ELISA/assay

Vero cell HCP ELISA Kit

DEIA-NS2311-7

96T

Vero cell HCP ELISA Kit

Vero Cell HCP

Vero cell HCP ELISA Kit

sELISA

Vero Cell HCP

Vero Cell

Quantitative

Biological samples

96T

The unopened kit is stored at 2-8°C and is valid for 12 months. Opened: The opened microplate strips, along with the desiccant, should be sealed in a self-sealing bag and stored under validated conditions at 2-8°C for up to 30 days. Reconstitution of calibration standards: The reconstituted calibration standards can be stored under -20°C. Do not repeatedly thaw and refreeze.

No cross-reactivity with engineered cell matrix host proteins such as Sf9, E.coli, and Pichia pastoris.

Vero Cell HCP

Vero cell host cell protein assay; HCP assay; Host cell protein assay; HCP ELISA kit

This kit is suitable for the quantitative detection of Vero host cell protein residues in intermediates, semi-finished products and finished products of various biological products in cell lysis processes.

The kit is based on Enzyme-linked Immunosorbent Assay (ELISA) and uses a double-antibody sandwich method to quantitatively detect residual Vero HCPs in the test sample. The polyclonal antibodies in this kit are obtained by lysing Vero cells and using HCPs as antigens. The serum is obtained by immunizing sheep, and then high-quality antibodies are obtained through affinity purification. Antibodies are evaluated for their coverage levels through current mainstream coverage analysis regulatory methods. This analysis method involves adding calibrators or test samples and HRP-labeled Vero HCP Antibodies to a microplate pre-coated with anti-Vero HCPs polyclonal antibodies for co-incubation; after washing, the added TMB Substrate is used to perform a color reaction. Finally, stop the reaction using Stop Solution. Use a microplate reader to read the absorbance value at a wavelength of 450 nm. The absorbance is positively correlated with the concentration of HCPs in the calibrator and sample, and can be calculated through the dose-response curve. Find the concentration of Vero HCPs in the sample. This kit requires no special processing of the actual sample and can be used directly only through appropriate dilution ratios for suitability verification.

1. Vero HCP calibrator, 2 vials. 2. Anti-Vero HCP microplate strips, 8×12 wells 3. Calibrator Buffer, 1.5 mL 4. Dilution buffer, 25 mL×2 5. Wash Buffer 10×, 25 mL×2 6. HRP-labeled Vero HCP Antibodies 100×, 120 μL 7. TMB Substrate, 12 mL 8. Stop Solution, 6 mL 9. Plate sealers, 3

1. Microtiter plate reader fitted with appropriate filters (450 nm required with optional 620 nm reference filter) 2. Microtiter plate washer or wash bottle 3. 10, 50, 100, 200 and 1000 μl adjustable single channel micropipettes with disposable tips 4. 50-300μl multi-channel micropipette with disposable tips 5. Multichannel micropipette reagent reservoirs 6. ddH 2 O 7. Vortex mixer 8. Orbital shaker 9. Miscellaneous laboratory plastic and/or glass, if possible sterile 10. Absorbent pads (tissue)

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 HCPs 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.

1. Remove the pre-sealed microplate from the packaging and let it equilibrate at room temperature for approximately 20 minutes. Other reagents should also be taken out in advance and allowed to equilibrate at room temperature before use. After use, promptly return them to 2-8°C storage. 2. Calculate the number of wells needed based on the number of samples to be tested. Take out the corresponding number of microplate strips, and seal the remaining strips along with the desiccant in a self-sealing bag. Place the bag back into the kit box and store it in a 2-8°C refrigerator until the expiration date. Note: Crystallization on some well walls of the microplate is a normal phenomenon and does not require any special treatment.

1. Vero HCP calibrator solution: Accurately measure 500 μL of Calibrator Buffer, add it to a stand-up cryovial, mix gently by inverting, and let stand for 5 minutes. The concentration of the reconstituted Vero HCP calibrator solution is the labeled concentration. Please store the dissolved calibrator at ≤-18°C and freeze and thaw repeatedly no more than 3 times. Note: If you need to use multiple bottles of calibrator at the same time, please dissolve them separately and transfer them to a 1.5 mL sterile centrifuge tube, shake and mix before use. If each bottle of calibration solution involves multiple freezing and thawing, it is recommended to use 1.5 mL sterile centrifuge tubes to appropriately aliquot and store at ≤ -18°C. 2. 1×Wash Buffer: Wash Buffer (10×) is diluted 10 times with ultrapure water. For example, take 25 mL Wash Buffer (10×) and add 225 mL ultrapure water and mix well, which is 1×Wash Buffer for washing. plate. Recommended for immediate use. If a plate washer is used for washing, the amount of reagent may not be enough. Wash Buffer with the same product number can be purchased separately. Note: Take out the Wash Buffer (10×) and diluent, observe if there are crystals, which is normal, and incubate at 37°C until completely dissolved. 3. Detect antibodies: Dilute 100× HRP-labeled Vero HCP Antibodies 100 times with dilution buffer in a sterile centrifuge tube, mix gently by inverting, and the result is 1× HRP-labeled Vero HCP Antibodies. Prepare appropriate volume to ensure sufficient margin when adding liquid. Configure before use and use immediately. 4. Calibration curve: Refer to the figure and table to perform the calibrator. Note: 1ng/mL serves as the anchor point to participate in the fitting of the calibration curve, but its concentration CV and relative deviation are not required in the method validation.

