ELISA/assay

E.coli HCP (Expression strain) ELISA

DEIA-JY2303

96T

E.coli HCP (Expression strain) ELISA

E. coli HCP

E.coli HCP ELISA

sELISA

E. coli HCP

Quantitative

Biological samples

96T

Store the unopened reagents at 2–8°C until expiration date. Do not use components beyond the expiry date indicated on the kit labels. Once opened the reagents are stable for 2 months when stored at 2–8°C. The dissolved standard can be stable for 30 days at 2-8°C. Once the resealable pouch has been opened, care should be taken to close it tightly again.

No cross-reactivity was observed with CHO, Vero, HEK293T, and Hansen yeast host proteins.

The E.coli Expression Strain HCP ELISA kit is designed for quantifying host cell proteins (HCPs) in Escherichia coli expression strain BL21. It is suitable for quantifying HCPs in intermediate and crude samples of recombinant protein products, including interleukins (IL), interferons (rhIFN), granulocyte-macrophage colony-stimulating factors (rhGM-CSF), necrosis factors (rhTNF), growth factors (EGF/FGF/PDGF), etc.

This assay kit is based on the Enzyme-linked Immunosorbent Assay (ELISA) using a sandwich format to detect residual levels of E.coli expression strain HCPs in samples. The method involves capturing HCPs using specific polyclonal antibodies, along with the addition of HRP-labeled anti-E.coli expression strain HCP antibodies. After incubation and washing, the addition of TMB substrate allows HRP to generate a blue product (maximum absorption at 655 nm). The reaction is then stopped and turns yellow (maximum absorption at 450 nm). The absorbance at 450 nm is directly proportional to the HCP concentration, which can be determined through a dose-response curve. No special treatment is required for actual samples, and the kit offers simplicity, speed, strong specificity, and reliable performance.

1. E.coli HCP-E Standard: 2 vials. Lyophilized. Dilute with 0.5 ml of Standard Dilution. Refer to the label on the vial for concentration. 2. Pre-coated microtitre strips: 1 × 12 × 8. Sheep anti-E. coli expression strain HCPs antibodies have been coated and packaged in aluminum foil bags with a drying agent. Seal the bags tightly after use for proper storage. 3. Standard Dilution: 1 × 1.5 ml. Dedicated to dissolving the E.coli HCP-E standard. 4. Samples/Antibody Dilution Buffer: 2 × 25 ml. Dilute samples and detection antibody-HRP. It is advised to perform a sample suitability verification to determine the optimal dilution ratio for primary test samples. 5. Washing Buffer (10× conc.): 2 × 25 ml. Crystization may occur at low temperatures. Prior to use, warm the reagent in a 37°C water bath and dilute it with freshly prepared ultrapure water at a 10-fold dilution to obtain a 1× buffer solution for plate washing. 6. Detection Antibody-HRP (100× conc.): 1 × 120 μl. HRP-labeled Antibody against E.coli expression strain host cell proteins (HCPs). Dilute 100-fold with the dilution buffer before use. 7. TMB Substrate: 12 ml. Allow the reagents to equilibrate at room temperature for a minimum of 20 minutes prior to use. Store the reagents in a light-protected and tightly sealed container. 8. Stop Solution: 6 ml. 9. 3 Cover foil.

1. Microplate reader capable of measuring absorbance at 450 nm and 620-650 nm 2. Incubator 37°C 3. Shaker 4. Precision pipettes to deliver 0.5 μl to 1 ml volumes 5. Absorbent paper 6. Distilled or deionized water 7. Log-log graph paper or computer and software for ELISA data analysis 8. Tubes to prepare the standard or sample dilutions

Samples: These include expressing purified process samples, stock solutions and other solutions. The samples should be clear and transparent, with insoluble components removed through methods like centrifugation or filtration. Storage: Prior to storing the samples, it is crucial to establish their stability and identify the optimal storage conditions. For long-term storage, it is generally recommended to keep samples at -65°C or below, while minimizing freeze-thaw cycles. For initial use or situations where the HCP content in the sample is unknown, it is highly advisable to conduct sample suitability verification to determine the suitable sample dilution ratio for accurate subsequent routine testing. Dilute the samples appropriately with a 1× dilution buffer based on their estimated concentration of HCPs, ensuring that the detection values fall within the quantification range of the calibration curve.

Bring all kit components and samples to room temperature (18-25°C) before use. If the kit will not be used up in 1 time, please only take out strips and reagents for present experiment, and save the remaining strips and reagents as specified. 1. 1× Wsahing Buffer : Prior to use, warm the reagent in a 37°C water bath and dilute it with freshly prepared ultrapure water at a 10-fold dilution to obtain a 1× Washing Buffer for plate washing. 2. E.coli HCP-E Standard : Centrifuge the Standard at 1000 ×g for 1 minute. Reconstitute the Standard with 0.5 mL of Standard Diluent Buffer, kept for about 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is on the label of vial. 3. Standard Working Solution - Please prepare 7 tubes containing 0.6 mL Standard Diluent Buffer and use the Diluted Standard to produce a double dilution series according to the picture shown below. To mix each tube thoroughly before the next transfer, pipette the solution up and down several times. Set up 7 points of Diluted Standard such as 243 ng/mL, 81 ng/mL, 27 ng/mL, 9 ng/mL, 3 ng/mL, 1.5 ng/mL and the last EP tubes with Standard Diluent is the Blank as 0 ng/mL. In order to guarantee the experimental results validity, please use the new Standard Solution for each experiment. When diluting the Standard from high concentration to low concentration, replace the pipette tip for each dilution. Note: the last tube is regarded as the Blank and do not pipette solution into it from the former tube. 4. Detection Antibody-Biotin (100× conc.): Dilute 100-fold with the 1× Dilution buffer.

