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Scientific Background
This ELISA kit applies to the in vitro quantitative determination of Human ACTH concentrations in serum, plasma and other biological fluids.
Assay Principle
This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with Human ACTH. During the reaction, Human ACTH in the sample or standard competes with a fixed amount of Human ACTH on the solid phase supporter for sites on the Biotinylated Detection Ab specific to Human ACTH. Excess conjugate and unbound sample or standard are washed away, and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns from blue to yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Human ACTH in tested samples can be calculated by comparing the OD of the samples to the standard curve.
Performance Specifications
| Sensitivity | 9.38 pg/mL |
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| Detection Range | 15.63-1000 pg/mL |
| Total Assay Time | 2 h 30 min |
| Compatible Sample Types | Serum, plasma and other biological fluids |
| Species Reactivity | Human |
| Detection Method | Competitive |
| Precision (CV) | Both intra-CV and inter-CV are < 10%. |
| Recovery Rate | 80%-120% |
| Storage | 2-8℃,12 months |
✓ Research-Grade Validation
Specificity
This kit recognizes Human ACTH in samples.No significant cross-reactivity or interference between Human ACTH and analogues was observed
Safety & Regulatory
Handle reagents in accordance with institutional biosafety guidelines. Refer to the Safety Data Sheet (SDS) for complete hazard and handling information. Contains components that may require special disposal procedures per local regulations.
This kit is validated for use with Serum, plasma and other biological fluids. For unlisted matrices (e.g., tissue lysate, urine), perform a spike-and-recovery experiment to confirm assay performance before generating reportable data. Sample dilution in the kit's provided diluent is recommended to minimize matrix interference.
The minimum detectable concentration (sensitivity) of this kit is 9.38 pg/mL. Values below this threshold should be reported as below the limit of detection (<LOD) and should not be extrapolated from the standard curve.
The total assay time from sample addition to absorbance reading is approximately 2 h 30 min, including all incubation, wash, and substrate steps. Hands-on time is typically 1–2 hours; most steps involve passive plate incubation. Plan the assay as a single uninterrupted session for best results.
Standard components of this Competitive ELISA Kit typically include: pre-coated microplate (96-well strip format), lyophilized or liquid recombinant ACTH standard, detection antibody, streptavidin-HRP conjugate, TMB substrate, stop solution, wash buffer concentrate, and sample/standard diluent. Refer to the kit insert or datasheet for the exact component list and storage requirements.
This kit uses colorimetric (TMB/HRP) detection and requires a standard microplate absorbance reader capable of measuring at 450 nm. A reference wavelength of 570 nm or 630 nm is recommended to reduce background. No specialized fluorescence or luminescence reader is needed. Ensure the instrument is calibrated and the plate is clean and free of condensation before reading.
Tissue samples may contain endogenous or exogenous proteases during processing, leading to degradation of extracted proteins. Therefore, it's necessary to add protease inhibitors during processing to ensure the integrity of target proteins. If customers can keep samples cold and handle them quickly during processing, omitting the protease inhibitor may not have a significant effect. After preparation, samples should be tested promptly or immediately aliquoted and frozen at -20°C or -80°C.
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