1. Add 100 μL of the 1×HRP-labeled Vero HCP Antibodies 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 Vero HCP calibrator solution, 1× Dilution Buffer (NCS) and sample into the corresponding microplate wells. Prepare 2-3 replicate wells for each concentration and record the location of each concentration well. 3. 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 500-600 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 spike 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. 6. Add 100 μl TMB substrate into each well. 7. Do not cover the strips! Incubate for 10 minutes at room temperature (15-30°C) * in the dark. 8. Add 50 μl stop solution (STOP) into each well and mix well. Immediately put it into the microplate reader for measurement. Note: The order of adding the stop solution must be consistent with the order of adding TMB. The pipette tip should be suspended during sample addition to avoid contact with the solution in the microplate and avoid generating bubbles. 9. Determine absorption 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.

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 2 or 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. For samples whose absorbance value exceeds the calibrator ST1, it can be diluted with a dilution buffer to an appropriate multiple before measurement, then the HCPs antigen concentration value in the sample = measured value after dilution × dilution multiple. If a suitable spiked sample at this dilution is also set, the recovery rate meets the method verification requirements of the corresponding regulations.

LLOQ: 3 ng/mL

3-243 ng/mL, R 2 >0.990

1. All reagent preparations must use sterile disposable pipette tips, test tubes, and sampling tanks, and do not mix them. To avoid contamination of the micropipette tip connection part, it is recommended to wipe it with 75% alcohol before and after each experiment. Standardize pipetting operations. It is strictly forbidden to suck liquid back into the pipette, or place it horizontally on the table without removing the pipette tip. 2. The dilution and mixing of the calibrator and sample should be gentle and thorough, and avoid generating a lot of foam. 3. The stop solution is an acidic solution. Pay attention to the protection of your eyes, face, hands and clothes during use. 4. It is not recommended to mix kits from different batches. 5. The water used to prepare the buffer solution must be sterile water or freshly prepared ultrapure water, and the water temperature must not exceed 37°C. 6. When adding the sample, add the sample to the bottom of the enzyme plate and try not to touch the well wall. Be careful not to have air bubbles, shake gently to mix. If there are bubbles before testing on the machine, they need to be punctured with a clean 10 μL pipette or needle. Be careful not to suck away the liquid in the hole, which will lead to large error in the results. 7. During the incubation reaction, the enzyme plate needs to be covered with a film to prevent the sample from evaporating. 8. After pouring out the buffer, add the subsequent solution immediately. Do not let the enzyme label wells dry to avoid affecting the detection performance of the kit. 9. Unused enzyme labeling strips must be stored in the self-sealing aluminum foil bag provided with the kit to avoid being contaminated by other samples and causing the kit to be scrapped. 10. Calibrator preparation, sample dilution, etc. must be accurate. The minimum sampling volume during preparation should not be less than 5 μL to prevent large errors in the results. 11. Vero HCP enzyme-labeled antibody (100×) should be centrifuged quickly before use, and the remaining reagents in the tube cap should be thrown to the bottom of the tube to prevent contamination and loss of reagents. 12. Calibrators, 1× Vero HCP HRP-labeled antibodies, etc. that have been diluted to working concentrations are not recommended to be reused because their stability cannot be guaranteed. 13. The chromogenic solution should be a colorless and transparent liquid. Be sure to replace the clean tip when sucking to prevent HRP contamination. If you find that there is already light blue, please discard it. 14. Since sodium azide can inhibit HRP activity and have a great impact on the test results, sodium azide cannot be added to the sample.

1. This product is for research use only and is not for clinical diagnosis. 2. The pH value of the sample should be 6.5~8.5. A pH value that is too low or too high may cause abnormal measurement values.

1. For samples whose absorbance value exceeds the calibrator ST1, it can be diluted with a dilution buffer to an appropriate multiple before measurement, then the HCPs antigen concentration value in the sample = measured value after dilution × dilution multiple. If a suitable spiked sample at this dilution is also set, the recovery rate meets the method verification requirements of the corresponding regulations.

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet