BioQuantiPro™ CHO HCP ELISA Kit

Details for Product No. ABIN6254804
Antigen
Reactivity
Chinese Hamster
Detection Method
Colorimetric
Method Type
Sandwich ELISA
Detection Range
2 ng/mL - 200 ng/mL
Minimum Detection Limit
2 ng/mL
Options
Purpose

BioQuantiPro CHO-HCP ELISA Kit for sensitive detection of host cell protein impurities.

The HCP Kit from Rockland meets industry standards for specificity and sensitivity. This kit is specifically designed for standard to high throughput applications and can be easily adapted for automation.

  • Broadest CHO-HCP contaminant coverage on the market
  • Low variability
  • High reproducibility/low lot-to-lot variation
  • Rapid time to result
  • Ease of use
Brand BioQuantiPro™
Sample Type Cell Culture Samples
Analytical Method Quantitative
Detection Method Colorimetric
Specificity The key component of this kit is a broad coverage antibody produced against a spectrum of host cell contaminants including both low abundancy and high abundancy proteins, as well as poorly immunogenic and highly immunogenic proteins.
  • Coverage: 89 % total coverage across LMW and HMW
  • Precision: Intra variability < 10 %, Inter variability < 15 %
  • Sensitivity: LLD = 2 ng/mL, LLQ = 4 ng/mL
  • Accuracy: Within 15 % exept at LLQ within 20 %
  • Linearity: R2 = 0.99
  • Assay Range: 2-200 ng/mL
  • Matrix Effect: Most buffer systems yielded 90-120 % recovery
Characteristics The CHO-HCP ELISA Kit is a sandwich ELISA designed to detect CHO-HCP contamination in process-derived samples. The kit uses purified antibodies generated against CHO-K1 cell lysates that were derived from cells grown in chemically-defined media. These antibodies react strongly with CHO-K1 cell lysates, showing the highest sensitivity for cells harvested at 60-85 % viability. Detection and quantification levels of samples derived from cell culture fluid (CCF) will vary and are sometimes limited. If a sample matrix is used in the assay, it is recommended to validate accurate recovery by first diluting the standard provided in the kit into the sample matrix.
Components
  • CHO-HCP Detection Antibody
  • CHO-HCP Antibody-coated 96-Well Strip Plate
  • CHO-HCP Protein Standard
  • HCP Kit Sample Buffer
  • HCP Kit Wash Buffer (10X)
  • HCP Kit TMB Buffer
  • HCP Kit Stop Buffer
  • Plate Sealer
Material not included
  1. 96 titer tubes (1.2 mL) compatible with multi-channel pipettes, arranged 12 x 8
  2. Serological pipettes
  3. Single-channel pipettes (2-20 μL and 10-100 μL) and tips
  4. Multi-channel pipette (200 μL) and tips
  5. Spectrophotometer (microplate reader) capable of reading 96-well plates at 450 nm with a reference filter at 630-650 nm
  6. Reagent reservoirs
  7. Plate shaker
  8. Vortex mixer
  9. Stir plate and magnetic stir bar
  10. Microplate washer, either 96-well or strip (optional)
  11. Absorbent paper
  12. Timer
  13. Polypropylene centrifuge tubes (15 mL)
  14. Deionized water
  15. Disposable gloves
Target Name (Antigen)
Alternative Name Host Cell Proteins
Application Notes The antibodies in the kit have been specifically designed to detect HCP contaminants and to measure analytes from crude cellular harvests, cell culture lysates, and process intermediates. The BioQuantiPro CHO-HCP ELISA Kit uses purified antibodies generated against a CHO-K1 cell lysate grown in chemically defined media. These antibodies react strongly with CHO-K1 cell lysates, showing the highest sensitivity for cells harvested at 60-85 % viability. Detection and HCP quantification of high-viability cell culture fluid (CCF)-derived samples will vary.
Comment

Validity of the kit tested in various buffer matrices used for protein purification.

In quality control applications, subject to thorough validation and qualification by each laboratory, these kits may also be useful for lot release testing.

ABIN6254804 from Rockland is able to provide relevant and specific data guiding process improvement and decreasing the level of HCP contamination.

Plate Pre-coated
Protocol The CHO-HCP ELISA Kit is a sandwich, enzyme-linked immunosorbent assay. The impurities in process-derived samples containing CHO-K1 proteins are captured and detected in a microtiter-type plate by using pre-coated, purified, rabbit polyclonal, CHO-HCP-specific antibodies. The immunological reaction after the addition of TMB results in a chromogenic change that can be measured photometrically at 450 nm with reference wavelength at 630-650 nm. The O.D. in the wells is directly proportional to the amount of HCP concentration and correlates to the amount of HCP present in the original sample. The HCP concentration can be calculated based on the calibration curve generated with the provided CHO-HCP standard.
Reagent Preparation

Before beginning the assay, allow the buffer reagents and the plate to equilibrate to room temperature (15-25 °C). The detection antibody must be kept refrigerated until 10 minutes before use.

