Powered by BiozThis GENLISA Mouse Interleukin-6 (IL6 / IL-6) 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 GENLISA ELISA kits are used for assessing the specific biomarker in samples analytes which may be serum, plasma, tissue homogenates and other biological fluids. The kit uses monoclonal antibodies to ensure a high degree of sensitivity and specificity in the estimation of the analyte.
Introduction:
This document presents a discussion of the characteristics of our GENLISA® Mouse Interleukin-6 (IL-6/ IL6) ELISA (Catalog No KB2068) kit considered by us during the validation of this kit in accordance with ICH Q2 (R1) guidelines. The document is prepared based on tests run in our laboratory and does not necessarily seek to cover the testing that may be required at user’s end for registration in, or regulatory submissions. The objective of this validation is to demonstrate that it is suitable for its intended purpose – detection of Mouse IL-6.
Background: Interleukin-6 (IL-6) is a pleiotropic pro-inflammatory cytokine produced by a variety of cells including macrophages, monocytes, T lymphocytes, B lymphocytes, fibroblasts, endothelial cells, and other immune and non-immune cells in response to infection, tissue injury, and immune stimulation. IL-6 plays a central role in the regulation of immune responses, inflammation, hematopoiesis, and acute-phase reactions. In mice, IL-6 is widely studied as a key mediator of inflammatory and autoimmune diseases, infectious disorders, cancer progression, and metabolic dysfunction. Elevated levels of Mouse IL-6 are associated with enhanced inflammatory activity and disease severity in numerous experimental models. Quantification of Mouse IL-6 is therefore important for investigating cytokine networks, monitoring inflammatory responses, evaluating disease progression, and assessing the efficacy of therapeutic interventions in preclinical research involving murine models.
Validation characteristics considered by us in accordance with the guidelines are listed below:
o Assay Validation
o Standard Curve
o Pharmacokinetic Relevance
o Precision and Reproducibility
o Linearity
Background
Interleukin-6 (IL-6) is a pleiotropic pro-inflammatory cytokine produced by a variety of cells including macrophages, monocytes, T lymphocytes, B lymphocytes, fibroblasts, endothelial cells, and other immune and non-immune cells in response to infection, tissue injury, and immune stimulation. IL-6 plays a central role in the regulation of immune responses, inflammation, hematopoiesis, and acute-phase reactions. In mice, IL-6 is widely studied as a key mediator of inflammatory and autoimmune diseases, infectious disorders, cancer progression, and metabolic dysfunction. Elevated levels of Mouse IL-6 are associated with enhanced inflammatory activity and disease severity in numerous experimental models. Quantification of Mouse IL-6 is therefore important for investigating cytokine networks, monitoring inflammatory responses, evaluating disease progression, and assessing the efficacy of therapeutic interventions in preclinical research involving murine models.
1. Purpose
To assess the specificity, assay performance, and clinical relevance of the GENLISA® Mouse Interleukin-6 (IL-6/IL6) ELISA developed using Anti Mouse IL-6 as capture protein.
2. Experimental Design
– `A sandwich ELISA was performed using anti-mouse IL-6 as capture protein.
– Standards prepared for Mouse IL-6.
– Assay Concentration Range: 0 – 2000 pg/ml.
– Signal (% absorbance) plotted versus concentration.
The GENLISA® Mouse Interleukin-6 (IL-6) ELISA employs a highly specific sandwich immunoassay strategy to ensure selective capture and detection of Mouse IL-6 present in biological samples. Affinity-purified anti-Mouse IL-6 antibodies immobilized on the microplate surface specifically bind Mouse IL-6 while preserving its native antigenic structure and epitope integrity. Following capture, an HRP-conjugated anti-Mouse IL-6 detection antibody binds to a distinct epitope on the cytokine, resulting in the formation of a stable antibody–antigen–antibody sandwich complex. Upon addition of the TMB substrate, the bound HRP enzyme catalyzes the development of a colored product, the intensity of which is directly proportional to the concentration of Mouse IL-6 present in the sample. Other cytokines and unrelated proteins exhibit minimal or no binding under these assay conditions, reflecting the high specificity of the antibody pair and ensuring reliable quantification of Mouse IL-6 in biological samples.
ELISA kits for Mouse IL-6 estimation offered by KRISHGEN uses Anti Mouse IL-6 capture proteins as above
3. Assay Validation
– IC50 Value : ~ 294.3 pg/ml (within 0-2000 pg/ml assay range). – LLOQ : ~ 31.25 pg/ml.
