Vedolizumab Specific ELISA Kit
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- Target
- Vedolizumab Specific
- Reactivity
- Chemical, Human
- Detection Method
- Colorimetric
- Method Type
- Sandwich ELISA
- Application
- ELISA
- Purpose
- Enzyme immunoassay for the specific quantitative determination of free Vedolizumab in serum and plasma
- Sample Type
- Plasma, Serum
- Analytical Method
- Quantitative
- Specificity
- There is no cross reaction with any other proteins present in native human serum. A screening test was performed with 24 different native human sera. All produced OD450/620 nm values (ranged from 0.097 to 0.166) less than the mean OD (0.311) of standard D (6 ng/mL). In addition, binding of Vedolizumab to the solid phase is inhibited by recombinant human 47 integrin protein in a concentration dependent manner. Therefore, the ImmunoGuide Vedolizumab ELISA (mAb-based) measures the biologically active free form of Vedolizumab, i.e. not pre-occupied by human 47 integrin antigen. No cross reaction was observed with sera spiked with the other therapeutic antibodies including Infliximab, Adalimumab, Etanercept, Bevacizumab, Cetuximab, Tocilizumab, Rituximab, and Nivolumab at concentrations up to 100 μg/mL. All produced mean OD450/620 nm values (ranged from 0.035 to 0.058) less than standard D.
- Sensitivity
- 5 ng/mL
- Components
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Microtiter ELISA Plate
Vedolizumab Standards (A-E, 10X)
Assay Buffer
Dilution Buffer
Enzyme Conjugate
TMB Substrate Solution
Stop Solution
Wash Buffer
Adhesive Seal - Material not included
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1. Micropipettes (< 3 % CV) and tips to deliver 5-1000 μL.
2. Bidistilled or deionised water and calibrated glasswares (e.g. flasks or cylinders).
3. Wash bottle, automated or semi-automated microtiter plate washing system.
4. Microtiter plate reader capable of reading absorbance at 450 nm (reference wavelength at 600-650 nm is optional).
5. Absorbent paper towels, standard laboratory glass or plastic vials, and a timer.
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- Application Notes
- Optimal working dilution should be determined by the investigator.
- Comment
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The Vedolizumab ELISA (mAb-based) is suitable also for being used by an automated ELISA processor.
- Sample Volume
- 10 μL
- Plate
- Pre-coated
- Protocol
- This ELISA is based on a Vedolizumab-specific monoclonal antibody (catcher Ab clone19F3). Diluted standards and samples are incubated in the microtiter plate coated with mAb. After incubation, the wells are washed. A horseradish peroxidase (HRP)-conjugated anti-human IgG antibody is added and binds to the Fc part of Vedolizumab. Following incubation, wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The color developed is proportional to the amount of Vedolizumab in the sample or standard. Results of samples can be determined by using the standard curve.
- Reagent Preparation
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Prepare Wash and Dilution Buffers before starting the assay procedure
- Sample Preparation
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Standards and samples should be properly diluted as homogenous mixture before starting the assay procedure. It is recommended mixing the standards and samples well to homogenous solution at each dilution step.
- Calculation of Results
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A standard curve should be calculated using the standard concentration (X-axis) versus the OD450 (or OD450/620) values (Y-axis). This can be done manually using graph paper or with a computer program. Concerning the data regression by computer, it is recommended to primarily use the "4 Parameter Logistic (4PL)" or alternatively the "point-to-point calculation". In case of manual plot there are 2 options: Semilog graph (see Fig. A) or linear graph (see Fig. B).
The concentration of the samples can be read from this standard curve as follows. Using the absorbance value for each sample, determine the corresponding concentration of the drug from the standard curve. This value always has to be multiplied by the individual dilution factor (usually 200). If any diluted sample is reading greater than the highest standard, it should be further diluted appropriately with 1X Dilution Buffer and retested. Also this second dilution has to be used for calculation the final result.
It is proposed that a sample with an OD equal or lower than the standard D should NOT be plotted on the standard curve, instead should be reported as "below the detection limit". - Assay Precision
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Intra-assay CV: < 10%.
Inter-assay CV: < 10% - Restrictions
- For Research Use only
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- Storage
- 4 °C
- Storage Comment
- The kit is shipped at ambient temperature and should be stored at 2-8°C. Keep away from heat or direct sun light. The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2-8°C.
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- Target
- Vedolizumab Specific
- Background
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The drug Vedolizumab (trade name Entyvio®) is a humanised immunoglobulin G1 monoclonal antibody that binds exclusively to the lymphocyte integrin α4β7, is indicated for the treatment of adult patients with moderately to severely active ulcerative colitis [UC] or Crohn's disease [CD]. According to the prescribing information, the mean ± SD of Vedolizumab steady-state trough serum concentrations in patients with UC and CD are reported to be 11.2±7.2 μg/mL (n=77) and 13.0±9.1 μg/mL (n=72) respectively. It was also reported in an other study that higher Vedolizumab trough concentrations were associated with greater efficacy in patients with both UC and CD. Higher Vedolizumab trough concentrations also were associated with numerically higher rates of clinical response and mucosal healing at week 6 in patients with UC. As with all therapeutic proteins, there is a potential for immunogenicity. According to the prescribing information, in patients who received Vedolizumab, the frequency of antibodies detected in patients was 13 % at 24 weeks after the last dose of study drug. During treatment, 56 of 1434 (4 %) of patients treated with Vedolizumab had detectable anti-Vedolizumab antibodise at any time during the 52 weeks of continuous treatment. Nine of 56 patients were persistently positive for anti-Vedolizumab antibodies and 33 of 56 patients developed neutralizing antibodies to Vedolizumab. The presence of persistent anti-Vedolizumab antibodies was observed to substantially reduce serum concentrations of Vedolizumab, either to undetectable or negligible levels at weeks 6 and 52.
Identification of biomarkers might be beneficial for (non-) response and risk factors for adverse drug reactions that might be related to serum drug levels and maintaining the effective concentration in order to potentially avoid some side effects with a reliable method.
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