HEK293 HCP Elisa Kit (One-step ELISA) HYI110
This kit utilizes a solid-phase Enzyme-linked Immunosorbent Assay (ELISA) with a double-antibody sandwich technique to detect residual host cell proteins (HCPs) from HEK293 cells in the sample. A polyclonal antibody specific to HEK293 HCPs was employed in the assay to capture any remaining HCPs in the sample. Both the Calibration Standard (or test sample) and the HRP (Horseradish Peroxidase) labeled anti-HEK293 HCP antibody were simultaneously added to the microtiter plate, which coated with the affinity purified capture antibody and followed by incubation and washing. Then TMB (3,3',5,5' -tetramethylbenzidine) substrate was added into reaction, HRP catalyzed the oxidation of TMB by H2O2 to produce a blue product (maximum absorption peak at 655nm). Then the stop solution was added to terminate the enzymatic reaction, resulting in a yellow colored product (maximum absorption peak at 450nm). The absorbance values at 450nm wavelength were positively correlated with the HCPs concentration in the Calibration Standard and the sample. The concentration of HCPs in the sample can be calculated using the dose-response curve. No special treatment is required for the test sample, and its suitability could be verified by the appropriate dilution ratios with the kit.
Preparation
A. Equilibration
1. Before use, allow the kit to equilibrate at room temperature for 20 minutes; Return to 2-8℃ after use.
2. Take the appropriate amount of strips to a strip holder according to the experiment design and store the remaining strips in the bag with desiccant at 2-8℃.
B. Preparation of Reagents
1. HEK293 HCP Calibration Standard solution: Pipette 500 μL of Reconstitution Solution into the bottle containing HEK293 HCP Calibration Standard. Gently invert to mix and let it stand for 5 minutes. Save the remaining solution under the recommended condition.
Note: Do not use other volumes of Reconstitution Solution to dissolve the Calibration Standard.
2. 1 × Wash Buffer: Dilute the Wash Buffer Concentrate (10×) at 1:10 with ultra-pure water. For example, add 25mL Wash Buffer Concentrate (10×) to 225 mL of ultra-pure water to make 250 mL of 1 × Wash Buffer. Prepare fresh and mix well before use. It can be purchased separately.
Note: If the Wash Buffer Concentrate (10×) or Diluent is cloudy or contains precipitates, heat at 37℃ until it clears.
3. 1×Anti-HEK293:HRP: Prepare the 1×HEK293:HRP by diluting the Anti-HEK293:HRP (100×) with Diluent in a sterile centrifuge tube. Gently mix the solution and use it immediately.
Note: Anti-HEK293:HRP (100×) need to equilibrate at room temperature for 20 minutes, and return to 2-8℃ after use.
C. Preparation of Calibration Standard Solutions
1. Please prepare HEK293 HCP Calibration Standard Solutions according to following figure and table
D. Sample preparation
1. Test samples: In-process samples, drug substance and drug product. Samples should be clear and transparent, and insoluble substances need to be removed from samples through centrifugation or filtration.
2. Conduct sample stability studies to prevent degradation or denaturation during the experiment. Long-term storage at -70°C is recommended to avoid degradation, and avoid repeated freeze-thaw cycles.
3. Dilute the samples with a suitable diluent to achieve a concentration of host cell proteins (HCPs) within the quantification range of the calibration curve.
4. Recommend to verify sample suitability by determining the appropriate sample dilution factor before the initial test and facilitating the subsequent routine testing. Note: Please contact us for support of validation protocol.