Powered by BiozThe precisionbind Human TNF-Alpha (TNFa / TNA-alpha / TNFA) ELISA includes features like:
– Ready to use protocol with break-apart wells for ease of use
– Standardisation and High Reproducibility
– Lot to Lot Consistency
– Accuracy and Precision
Principle Of Assay: This ELISA is a sandwich immunoassay. Antibodies are coated on 96 well plates. The antigen protein present in sample and standard respectively bind to the coated wells. The wells are washed and an antibody:HRP Conjugate is added which binds to the bound complex in the well. Washing is performed to remove any unbound material. TMB substrate is added and the enzyme reaction is stopped by dispensing of stop solution into the wells. The optical density (OD) of the solution at 450 nm is directly proportional to the amount of antigen protein present in the standard or samples.
Disclaimer: The data indicated herein with specifications are changed from time to time at time of production of the assay. We request you to confirm the specifications including the assay range and procedure as per the most current IFU (instructions for use) accompanying the assay kit.
Validated against seven points for a “GOLD RING” Standard Quality ELISA – the benchmark sign for Krishgen Quality. The The precisionbind ELISA kits are used for assessing the specific biomarker in samples analytes which may be human serum, plasma, biological fluids and cell culture supernatant. The kit uses indirect sandwich assay with double antibodies – capture and detection to ensure a high degree of sensitivity and specificity in the estimation of TNF-Alpha (TNFa / TNA-alpha / TNFA);
About PrecisionBind:
PrecisionBind is Krishgen`s cytokine ELISA platform developed for bioanalytical robustness in translational research, including immuno-oncology and cell & gene therapy programs. Where applicable, kit standards are calibrated against NIBSC/WHO international standards to support unit traceability and inter-study comparability.
Assays are characterized following ligand-binding assay performance expectations, including:
- Defined assay range and sensitivity (LOD/LOQ where reported)
- Intra- and inter-assay precision
- Cross-matrix performance characterization (serum, plasma, CSF where applicable), including parallelism and recovery assessment, to support quantitative comparability across biological matrices.All kits are Research Use Only (RUO) and not intended for diagnostic use.
Validation Parameters and Acceptance Criteria
1. TNF-α Cmax Values and Recommended ELISA Range
This table summarizes TNF-α Cmax levels across diseases and suggests corresponding ELISA working ranges.
Note: Assay sensitivity < 5 pg/mL recommended for baseline detection; upper limit ≥ 5,000 pg/ml advised for CRS monitoring.
2. Specificity and Selectivity
2.1 Specificity
The capture antibody and detection antibody are both specific to TNFa and are monoclonal antibodies. They show a high affinity to bind to native as well as recombinant TNFa.
2.2 Selectivity
The ELISA has no or low cross reactivity to IL‑1β (IL-1beta), IL‑6, or TNF‑β (TNFbeta).
2.3 NIBSC validation
The standard used in the kit is calibrated against an international standard from the National Institute of Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire EN6 3QG, UK. 1 ng of supplied standard equals 46 U of 12/154 NIBSC-standard.
Therefore 1000 pg/ml is equivalent to 46 U of TNFα as per NIBSC.
2.4 LOD, LOQ and IC50
LOD (Limit of Detection)
The lowest analyte concentration that can be reliably distinguished from blank/background noise but not necessarily quantified precisely.
Statistically:
LOD = Mean of Blank + 3X SD of Blank
(3σ criterion is most common).
LOD for PrecisionBind Human TNFalpha ELISA = 3.1 pg/ml
LOQ (Limit of Quantitation)
The lowest analyte concentration that can be quantified with acceptable accuracy and precision.
Statistically:
LOQ = Mean of Blank + 10X SD of Blank
(10σ criterion is most common).
LOQ for PrecisionBind Human TNFalpha ELISA – 9.3 pg/ml
IC50 in ELISA (Half Maximal Inhibitory Concentration)
IC50 = The concentration of an inhibitor (drug, antibody, compound) required to reduce the signal (e.g., binding, enzymatic activity) by 50% compared to the maximum signal in the assay.
In ELISA, this is commonly used for:
Neutralization ELISA: Quantifies potency of antibodies inhibiting target–ligand interaction.
Drug Potency Testing: Measures concentration at which drug inhibits 50% of target activity.
IC50 for PrecisionBind Human TNFalpha ELISA = ~173 pg/ml
Regulatory Note:
LOD *S/N ≥ 3:1, LOQ ≥ 10:1, %CV ≤ 20% *S/N = Signal / Noise Ratio
2. Linearity and Range
3. Accuracy and Precision (Intra / Inter-Assay)
A) Intra-Assay:
B) Inter Assay:
4. Parallelism and Matrix Effect
Sample Dilution factor – Human Serum, Human Plasma and Human CSF samples have been tested. Sample dilution Factor for all three matrices is 1:50 dilution.
Neat Human Serum, Human Plasma and Human CSF were spiked with 250 pg/ml Human TNF alpha and ELISA assay was run.
A) Serum:
B) Plasma:
C) Cerebrospinal Fluid (CSF):
Results:
i) Parallelism is maintained across the 1:100 to 1:6400 dilutions.
ii) % Recovery for most dilutions falls within the acceptable range of 80%–120%.
iii) No significant matrix effect observed at higher dilutions.
iv) The PrecisionBind Human TNFalpha ELISA kit was tested for matrix effect on human serum, plasma, CSF and physiological buffer 7.4 to mimic tear fluid samples.
5. Sample Handling and Storage Conditions.
A.) Sample collection and handling:
Specimens should be clear and non-hemolyzed. Samples should be run at a number of dilutions to ensure accurate quantitation.
Cell Culture Supernatant: If necessary, centrifuge to remove debris prior to analysis.
Samples can be stored at temperature <-20°C. Avoid repeated freeze/thaw cycles.
Serum: Use a serum separator tube and allow clotting for 30 minutes, then centrifuge for 10 minutes at 1000 x g. Remove serum layer and assay immediately or store serum samples at temperature <-20°C. Avoid repeated freeze/thaw cycles.
Plasma: Collect blood sample in a citrate, heparin or EDTA containing tube. Centrifuge for 10 minutes at 1000 x g within 30 minutes of collection. Assay immediately or store plasma samples at temperature <-20°C. Avoid repeated freeze/thaw cycles.
B) Storage conditions:
Store main kit components at 2-8°C.
Store recombinant lyophilized standard at 2-8°C. Upon reconstitution aliquot standards into polypropylene vials and store at -20°C as per assay requirements. Do not freeze thaw for more than two times.
Before using, bring all components to room temperature (18-25°C). Upon assay completion return all components to appropriate storage conditions.
Graphs, Maps and Appendices:
Calibration of Standard Used in the KIT
Matrix Effect Heat Map
Determined Limits for Acceptance according to EMA/FDA and CLSI regulations
References
Aggarwal, B. B. (2003). Signalling pathways of the TNF superfamily: a double-edged sword.
Nature Reviews Immunology, 3(9), 745–756.
Tracey, K. J., & Cerami, A. (1994). Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annual Review of Medicine, 45, 491–503.
FDA Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products (2014).
EMA Guideline on Biosimilar Monoclonal Antibodies (2012).
