Description
Anti-Risankizumab Free Drug ELISA Kit
Enzyme immunoassay for the quantitative determination of free Risankizumab in serum and plasma. The Assay Genie Risankizumab ELISA has been especially developed for the quantitative analysis of free Risankizumab in serum and plasma samples.
Solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. Standards and samples (serum or plasma) are incubated in the microtiter plate coated with the reactant for Risankizumab. After incubation, the wells are washed. Then, horse radish peroxidase (HRP) conjugated probe is added and binds to Risankizumab captured by the reactant on the surface of the wells. Following incubation wells are washed and the bound enzymatic activity is detected by addition of tetramethylbenzidine (TMB) chromogen substrate. Finally, the reaction is terminated with an acidic stop solution. The colour developed is proportional to the amount of Risankizumab in the sample or standard. Results of samples can be determined directly using the standard curve.
| Application | Free drug |
| Required Volume (µL) | 10 |
| Total Time (min) | 70 |
| Sample Type | Serum, Plasma (EDTA, Heparin) |
| Number of Assays | 96 |
| Sensitivity (LOD, ng/mL) | 10 |
| Functional Sensitivity (LOQ, ng/mL) | 6.125 |
| Linearity (r²) | >0.95 |
| Recovery | <100±30% |
| Precision (Intra/Inter-assay CV) | <30% |
| Component | Size | Description |
| Microtiter Plate | 1 x 12 x 8 | Break apart strips. Microtiter plate with 12 rows each of 8 wells coated with reactant. |
| Standards A-E | 1.0 mL (each) | Standard A: 300 ng/mL | Standard B: 100 ng/mL | Standard C: 30 ng/mL | Standard D: 10 ng/mL | Standard E: 0 ng/mL. Ready to use. Contains Risankizumab, human serum and stabilizer, <0.1% NaN3 |
| Control Low & High | 1.0 mL (each) | Ready to use. Contains human serum and stabilizer, <0.1% NaN3. Control concentrations given in Quality Control Certificate. |
| Assay Buffer | 1 x 50 mL | Ready to use. Blue coloured. Contains proteins, <0.1% NaN3. |
| Conjugate | 1 x 12 mL | Horse radish peroxidase conjugated probe. Ready to use. Red coloured. Contains HRP conjugated probe, stabilizer and preservatives. |
| Substrate | 1 x 12 mL | TMB substrate solution. Ready to use. Contains 3,3′,5,5′-Tetramethylbenzidine (TMB). |
| Stop Buffer | 1 x 12 mL | TMB stop solution. Ready to use. 1N HCl. |
| Wash Buffer | 1 x 50 mL | Wash buffer (20x). Prepared concentrated (20x) and should be diluted before the test. Contains buffer with tween 20. |
| Adhesive Foil | 2 x 1 | For covering microtiter plate during incubation. |
| Parameter | Conditions |
| Specimen Type | Serum, Plasma (EDTA, Heparin) |
| Storage (2-8°C) | 2 days |
| Storage (-20°C) | 6 months |
| Precautions | Do not use grossly haemolytic, icteric or lipemic specimens. Samples appearing turbid should be centrifuged to remove particulate material. Avoid repeated freeze-thaw cycles. |
Total Assay Time: 70 minutes
Pre-test Setup - Wash Buffer Preparation (must be prepared before starting assay):
| Dilute | 10 mL |
| With | Up to 200 mL distilled water |
| Dilution Ratio | 1/20 |
| Remarks | Warm up to 37°C to dissolve crystals. Mix vigorously. Storage: 2-8°C. Stability: 2 weeks. |
Sample Dilution:
| Sample | Serum/Plasma |
| Diluent | Assay Buffer |
| Dilution Ratio | 1/100 |
| Procedure | 5 µL sample + 495 µL assay buffer |
Assay Steps:
- Pipette 100 µL Assay Buffer into each well to be used.
- Pipette 10 µL of each Standards (A-E), Low level control, High level control and diluted samples into respective wells (A1: Standard A, B1: Standard B, C1: Standard C, D1: Standard D, E1: Standard E, F1: Low level control, G1: High level control, H1 and on: Samples).
- Cover plate with adhesive foil. Briefly mix by gently shaking. Incubate 30 minutes at room temperature (18-25°C).
- Remove foil. Discard incubation solution. Wash plate three times each with 300 µL Wash Buffer. Remove excess by tapping inverted plate on paper towel.
- Pipette 100 µL Conjugate into each well.
- Cover plate with adhesive foil. Incubate 30 minutes at room temperature (18-25°C).
- Remove foil. Discard incubation solution. Wash plate three times each with 300 µL Wash Buffer. Remove excess by tapping inverted plate on paper towel.
- Pipette 100 µL Substrate into each well.
- Incubate 10 minutes without foil at room temperature (18-25°C) in the dark.
- Stop substrate reaction by adding 100 µL Stop Solution into each well. Briefly mix by gently shaking. Colour changes from blue to yellow.
- Measure optical density with photometer at OD 450nm with reference wavelength 650 nm (450/650 nm) within 30 minutes after pipetting Stop Solution.
Quality Control: Test results are only valid if performed following instructions and in compliance with GLP. For valid study: OD 450/650 of highest standard should be >1.000 and OD 450/650 of lowest standard should be <0.200.
Calculations: Create standard curve using standards. OD 450/650 nm (vertical/Y-axis) versus drug concentration (horizontal/X-axis). Determine sample concentration directly from standard curve. If computer data is used, recommend "Four Parameter Logistic (4PL)" or "point-to-point calculation". Multiply determined concentration by dilution factor (100x for standard 1/100 dilution). Any sample reading greater than highest standard should be further diluted and retested.
| Balmer, C., et al. | Limited placental transfer and clearance of risankizumab by 6 months following in utero exposure | Journal of Crohn's and Colitis 2026 | View Citation |
