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| Product Name | p53 Autoantibody ELISA Kit, Quantitative, Human Serum and Plasma, Research Use |
| Catalog No. | TMTR-HMM-0087 |
| Description | Indirect ELISA kit for quantitative detection of circulating autoantibodies against the tumor suppressor protein p53 (TP53) in human serum and plasma. p53 autoantibodies are produced as part of the humoral immune response to accumulated mutant or overexpressed p53 protein in tumor cells, and are detected in approximately 10-40% of cancer patients depending on tumor type and stage (highest prevalence in ovarian, colorectal, lung, breast, and head and neck cancers). p53 autoantibodies appear early in tumor development and can precede clinical diagnosis by months to years, making them a research biomarker for early cancer detection, monitoring, and prognosis. This ELISA uses recombinant wild-type human p53 protein coated on the microplate to capture anti-p53 autoantibodies (primarily IgG class) from patient serum, followed by anti-human IgG-HRP detection and TMB color development. |
| Intended Use | Qualitative and semi-quantitative detection of IgG autoantibodies against p53 in: human serum and plasma samples for cancer biomarker research; longitudinal cohort studies of cancer development and screening; monitoring of tumor burden and treatment response; prognostic stratification (p53 autoantibody-positive status associated with poor prognosis in colorectal, esophageal, and lung cancers); and research into p53 mutations and tumor immunology. |
| Principle / Technology | Indirect ELISA: recombinant wild-type human p53 protein (full-length, expressed in E. coli or baculovirus) is coated on the microplate. Diluted patient serum is added; anti-p53 IgG autoantibodies, if present, bind to the immobilized p53. After washing, HRP-conjugated anti-human IgG (Fc-gamma specific) detection antibody is added, binding to captured autoantibodies. TMB substrate produces a colorimetric signal proportional to anti-p53 IgG concentration. Results are reported as arbitrary units/mL (AU/mL) relative to a calibrator serum with known anti-p53 reactivity. |
| Detection Method | 1) Dilute serum/plasma 1:100 in sample diluent; 2) Add 100 uL diluted sample, calibrator, and controls to wells; incubate 1 h at RT; 3) Wash 3x; 4) Add 100 uL anti-human IgG-HRP conjugate; incubate 30 min at RT; 5) Wash 3x; 6) Add 100 uL TMB; incubate 15 min RT in dark; 7) Add 100 uL stop solution; 8) Read A450; 9) Calculate AU/mL from calibrator curve; interpret positive/negative based on cut-off value (typically mean of healthy controls + 2-3 SD). |
| Sample Type | Human serum (preferred — collected in SST, allowed to clot 30 min, centrifuge, aliquot, store at -80 C); plasma (EDTA or citrate — avoid heparin which may cause background); avoid lipemic, hemolyzed, or icteric samples; freeze-thaw cycles should be minimized (<3 cycles). |
| Performance Range / Specifications | Measuring range: 1-100 AU/mL (arbitrary units relative to internal calibrator); cut-off for positivity: typically 5-15 AU/mL (determined by ROC analysis in target population); healthy donor range: 0.5-8 AU/mL; positive cancer patient sera: 10-200+ AU/mL. |
| Sensitivity / LOD | Analytical sensitivity: <1 AU/mL; diagnostic sensitivity (cancer detection): 15-40% depending on tumor type and stage (highest for colorectal, ovarian, lung cancers); diagnostic specificity: >95% vs healthy controls. |
| Specificity | Recombinant p53 protein specifically captures anti-p53 autoantibodies; <1% false-positive rate in healthy donor sera; cross-reactivity: anti-p63 and anti-p73 autoantibodies may show limited cross-reactivity due to p53 family protein homology (approximately 20% sequence identity) — confirm with p53-specific competition ELISA if needed. |
| Reaction Conditions / Protocol | Sample incubation 1 h RT; conjugate 30 min RT; TMB 15 min RT; total assay time ~2 h. |
| Components / Formulation | p53 Antigen Coated 96-Well Microplate, Calibrator Serum (lyophilized, anti-p53 positive, calibrated in AU/mL), Positive Control (lyophilized), Negative Control (lyophilized), Sample Diluent (100 mL), Anti-Human IgG-HRP Conjugate (100x, 150 uL), Wash Buffer (20x, 50 mL), TMB Substrate (12 mL), Stop Solution (12 mL), Plate Sealers, Protocol. |