  1. Wash buffer (1X) preparation: using a magnetic stir bar, mix 60 mL of wash buffer (10X) and 540 mL of deionized water. Mix well for at least 10 minutes. The wash buffer (1X) may be stored at room temperature (15-25 °C) for up to 2 weeks, after which it should be discarded.
    Note: Crystals may appear in wash buffer (10X) when stored at 2-8 °C.
  2. Assay standard preparation: a standard curve must be generated for every assay. The provided CHO-HCP protein standard is lyophilized. Reconstitute the standard with 1 mL deionized water to obtain a final concentration of 1 μg/mL. It is recommended to aliquot the standard solution and store it at -20 °C to avoid freeze/thaw cycles. The stock solution provided in the kit is enough to run 10 sets of assay standard duplicates at the described dilutions.
  3. Detection antibody preparation: the working solution of the detection antibody must be freshly prepared each time an assay is performed. This is done by diluting the CHO-HCP detection antibody (500X) into the sample buffer. Remove the vial from the refrigerator and allow it to reach room temperature for 10 minutes prior to use. For a complete 96-well plate, dilute 24 μL of the detection antibody into 12 mL of the sample buffer to prepare a 1X working solution.
Sample Preparation

The performance of the kit was tested in various buffer matrices commonly used in the purification of biotherapeutics. Some buffer systems may greatly affect the assay, causing low recovery because of suboptimal conditions such as high salt concentrations or low  pH . It is recommended that users dilute their assay samples to a minimum ratio of 1:1 into the provided sample buffer, then begin a series of dilutions into the sample buffer. Note: Optimal dilution factors for each assay sample must be determined by the user. If a test sample is not diluted into the provided sample buffer, specific matrix effects need to be validated by the user for accurate recovery of a spiked sample. If precipitates or aggregates are discovered in the test samples, use a centrifuge to remove insoluble proteins and avoid any unexpected complications.

Assay Procedure

This kit can be run as a standard (VI-A) or rapid (VI-B) protocol. The standard protocol takes approximately 3.5 hours with a detection range of 2-200 ng/mL and the rapid protocol takes approximately 2.5 hours with a detection range of 6-400 ng/mL. The plate and all reagents (except detection antibody) should be brought to room temperature prior to running the assay. Samples and reagents must be mixed thoroughly before use. It is recommended to assay all standards and samples in duplicate or triplicate. A thorough washing procedure using abundant wash buffer to perform the wash cycle is essential for reproducibility.

VI-A. Standard Procedure

  1. Add 100 μL of standards and assay samples in serial dilutions into the designated wells. Cover the plate and incubate it at room temperature for 2 hours with continuous shaking.
  2. Discard the solutions and wash the plate 4 times with wash buffer (1X) (no less than 300 μL) allowing a 30-second soak between each wash. If a plate washer is used, soaking cycle is not necessary. After the final wash, tap the plate upside down against absorbent paper to remove any remaining wash buffer. Complete removal of the final wash buffer is critical for an optimal assay performance.
    Caution: DO NOT let the wells dry completely at any time.
  3. Add 100 μL of the detection antibody working solution to each well, cover the plate and incubate at room temperature for 1 hour with continuous shaking.
  4. Wash the plate as described in step 2.
  5. Add 100 μL of TMB buffer to each well. Cover the plate while keeping it in the dark and incubate at room temperature for 20-30 minutes. Gently tap the plate to ensure a thorough mix.
    Note: An optimal incubation time should be determined by the user.
  6. Add 100 μL of the stop buffer to each well to stop the reaction. Gently tap the plate to ensure a thorough mix.
  7. Read plate using a spectrophotometer set at 450 nm wavelength with reference wavelength at 630-650 nm.
    Note: The assay must be read within 30 minutes after addition of stop solution. The signal may be reduced if prolonged incubation is allowed.



VI-B. Rapid Procedure

  1. Add 50 μL of the standards (adjust the standard preparation to 20 ng in 50 μL volume, then prepare a set of standard dilutions) and the assay samples in serial dilutions into the designated wells. Add 50 μL of working solution of the detection antibody (adjust detection antibody dilution to 1:250) to each well, cover plate and incubate it at room temperature for 2 hours with continuous shaking.
  2. Discard the solutions and wash the plate 4 times with wash buffer (1X) (not less than 300 μL) allowing a 10-second soak between each wash. If a plate washer is used, the soak cycle is not necessary. After the final wash, wrap the plate in absorbent paper and tap upside down to remove any remaining wash buffer. Complete removal of liquid at each step is critical for an optimal assay performance.
    Caution: DO NOT let the wells dry completely at any time.
  3. Add 100 μL of TMB buffer to each well. Cover plate and incubate it at room temperature for 20-30 minutes without shaking.
    Note: An optimal incubation time should be determined by the user.
  4. Add 100 μL of stop buffer to each well to stop the reaction. Gently tap the plate to ensure a thorough mix. The assay can be read immediately.
  5. Read plate using a spectrophotometer set at 450 nm wavelength with reference wavelength at 630-650 nm.
Calculation of Results

Calculate the Relative O.D. 450 using the following formula:
Relative O.D. 450 = (O.D. 450 of well) - (O.D. of the blank well)

Standard curve is plotted as the Relative O.D. 450 of each standard solution (Y) vs. the respective concentration of the standard solutions (X). The CHO-HCP concentration of the samples are estimated using linear interpolation from the standard curve. Note: If assay samples are from dilutions, multiply concentrations obtained from interpolations by the dilution factor.

Assay Precision <20% intra and inter assay variability
Restrictions For Research Use only
Storage 4 °C
Storage Comment Store all kit components except protein standard at 2-8°C upon arrival.
Supplier Images
Two Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) image for BioQuantiPro™ CHO HCP ELISA Kit (ABIN6254804) Quadrant Analysis for Antibody Coverage: The antibodies developed for this kit were v...
Two Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) image for BioQuantiPro™ CHO HCP ELISA Kit (ABIN6254804) Competitor Coverage Comparison: CHO-HCP proteins were detected by a competitor's gene...
 image for BioQuantiPro™ CHO HCP ELISA Kit (ABIN6254804) A representative standard curve
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