E.coli HCP ELISA Kit (One-step ELISA) HYI112
This kit employs a solid-phase Enzyme-linked Immunosorbent Assay (ELISA) with a double-antibody sandwich technique to detect residual E.coli (Protein expression strains) host cell proteins (HCPs) in the sample. Polyclonal antibody specific to E.coli (Protein expression strains) 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 with anti-E.coli (Protein expression strains) HCP antibody were simultaneously added to the pre-coated microtiter plate, and followed by incubation and washing steps. Then TMB (3,3',5,5'-tetramethylbenzidine) substrate added for reaction, HRP catalyzed the oxidation of TMB by H2O2 to produce a blue product (maximum absorption peak at 655nm). Afterwards the stop solution is added to terminate the enzymatic reaction, resulting in a yellow color 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 E.coli (Protein expression strains) 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, using this kit.)
Applications
This kit is intended for use in determining the presence of host cell proteins (HCPs) in products manufactured by expression in E.coli originated from BL21, such as interleukin (IL), recombinant human interferon (rhIFN), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), recombinant human tumor necrosis factor (rhTNF), growth-promoting factor (EGF/FGF/PDGF), et.al. The kit is for RESEARCH USE ONLY and not intended for clinical use.
Figure 1. Schematic diagram
Components&Storage Conditions
|
Reagent |
Quantity |
Notes |
|
E.coli HCP-E Calibration Standard |
2 bottles |
Lyophilized powder. Dissolve it with 500μL Reconstitution Solution and let it stand for about 5 minutes until transparent. Please refer to the details on the label of the tube. |
|
Anti-E.coli HCP-E Microtiter Strips |
8 well ×12 strips |
Strips pre-coated with sheep anti-E.coli(Protein expression strains) HCP affinity antibody in a vacuumed bag with desiccant. Seal and store immediately after use. |
|
Reconstitution Solution |
1×1.5 mL |
Only used for dissolving E.coli HCP-E Calibration Standard. |
|
Diluent |
2×25 mL |
For dilution of Calibration Standard, Anti-E.coli-E:HRP (100×) and samples. |
|
Wash Buffer Concentrate (10×) |
2×25 mL |
It is easy to crystallize at low temperatures, and redissolved in 37 ℃ water bath before use. Dilute 10 times with freshly prepared ultra-pure water for plate washing. |
|
Anti-E.coli HCP-E:HRP (100×) |
1×120 μL |
Affinity purified sheep antibody conjugated to HRP need to be 100 times diluted in diluent. |
|
TMB Substrate |
1×12 mL |
Equilibrate to room temperature for 20 minutes before use. Sealed and keep away from light. |
|
Stop Solution |
1×6 mL |
1 M hydrochloric acid. Avoid direct contact with eyes, skin, and clothing. Wear goggles while handling. |
|
Sealing Film |
3 pieces |
Cover the strips with it during incubation to prevent contamination and liquid evaporation. |
Note: Room temperature refers to 25±3℃.
Storage Conditions
Store the kit at 2-8℃. Use within the expiration date labeled upon the kit package. The opened components should be stored as follows.
Table 2. Recommended storage conditions for opened components
|
Component |
Stability |
|
Anti-E.coli HCP-E Microtiter Strips |
Store in the bag with desiccant at 2-8℃ for up to 60 days. |
|
Reconstituted E.coli HCP-E Calibration Standard |
Store at 2-8℃ for up to 30 days |
Required materials not included in the kit
1.Sterile centrifuge tubes for dilution
2.Clean tissue paper for plate drying
3.Pipette Tips
4.Multi-channel reagent reservoirs (50 mL)
5.Microplate thermoshaker
6.Incubator (optional)
7.Plate washer (optional)
Related equipment
1.Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 620 nm to 650 nm.
2.Single or multi-channel pipettes