| Storage Conditions | Plate, sample diluent, wash, TMB, stop at 2-8 C; Calibrator, Controls, Conjugate at -20 C; protect TMB and conjugate from light. |
| Shelf Life | 18 months from manufacture. |
| Package Specifications | 96 determinations. |
| Product Form | Antigen-coated plate; liquid reagents; lyophilized calibrator and controls. |
| Quality Control | Calibrator: assigned value in AU/mL (calibrated against reference panel); positive control within assigned range; negative control below cut-off; intra-assay CV <10%; inter-assay CV <15%; p53 antigen coating uniformity <15% CV; stability of coated plate: 18 months at 2-8 C. |
| Key Features | Indirect ELISA for p53 autoantibody detection; recombinant wild-type p53 antigen; anti-human IgG-HRP detection; calibrator and controls included; semi-quantitative results in AU/mL; 2-hour assay; research-use cancer biomarker. |
| Purity | Recombinant p53: >95% by SDS-PAGE; functional p53 (DNA-binding competent); anti-human IgG: affinity-purified, Fc-gamma specific; analytical grade reagents. |
| Concentration | Calibrator: assigned in AU/mL (lot-specific); sample dilution 1:100; conjugate 100x. |
| Activity / Unit Definition | Anti-human IgG-HRP: >200 U/mg; reacts with all four human IgG subclasses. |
| Molecular Weight | p53: ~53 kDa (393 amino acids, homotetramer ~212 kDa); IgG: ~150 kDa; HRP: ~44 kDa. |
| Source / Origin | Recombinant human p53: expressed in E. coli, purified by DNA affinity and size exclusion; anti-human IgG: goat or rabbit polyclonal, affinity-purified; calibrator and controls: human serum pools (tested negative for HIV, HBV, HCV); HRP: plant-derived. |
| pH Range / Optimal pH | Sample diluent pH 7.2-7.4; optimal binding pH 7.0-8.0. |
| Shipping Conditions | Cold pack; calibrator, controls, conjugate on dry ice. |
| Expiration Date / Stability | 18 months; reconstituted calibrator/controls: 1 week at 2-8 C, 3 months at -80 C (aliquot). |
| Regulatory / Compliance | For research use only; not for clinical diagnostic use. Not FDA cleared or approved for cancer screening or diagnosis. Human serum-derived calibrator and controls tested negative for HIV, HBV, HCV — handle as potentially infectious per BSL-2 guidelines. |
| Compatibility | Serum strongly preferred over plasma; EDTA and citrate plasma acceptable but may have slightly different background. Lipemic samples may interfere; centrifuge at 10,000 x g before assay. Avoid samples from patients receiving biotin therapy (may interfere with streptavidin-based detection — not relevant here as detection is direct anti-human IgG-HRP). Hemolysis (hemoglobin) at visible levels may cause false elevation. |
| Recommended Buffer System | Sample Diluent: PBS pH 7.4, 1% BSA, 0.05% Tween-20, 0.05% ProClin 300; Wash Buffer: PBS pH 7.4, 0.05% Tween-20. |
| Application Notes / Precautions | p53 autoantibody testing is for research purposes only. Elevation is not diagnostic of cancer and normal levels do not exclude cancer. Autoantibody levels may fluctuate over time. Define a cut-off value for your specific study population using ROC analysis against an appropriate control group (age, sex, smoking status-matched). Include both positive and negative controls in each run. For serial monitoring studies: run all samples from the same patient in the same assay batch to minimize inter-assay variation. p53 autoantibodies are predominantly IgG1 and IgG3 subclasses; total IgG detection is appropriate for most studies. Confirm positive results with a competition ELISA (pre-incubating serum with soluble p53 should reduce signal by >70%). p53 autoantibodies can coexist with autoantibodies to other tumor-associated antigens (HER2, MUC1, NY-ESO-1) — multiplex panels may improve sensitivity. |
| Batch-to-Batch Consistency | Calibrator value within specified range for every lot; positive/negative control classification consistent; inter-lot CV <15% for calibrator; antigen coating CV <15%. |
For research use only, not for clinical use.
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