| Field | Specification |
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| Alternative Names | AGS-22CE, AGS-22M, AGS-22M6E, unconjugated : AGS-22C3 or AGSM6, CAS: 1346452-25-2 |
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| Cas No. | |
| Detection Method | |
| Detection Range | |
| Product Type | |
| Reactivity | |
| Sample Type(s) | Plasma, Serum |
| Sensitivity | |
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Overview
Enfortumab ELISA Kit is an ELISA-based immunoassay designed for quantitative measurement of Enfortumab in research samples. It is commonly used to generate traceable concentration data for biomarker discovery, pathway studies, and comparative analyses across experimental conditions.
Key elements and design rationale
- Assay format: Quantitative Colorimetric ELISA. The format defines how signal scales with analyte abundance and how results are interpreted across a standard curve.
- Working range and sensitivity: dynamic range 93.75 - 1,500 ng/mL; analytical sensitivity 47.39 ng/mL. Use these values to plan dilutions and keep readouts within the linear portion of the calibration curve.
- Sample compatibility: Intended for Plasma, Serum matrices. As with most immunoassays, matrix composition can influence apparent signal and should be evaluated with dilution linearity and spike-recovery concepts.
- Recovery reference: Typical recovery is reported as 80-120%. Recovery helps assess whether the sample matrix interferes with detection of spiked analyte.
PRINCIPLE OF THE ASSAY This assay employs the quantitative competitive enzyme immunoassay technique. Recombinant Human NECTIN4 has been pre-coated onto a microplate. Standards or samples are premixed with biotin-labeled antibody and then pipetted into the wells. Enfortumab in the sample competitively binds to the pre-coated protein with biotin-labeled Enfortumab. After washing away any unbound substances, Streptavidin-HRP is added to the wells. Following a wash to remove any unbound enzyme reagent, a substrate solution is added to the wells and color develops in inversely proportion to the amount of Enfortumab bound in the initial step. The color development is stopped and the intensity of the color is measured.
Biological background
Enfortumab vedotin is an antibody-drug conjugate (ADC) designed for the treatment of cancer expressing Nectin-4. It was developed through two main lines, hybridoma (ASG-22ME) and Chinese hamster ovary (ASG-22CE). Enfortumab refers to the fully humanized (from mouse) monoclonal antibody (mAb) created by scientists at Agensys (part of Astellas) using Amgen’s transgenic system (XenoMouse), it is the first agent to target Nectin-4 that expressed on many solid tumors especially on bladder cancers. Vedotin refers to the payload drug microtubule-disrupting agent monomethyl auristatin E (MMAE) and the linker. The linker technology holding the antibody and the toxin together was provided by and licensed from Seattle Genetics. Preclinical studies showed that enfortumab vedotin effectively binds to target cells, internalizes and induces cell-killing activity. Mouse and patient xenograft models were used to test enfortumab vedotin’s antitumor activity in human breast, bladder, pancreatic and lung cancers. In March 2018, Seattle Genetics and Astellas received the U.S. Food and Drug Administration (FDA) Breakthrough Therapy Designation for enfortumab vedotin based on interim results from the phase 1 study examining enfortumab vedotin as monotherapy treatment for patients with metastatic urothelial cancer who were previously treated with checkpoint inhibitors.
Research relevance and current trends
- Biomarker translation in RUO settings: Increasing use of quantitative immunoassays to stratify experimental cohorts, track longitudinal changes, and benchmark model systems.
- Matrix-aware assay design: Greater emphasis on dilution linearity, spike-recovery, and control concepts to reduce matrix-driven artifacts in serum/plasma and complex lysates.
- Integration with multi-omics: ELISA measurements are often used alongside transcriptomics and proteomics to connect abundance changes with pathway activity and phenotype.
Common research applications
- Comparative quantification: Measure relative changes in analyte levels across treatments, time points, or genotypes to support mechanistic hypotheses.
- Assay development and standardization: Generate reproducible concentration inputs for method qualification, inter-operator comparisons, or bridging studies across platforms.
- Model and sample characterization: Profile baseline and stimulated levels to help interpret immune, endocrine, neurodegenerative, or metabolic phenotypes (as relevant to the target).
Interpretation typically focuses on direction and magnitude of change in the context of controls and sample handling metadata, rather than single-point absolute values.
Notes for experimental interpretation
- Matrix effects: Hemolysis, lipemia, and high protein content can alter background and apparent concentration. Consider consistent collection/processing and evaluate dilution behavior.
- Isoforms and modified forms: Some targets exist as isoforms, fragments, or post-translationally modified species. Ensure the measured form aligns with the biological question and the kit’s intended analyte definition.
- Control concepts: Use negative/blank controls, replicate wells, and—when feasible—orthogonal confirmation (e.g., WB or MS) to strengthen conclusions.
What is this PK ELISA kit designed for?
This pharmacokinetic (PK) ELISA kit is designed to quantitatively measure Enfortumab (CAS No. 1346452-25-2) concentrations in Human biological matrices. It supports PK profiling studies including determination of Cmax, AUC, half-life (t«), clearance, and volume of distribution in preclinical or clinical research settings.
What biological matrices are validated for this assay?
This PK ELISA has been validated for Plasma and Serum. For PK studies, serum or plasma (EDTA or heparin-treated) are the most common matrices. Non-validated matrices (e.g., CSF, urine, tissue lysate) require additional matrix validation before use, including matrix effect assessment and dilutional linearity testing.
What is the quantification range and LLOQ for this kit?
The standard curve (LLOQ?ULOQ) spans 93.75 - 1,500 ng/mL. The lower limit of quantification (LLOQ) is 47.39 ng/mL. Samples with drug concentrations above the ULOQ should be diluted in drug-free matrix and re-assayed. The LLOQ is defined as the lowest concentration that can be measured with acceptable accuracy (ó20% RE) and precision (ó20% CV).
Does the assay distinguish between free and total Enfortumab?
The assay format (free vs. total Enfortumab measurement) depends on the capture and detection antibodies used. Please refer to the datasheet for information on whether the kit measures free (unbound), total (free + target-bound), or both forms of the drug. This distinction is critical for interpreting PK parameters and receptor occupancy.
Can this kit be used for GLP-regulated bioanalytical studies?
This kit is classified as research use only (RUO). For regulated PK studies submitted to regulatory agencies (FDA, EMA), the bioanalytical method must be fully validated per FDA Guidance (2018 Bioanalytical Method Validation) or EMA equivalent. This kit may be used as a platform for method development prior to GLP validation.
Is there cross-reactivity with endogenous proteins or related drugs?
The selectivity of this assay for Enfortumab over structurally related endogenous proteins or biosimilars depends on the antibody specificity described in the datasheet. Researchers should evaluate cross-reactivity with endogenous analogues (e.g., endogenous hormone counterparts) and co-administered drugs that share structural similarity when designing PK studies.
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