– Clinical Cmax Values* : NA – Mouse IL-6 is an endogenous cytokine produced by immune and non-immune cells and is not administered therapeutically; therefore, no fixed dose-dependent Cmax values are defined.
– Published studies indicate that Mouse IL-6 concentrations are generally low or undetectable under normal physiological conditions but can increase from low pg/mL levels to several hundred or thousand pg/mL in response to infection, inflammation, autoimmune disorders, tissue injury, or other immune stimuli.
– Precision:
– Intra-Assay CV: <10%.
– Inter-Assay CV: <12%.
– Inter-Operator CV: <10%.
4. Standard Curve
Below is the standard curve for Mouse IL-6 Sandwich ELISA assay:
Linearity and Range
5. LOD and LOQ
– LOD Absorbance : ~31 pg/ml
– LOQ Absorbance : ~31.25 pg/ml
6. Pharmacokinetic Relevance
The assay is designed to detect physiologically and pathologically relevant levels of Mouse Interleukin-6 (IL-6) in biological samples, making it suitable for cytokine profiling, inflammation studies, and immunological research applications.
The Mouse IL-6 ELISA demonstrates high sensitivity, typically within the pg/mL range, ensuring accurate detection and quantification of IL-6 across biologically meaningful concentrations.
Published studies evaluating Mouse IL-6 expression indicate variable cytokine levels depending on the disease model, inflammatory stimulus, and sample type:
• IL-6 concentrations are influenced by the degree of immune activation, infection status, inflammatory response, and experimental treatment conditions.
• Baseline IL-6 levels in healthy mice are generally low or undetectable, whereas significantly elevated concentrations may be observed in inflammatory, autoimmune, infectious, and tumor-associated models.
• Increased IL-6 levels are often associated with enhanced inflammatory activity, acute-phase responses, and disease progression.
Thus:
• Mouse IL-6 concentrations in serum, plasma, cell culture supernatants, and tissue homogenates typically fall within the measurable range of the ELISA.
• The assay working range enables reliable differentiation between basal, moderate, and elevated cytokine expression levels.
• In samples exhibiting high IL-6 concentrations, appropriate sample dilution may be required to ensure measurements fall within the linear dynamic range of the assay.
• The assay is therefore suitable for cytokine profiling, assessment of inflammatory responses, therapeutic efficacy studies, and biomarker research in mouse models.
7. Precision and Reproducibility
Precision was assessed by analysing three standard concentrations (62.5 pg/ml, 250 pg/ml, and 2000 pg/ml). Each concentration was tested in triplicate across three independent assay runs. %CV (Coefficient of Variation) was calculated within runs (intra-assay precision) and across runs (inter-assay precision).
Acceptance Criteria:
Intra-assay %CV should be ≤15% for samples.
Inter-assay %CV should be ≤15% for samples.
%CV at LLOQ (Lower Limit of Quantitation) allowed up to 20%.
Precision Results Summary:
Observations:
Intra-assay precision was consistently less than 10% across all levels tested.
Inter-assay precision was consistently less than 12%.
All precision values met the acceptance criteria for ELISA validation.
Conclusion:
The GENLISA® Mouse Interleukin-6 (IL-6/IL6) ELISA demonstrates excellent intra- and inter-assay precision. These results support the assay’s reliability and reproducibility for routine use in pharmacokinetic and bioanalytical studies.
8. Linearity
The linearity study was performed to evaluate the ability of the GENLISA® Mouse Interleukin-6 (IL-6/IL6) ELISA ELISA to generate results that are directly proportional to the concentration of Mouse IL-6 across a range of sample dilutions.
Observations:
Mouse IL-6 -spiked serum samples demonstrated recoveries ranging from 82–112% across the tested dilution series.
Mouse IL-6 -spiked EDTA plasma samples demonstrated recoveries ranging from 83–118% across the tested dilution series.
Mouse IL-6 -spiked heparin plasma samples demonstrated recoveries ranging from 80–99% across the tested dilution series.
All matrices exhibited acceptable dilution linearity with recoveries within the predefined acceptance criteria.
9. Conclusion
The GENLISA® Mouse Interleukin-6 (IL-6/IL6) is validated for sensitivity, specificity, precision, and accuracy, and is appropriate for pharmacokinetic applications in regulatory settings.
