Standard Curve
Example of Rat IgM standard curve in Assay Diluent H1. Plotted are the background-subtracted data.
This standard curve is provided for demonstration only.
Rat IgM Serum & Plasma OneStep ELISA Kit
Rat IgM Serum & Plasma OneStep ELISA Kit
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Product Details
Product Details
Product Specification
| Antigen | IgM |
| Immunogen | Recombinant Protein |
| Antibody Type | Recombinant mAb |
| Reactivity | Rt |
| Purification | Protein A |
| Stability & Storage | 12 months from date of receipt / reconstitution, 2 to 8°C as supplied. |
Kit
| Precision | Intra-assay: 3.5% Inter-assay: 4.9% |
| Sample type | Serum; Plasma |
| Assay type | Sandwich (quantitative) |
| Sensitivity | 0.156 ng/mL |
| Range | 1.6 ng/mL – 100 ng/mL |
| Recovery | SD Rat Serum: 95% SD Rat EDTA Plasma: 93% SD Rat Heparin Plasma: 105% SD Rat Citrate Plasma: 102% SHR Rat Serum: 95% LEWIS Rat Serum: 102% LEWIS Rat EDTA plasma: 95% |
| Assay time | 60 minutes |
| Species reactivity | Ms |
Background
Rat immunoglobulin M (IgM) is a primary antibody isotype that plays a critical role in the early immune response of rats, being the first antibody produced upon initial antigen exposure. This protein is predominantly synthesized in the spleen and, due to its large size, remains largely confined to the bloodstream. Structurally, rat IgM in its secreted form is a pentamer, covalently linking five immunoglobulin units (monomers) along with a single joining (J) chain to create a complex with a molecular weight of approximately 900 kDa. Each individual monomer has a molecular weight of about 190 kDa and is composed of two μ heavy chains and two light chains. This pentameric structure theoretically provides up to ten antigen-binding sites, giving it high avidity for multivalent antigens, though not all sites may be able to bind simultaneously due to steric constraints. While the monomeric form is expressed as a membrane-bound receptor on the surface of B cells, the pentameric form is secreted by plasma cells and is highly efficient at activating the complement pathway and promoting agglutination. As such, rat IgM is a crucial component of the humoral immune system and a valuable research tool, often used as a control reagent or a standard in immunological assays like ELISA and Western blotting.
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ELISA
Spike Recovery
Recovery of Rat IgM was assessed by spiking a 12.5 ng/mL standard into 0.003% serum (or 0.005% EDTA plasma, or 0.005% heparin plasma, or 0.0125% citrate plasma). Serial dilutions of the spiked sample were prepared, and the percent recovery was calculated for each dilution relative to the expected concentration.
Spike Recovery
Recovery of Rat IgM was assessed by spiking a 100 ng/mL standard into 0.01% serum (SHR). Recovery of Rat IgM was assessed by spiking a 100 ng/mL standard into 0.01% serum (LEWIS). Recovery of Rat IgM was assessed by spiking a 100 ng/mL standard into 0.02% EDTA plasma (LEWIS). Serial dilutions of the spiked sample were prepared, and the percent recovery was calculated for each dilution relative to the expected concentration.
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.
Cross-reactivity
The cross-reactivity was checked by testing Rat IgM, Human IgM, Mouse IgM, Monkey IgM, and Rabbit IgM in the same assay system. Each protein was tested with serial dilutions, and the dose-response curves were compared. A noticeable cross-reactivity was seen when mouse IgM was tested in the rat IgM assay. No significant cross-reactivity was observed when other IgM was tested in the Rat IgM assay, as the signals remained within the background range of the assay.
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
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.
Dilution Linearity
The concentrations of Rat IgM 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 0.025% concentration. "undiluted" was defined as 0.025% biological sample. The subsequent bars represent two-fold serial dilutions of this reference. The mean target concentration was determined to be 303.8μg/mL in Rat SD citrate plasma.
Dilution Linearity
The concentrations of Rat IgM 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 0.01% concentration. "undiluted" was defined as 0.01% biological sample. The subsequent bars represent two-fold serial dilutions of this reference. The mean target concentration was determined to be 227.1 μg/mL in Rat SD EDTA plasma. The mean target concentration was determined to be 310.4 μg/mL in Rat SD heparin plasma. The mean target concentration was determined to be 950.2 μg/mL in Rat SHR EDTA plasma. The mean target concentration was determined to be 693 μg/mL in Rat LEWIS EDTA plasma.
Dilution Linearity
The concentrations of Rat IgM 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 0.005% concentration. "undiluted" was defined as 0.005% biological sample. The subsequent bars represent two-fold serial dilutions of this reference. The mean target concentration was determined to be 242 μg/mL in Rat SD serum. The mean target concentration was determined to be 1366.5 μg/mL in Rat SHR serum. The mean target concentration was determined to be 625.8 μg/mL in Rat LEWIS serum.
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 Three Kits
Standard curves from Company A and Starter Onestep were compared.
Protocol Diagram