1. Add 100 μL of the Detection Antibody-HRP into the corresponding microplate wells. Ensure that the solution is added to the bottom of the wells and avoid generating bubbles during the operation. 2. Add 100 μL of the Standard Work Solution and 1× Dilution Buffer (NCS) into the corresponding microplate wells. Prepare 2-3 replicate wells for each concentration and record the location of each concentration well. 3. Add the prepared test samples into the corresponding microplate wells. After adding the samples, seal the microplate with a sealing film and place it on a microplate constant temperature shaker. Incubate at room temperature at 600 rpm for 3 hour. 4. Refer to the layout in Table below to arrange the 96-well plate based on the number of samples. This example shows a calibration of 6 concentrations (ST1-ST6), a negative control (NCS), 3 test samples (S1-S3), and sample recovery control (S1 SRC-S3 SRC) for each test. 2-3 replicate wells for each test. 5. After incubation, remove the plate sealer, discard the contents and wash the strips 5 times with 340 μL 1× Washing Buffer, ensuring every well is filled. When washing is completed, tap the strips firmly on absorbent tissue to remove residual Washing Buffer. 12. Add 100 μl TMB substrate into each well. 13. Do not cover the strips! Incubate for 15 minutes at room temperature (15-30°C) * in the dark. 14. Add 50 μl stop solution (STOP) into each well and mix well. 15. Determine absorption after 5 minutes with an ELISA reader at 450 nm and 620 nm-650 nm as a reference. If no reference wavelength is available, read only at 450 nm. * The intensity of the color change is temperature sensitive. We recommend observing the color change and stopping the reaction upon good differentiation.

For samples with absorbance values exceeding the calibration point ST1, they can be further diluted with 1× dilution buffer and retested. The concentration of HCP antigen in the sample is determined by multiplying the measurement value after dilution by the dilution factor. At the same time, set appropriate spiked samples at this dilution level, and ensure that the accuracy (recovery rate) meets the methodological validation requirements according to the corresponding regulations.

1. The OD450 nm values of each well should be subtracted by the respective long wavelength OD value. If the plate reader is not equipped with a long wavelength filter, this step can be omitted. 2. After subtracting the OD value of the negative control from the OD values of each calibration point and sample, calculate the average of the 3 replicate wells. Perform a four-parameter fit of the concentration values and OD values of the calibration points to obtain the calibration curve equation. Substitute the average OD value of the sample into the equation to calculate the sample concentration. This concentration should be multiplied by the dilution factor to obtain the actual concentration of the sample. 3. The curve fitting software can be the one provided with the plate reader. If not available, it is recommended to use professional curve fitting software such as Curve Expert, ELISA Calc, etc.

1.5-243 ng/ml

1.5 ng/ml

The personnel using the kit must undergo training and be qualified before use. To obtain satisfactory test results, please pay attention to the following points: 1. Use sterile disposable tips, tubes, and sample wells for all reagent preparations, and do not mix them. Avoid contamination at the connection part of the pipette tip, and it is recommended to wipe it with 75% alcohol before and after each experiment. Standardize pipetting operations and strictly prohibit liquid backflow into the pipette or placing the pipette sideways on the table without removing the tip. 2. Gently and thoroughly mix the dilutions of calibration standards and samples, avoiding the generation of excessive foam. 3. The stop solution is an acidic solution, so when using it, pay attention to the protection of eyes, face, hands, and clothing. 4. It is not recommended to mix reagents from different kit batches. 5. Use sterile water or freshly prepared ultrapure water for buffer preparation, and the water temperature should not exceed 37 degrees Celsius. 6. When adding samples, add them to the bottom of the microplate wells and try to avoid touching the well walls. Be careful not to have any bubbles, and gently shake to mix. If there are bubbles present before testing, use a clean 10 μL pipette tip or needle to puncture them, taking care not to aspirate the liquid in the well, which could lead to significant result errors. 7. Cover the microplate with a film during the incubation process to prevent sample evaporation. 8. After discarding the buffer, immediately add the subsequent solution to prevent the microplate wells from drying, which could affect the kit's performance. 9. Unused microplate strips should be stored light-protected in the self-sealing aluminum foil bag provided with the kit to avoid contamination from other samples, which could render the kit unusable. 10. Ensure accurate preparation of calibration standards and sample dilutions, with a minimum sampling volume not less than 5 μL to prevent significant errors in the results. 11. Before use, centrifuge the biotinylated E.coli HCP-AC antibody (20×) and streptavidin-HRP conjugate (100×) quickly to remove any residual reagent on the tube cap, preventing contamination and loss of the reagent. 12. Pre-diluted calibration standards, biotinylated E.coli HCP-AC antibody, and streptavidin-HRP conjugate that have been diluted to working concentration are not recommended for reuse due to the inability to guarantee their stability. 13. The chromogenic substrate should be a colorless and transparent liquid. When aspirating it, be sure to use a clean tip to prevent HRP contamination. If a pale blue color is observed, it should be discarded. 14. Ensure a delay of 5-10 minutes after adding the stop solution before conducting the machine readout for more stable results and a time not exceeding 30 minutes. 15. Sodium azide can inhibit HRP activity, which significantly affects the test results. Therefore, sodium azide should not be added to the sample.

1. This product is exclusively for research purposes and should not be used for clinical diagnosis. 2. This product is specifically designed for detecting host cell protein content in E.coli BL21 expression strains. Compatibility testing is necessary for other types of expression strains. 3. This product is not suitable for samples derived from cloning strains or samples treated with alkaline lysis. Please contact our company for information on compatible products. 4. The pH of the sample should fall within the range of 6.5 to 8.5. Abnormally high or low pH values may result in measurement irregularities.

E.coli HCP

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See the individual product datasheet

See the individual product datasheet