The precisionbind Human Interleukin 2 (IL-2 / IL2) 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 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 Interleukin 2 (IL-2 / IL2);
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. IL-2 Cmax Values and Recommended ELISA Range
This table summarizes IL-2 Cmax levels across diseases and suggests corresponding ELISA working ranges.
Note: Assay sensitivity <2 pg/mL recommended for baseline detection; upper limit ≥2,000 pg/mL advised for CRS monitoring.
2. Specificity and Selectivity
2.1 Specificity
The capture antibody and detection antibody are both specific to IL-2 and are monoclonal antibodies. They show a high affinity to bind to native as well as recombinant IL-2.
2.2 Selectivity
The ELISA has no or low cross reactivity to IL-2 Rα IL-2 Rβ IL-2 Rγ
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 15 U of 86/504 NIBSC-standard.
Therefore 1000 pg/ml is equivalent to 15 U of IL-2 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 IL-2 ELISA = 2.01 pg/mlLOQ (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 IL-2 ELISA – 6.1 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 IL-2 ELISA = ~257 pg/ml
Regulatory Note:
LOD S/N ≥ 3:1, LOQ ≥ 10:1, %CV ≤ 20% *S/N = Signal / Noise Ratio
3. Linearity and Range
4. Accuracy and Precision (Intra/Inter-Assay)
A) Intra-assay:
B) Inter assay:
5. 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 1:10 dilution.
Neat Human Serum, Human Plasma and Human CSF were spiked with 500 pg/ml Human IL-2 and ELISA assay was run.
A) Serum:
B) Plasma:
C) Cerebrospinal Fluid (CSF):
Results:
i) Parallelism is maintained across the 1:20 to 1:1280 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 IL-2 ELISA kit was tested for matrix effect on human serum, plasma, CSF and physiological buffer 7.4 to mimic tear fluid samples.
6. 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
Chorro L, Suzuki M, Chin SS, Williams TM, Snapp EL, Odagiu L, Labrecque N, Lauvau G.
Interleukin 2 modulates thymic-derived regulatory T cell epigenetic landscape. Nat Commun. (2018). doi: 10.1038/s41467-018-07806-6. PMID: 30560927; PMCID:PMC6299086.
Wang, T., Hu, Y., Wangkahart, E., Liu, F., Wang, A., Zahran, E., Maisey, K. R., Liu, M., Xu, Q., Imarai, M., & Secombes, C. J. (2018). Interleukin (IL)-2 is a key regulator of T helper 1 and T helper 2 Cytokine expression in fish: Functional characterization of two divergent IL2 paralogs in salmonids. Frontiers in Immunology, 9.
https://doi.org/10.3389/fimmu.2018.01683
FDA Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products
(2014).
EMA Guideline on Biosimilar Monoclonal Antibodies (2012).
