ELISA/assay

HEK 293 HCP ELISA Kit

DEIA-JY2324

96T

HEK 293 HCP ELISA Kit

HEK 293 HCP

HEK 293 HCP ELISA Kit

sELISA

HEK 293 HCP

HEK 293 Cell

Quantitative

Biological samples

96T

Store at 2 - 8°C. Do not use the kit beyond the expiration date. 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.

It does not show significant cross-reactivity with commonly used host proteins such as E.coli, CHO, Vero, and Saccharomyces cerevisiae.

HEK 293 Cell

HEK 293 HCP; HEK 293; HEK 293T; HEK 293T HCP; HCP

The HEK 293 HCP ELISA kit (one-step enzyme-linked immunosorbent assay) is suitable for the quantitative detection of host cell proteins derived from HEK 293 and HEK 293T, such as biotherapeutics based on the insect cell-baculovirus expression system, including but not limited to recombinant proteins, vaccines, and gene therapy vectors.

This ELISA kit is based on the solid-phase enzyme-linked immunosorbent assay (ELISA) and utilizes a sandwich format with dual antibodies to quantitatively detect residual Host Cell Proteins (HCPs) from HEK 293 and HEK 293T in the test samples. The polyclonal antibodies in this ELISA kit are produced by immunizing sheep with HCPs obtained by lysing HEK 293T cells as antigens, followed by affinity purification to obtain high-quality antibodies. Add the calibrator or test sample along with HRP-labeled anti-HEK 293T HCPs polyclonal antibodies into an enzyme immunoassay plate precoated with antibodies against HEK 293T HCPs. After incubation, the plate is washed, and a colorimetric reaction is initiated by adding TMB substrate. Finally, the enzymatic reaction is terminated using a stop solution. The absorbance is measured at a wavelength of 450 nm using an ELISA reader. The absorbance is directly proportional to the HCPs concentration in the calibrator and samples, and the concentration of HCPs in the sample can be calculated using a dose-response curve. No special treatment is required for actual samples using this ELISA kit; it can be directly used after the appropriate dilution.

1. Anti-HEK 293T coated microtiter strips : The appropriate amount of sheep anti- HCPs polyclonal antibodies has been coated. It is sealed in an aluminum foil bag and contains a desiccant. It is normal to observe some crystallization on the inner walls of some wells of the microplate and does not require any special treatment. 2. HEK 293T HCPs Standard : 3 vials. Lyophilized. Accurately measure 500 μL of the reconstitution solution, dissolve it, and let it stand for 5-10 minutes. The solution should appear clear and transparent with no visible insoluble particles. The specific concentration can be found labeled on the bottle. 3. Standard Dilution Buffer: 1.5 mL × 2 vials. 4. Standard/Sample Dilution Buffer : 25 mL × 2. Dilutions are required for standard, test samples, and enzyme-labeled antibodies. For samples being tested for the first time, a sample suitability verification is necessary to determine the optimal dilution factor. 5. Washing Buffer 10× : 25 mL × 1. Crystallization may occur at low temperatures. To dissolve, the Washing Buffer can be placed in a 37°C water bath. Prior to use, dilute the Washing Buffer 10-fold with freshly prepared ddH2O. 6. HEK 293T HCP-HRP Antibody 100× : 120 μL × 1. The HRP-conjugated sheep polyclonal antibody against HEK 293T HCPs should be diluted 100-fold with a dilution buffer before use. Avoid light. 7. TMB Substrate : 12 mL×1. Equilibrate for at least 20 minutes at room temperature. Store in a dark and sealed tightly. 8. Stop Solution : 6 mL×1. Hydrochloric acid. When handling, please wear safety goggles and avoid contact with the skin. 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. ddH2O 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. HEK 293T HCPs Standard: Accurately measure 500 μL of the reconstituted standard solution and transfer it into a vial. Gently invert the vial to mix, and allow it to stand for 5-10 minutes. The concentration of HCPs in the reconstituted calibration standard is equivalent to the labeled concentration. The solution can be stored at 2-8°C after reconstitution and should be used within the expiration date. Note: If multiple vials of calibrator are to be used simultaneously, please dissolve them separately, transfer to a 1.5 mL sterile centrifuge tube together, shake well, and then use. If the reconstitution solution of each vial of calibrator is subjected to multiple freeze-thaw cycles, it is recommended to aliquot into 1.5 mL sterile centrifuge tubes and store at ≤ -20°C. 2. Washing Buffer (1× conc.) : Dilute Washing Buffer 1 + 9; e. g. 25 mL Washing Buffer (10× conc.) + 225 mL distilled water. In case crystals appear in the concentrate, warm up the solution to 37°C e.g. in a water bath. Mix well before dilution. 3. HEK 293T HCP-HRP Antibody: The HRP-conjugated sheep polyclonal antibody against HEK 293T HCPs should be diluted 100-fold with a dilution buffer before use.

1. Add 100 μL of the HEK 293T-HRP Antibody 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 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 15 minutes at room temperature (15-30°C) * in the dark. 8. Add 50 μl stop solution (STOP) into each well and mix well. 9. 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.

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.

6-540 ng/mL

6 ng/mL

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet