.80 pg/mL |
| Range | 3.9 pg/mL – 250 pg/mL |
| Recovery | Serum: 109% EDTA Plasma: 113% Citrate plasma: 110% Heparin Plasma: 97% Cell Culture Supernatant: 94% |
| Assay time | 60 minutes |
| Species reactivity | Hu |
Background
Human C3a is a 77-amino-acid peptide released by proteolytic cleavage of the complement component C3 during activation of the classical, lectin, or alternative complement pathways. As a potent anaphylatoxin, it binds to the G-protein-coupled receptor C3aR, which is expressed on mast cells, eosinophils, macrophages, neutrophils, and activated T lymphocytes. Upon receptor activation, C3a induces mast cell degranulation, histamine release, smooth muscle contraction, and increased vascular permeability, thereby contributing to acute inflammatory responses. It also acts as a chemoattractant for eosinophils and mast cells and modulates cytokine production in various immune cells. Beyond its proinflammatory role, C3a participates in antimicrobial defense and has been implicated in tissue regeneration and neural development. The biological activity of C3a is tightly controlled by carboxypeptidase N, which removes the C-terminal arginine to generate the less active C3a-desArg form. Dysregulated C3a signaling has been associated with several human diseases, including asthma, sepsis, and autoimmune disorders, making it an important target for therapeutic intervention.
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ELISA
Spike Recovery
The recovery of Human Complement C3a was evaluated in activated samples spiked with concentrations spanning the entire assay range.
Spike Recovery
Recovery of Human Complement C3a was assessed by spiking a 125 pg/mL standard into matrix diluted 56,000-fold. Serial dilutions of the spiked sample were prepared, and the percent recovery was calculated for each dilution relative to the expected concentration.
Dilution Linearity
The concentrations of Human Complement C3a were measured and interpolated from the target standard curves and corrected for sample dilution.
The leftmost bar in the graph shows biological samples diluted in Assay Diluent to 0.003% concentration. "undiluted" was defined as 0.003% biological sample. The subsequent bars represent two-fold serial dilutions of this reference.
The mean target concentration was determined to be 12.82 μg/mL in Human Serum.
The mean target concentration was determined to be 15.84 μg/mL in Human EDTA plasma.
The mean target concentration was determined to be 3.36 μg/mL in Human Heparin plasma.
The mean target concentration was determined to be 0.84 μg/mL in Human Citrate plasma.
Dilution Linearity
The concentrations of Human Complement C3a were measured and interpolated from the target standard curves and corrected for sample dilution.
The leftmost bar in the graph shows biological samples diluted in Assay Diluent to 2.5% concentration. "undiluted" was defined as 2.5% biological sample. The subsequent bars represent two-fold serial dilutions of this reference.
The mean target concentration was determined to be 15.56 ng/mL in HepG2 cell culture supernatant.
Serum levels in healthy individuals
Duplicate measurements were performed on serum samples obtained from eight individual healthy donors. Data are reported as dilution factor-corrected interpolated. The resultant mean Human Complement C3a concentration was 4768.9 ng/mL, with a distribution spanning 0.22 to 13027.8 ng/mL across the donor cohort.
Intra-Assay Precision (Precision within an assay)
Three samples of known concentration were tested fifteen times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays).
Three samples of known concentration were tested in separate assays to assess inter-assay precision. Assays were performed with at least three lots of components.
Determination of Minimum Detectable Dose (MDD)
The MDD was determined using three independent lots of assay components. For each lot, 19 replicate measurements of the diluent (zero calibrator) were performed. The mean (AVERAGE) and standard deviation (STDEV) of the 19 replicates were calculated. The MDD for each lot was then calculated according to the following formula:
MDD = 2 × STDEV + AVERAGE
Cross‑reactivity
The heatmap displays OD450 ratios for serum samples from multiple species. Color intensity is proportional to the O.D.450 ratio (test species / reference species). Darker colors indicate higher cross‑reactivity.
HOOK Effect Threshold
The upper limit of the HOOK effect was established at 100× the highest calibrator concentration (equivalent to a 2-log10 increase). No HOOK effect was observed below this threshold, confirming that samples within this concentration range yield reliable quantitative results without signal depression.
Comparison of Standard Curves from Two Kits
Standard curves from Company A and Starter Onestep were compared.
Protocol Diagram
