Universal Test Kit for Residual Protein A Detection-HYI135
This assay kit is used to detect residual Protein A in antibodies and Fc-fusion proteins. The principle of this assay kit is based on a sandwich immunoassay, utilizing an acidic buffer to dilute the sample and effectively separate residual Protein A from the sample's Fc portion, and the steps involved are simple. As shown in Figure 1, the provided assay plate in the kit is pre-coated with anti-Protein A mAb. The acid-diluted series of Protein A standards and samples are directly added to the pre-coated assay plate. After incubation at room temperature with shaking for 1 hour, unbound substances are washed away, resulting in the formation of a "coated antibody-Protein A" complex on the assay plate. Subsequently, biotinylated anti-Protein A detection antibodies are added, forming a "coated antibody-Protein A-biotinylated detection antibody" complex. After incubation at room temperature for 1 hour, unbound substances are washed away. Then, streptavidin-horseradish peroxidase (SA-HRP) complex is added and incubated at room temperature for 20 minutes, resulting in the formation of a "coated antibody-Protein A-biotinylated detection antibody-SA-HRP" complex on the assay plate. After washing away unbound substances, TMB substrate is added. The enzyme-catalyzed reaction of the substrate on the solid phase generates a color reaction. After approximately 6 minutes of color development, the reaction is terminated. The results are measured using an ELISA reader at wavelengths of 450 nm, with 650 nm as the reference wavelength. The results are calculated using the difference between OD450nm and OD650nm, which indirectly reflects the amount of Protein A bound to the assay plate. The residual amount of Protein A in antibodies and Fc-fusion proteins can be quantitatively analyzed based on the standard curve.
Kit Features
1.The coated and detection antibodies are both mAbs produced from monoclonal cell lines. They are purified using a two-step column chromatography process, ensuring batch-to-batch consistency and sustainable supply of the assay kit. The Fc regions and framework regions of both antibodies do not bind to Protein A, ensuring the scientific validity of the assay kit's designprinciple.
2.The kit is equipped with a universal Protein A ligand. When this ligand is used as a standard, it has been validated to show good recovery rates for various other ligands in actual samples, demonstrating the kit's versatility for multiple imported and domestic Protein A ligands. Additionally, the kit includes four commonly used imported and four domestic Protein A ligands commonly applied in the industry. Users can choose the corresponding ligand as a standard for testing according to their needs. If a Protein A standard not included in the kit is used, it can be diluted and prepared following the steps in section 6.2 (10) of this manual. Subsequent investigations on other Protein A ligands are ongoing and will be included in the kit if needed after evaluation.
3.The experimental procedure is simple, requiring only sample dilution and direct addition to the plate. It does not involve complex steps such as heating, denaturation, centrifugation, or pre-incubation before sample dilution and addition to plate. The entire experimental operation takes approximately 3 hours.
4.The kit offers high sensitivity compared to other similar products. The lowest point at the standard curve serves as the quantitation limit (10 pg/mL). The low quantitation limit ensures that users have a high degree of flexibility and selectivity in experimental design. Depending on the actual residual amount of Protein A in the test sample, the sample can be diluted to 1-0.01 mg/mL, corresponding to a final quantitation limit of 0.01-1 ppm. The ratio of OD between 10 pg/mL and 0 pg/mL on the standard curve is not less than 2 times, ensuring the stability and accuracy of experimental measurement data when the Protein A content in the sample is near the quantitation limit. It is important to note that the quantitation limit refers to the quantitation limit in a specific sample with a specific protocol. The quantitation limit of the kit is obtained through methodological validation on representative samples. When applying this kit, users should not directly adopt the quantitation limit of 10 pg/mL but should develop a methodological validation plan based on their own sample characteristics and needs of specification, and set a reasonable quantitation limit according to the results of the methodological validation.
5.The kit has undergone comprehensive and relatively rigorous methodological validation according to the latest version of ICH-Q2 (R2). It has been validated for accuracy, precision, linearity, quantitation limit, specificity, and other parameters, meeting regulatory requirements for residual Protein A detection in antibodies and Fc-fusion protein drugs in various countries. The kit comes with a recommended validation protocol, but users can also design their own validation plan according to their specific needs.
The kit demonstrates good stability. After being stored at 37℃ for 1 months, there are no significant changes in performance. Long-term stability assessment at room temperature is currently underway. Currently, the kit supports transportation at room temperature, but it is still recommended to store the kit at 2-8℃ for long-term storage.
Components
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Reagent |
Specifications |
Description |
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Anti-Protein A Antibody Pre-Coated Plate |
8 wells × 12 strips * 1 plate |
Removable, coated with mAb whose Fc that do not bind to Protein A |
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Universal Protein A Standards |
128 ng/mL 120 μL/vial |
Select the corresponding standard based on the sample being tested; dilute with "1× Wash Buffer 1" to 640, 320, 160, 80, 40, 20, 10 pg/mL. |
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Standard 1: MabSelect SuRe(Cytiva) |
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Standard 2: MabSelect PrismA(Cytiva) |
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Standard 3: TOYOPEARL AF-rProtein A(HC)-650F(Tosoh) |
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Standard 4: Eshmuno A(Merck) |
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Standard 5: NMab Pro(NanoMicro) |
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Standard 6: UniMab 50HC, NMab(NanoMicro) |
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Standard 7: MabPurixTM A/P(Sepax) |
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Standard 9：MaXtar® ARPA(BioLink) |
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Wash Buffer 1 |
50mL/bottle(10×) |
Dilute with deionized water as a 1× solution to be used as a dilution buffer for Protein A standards and samples, and as the wash buffer in the first step. |
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Biotin-labeled Detection Antibody |
150 μL/vial(100×) |
mAb with Fc subtype that does not bind to Protein A; dilute with "Detection Antibody and SA-HRP Diluent" for 1× use |
|
SA-HRP |
150 μL/vial(100×) |
Dilute with "Detection Antibody and SA-HRP Diluent" for 1× use. |
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Detection Antibody and SA-HRP Diluent |
30 mL/bottle(1×) |
Used to dilute "Biotin-labeled Detection Antibody" and "SA-HRP" |
|
Wash Buffer 2 |
30 mL/bottle(10×) |
Dilute with deionized water as a 1× solution to be used as the wash buffer in the second and third steps. |
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TMB Substrate Solution |
12 mL/bottle(1×) |
Solution containing 3,3',5,5'-Tetramethylbenzidine (TMB) and H2O2; used directly. |
|
Stop Solution |
8 mL/bottle(1×) |
Acid solution, used directly |
|
Plate sealing film |
1 piece |
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Notes: 1.Standard 8 for NGL-ImpactTM A ligand (No. PA-STD-NGL), is not included in this kit.
Please contact us if needed.
2.To meet market demand, the company will continue to introduce more specific protein A standards, each provided with a separate product number, and these are currently not included in the kits. Please contact the company separately if needed.
3.Crystallization may occur in wash buffer 1 (10×) and wash buffer 2 (10×). It can be heated and dissolved in a water bath of 40℃-50℃, and can be stored at room temperature for three months.
Storage
The assay kit should be stored at 2-8°C, and the shelf life under this condition is 3 years.
Other Required Reagents and Materials (not provided in the kit)
- Deionized water
- Sample reservoir
- Absorbent paper
- Low-binding pipette tips with volumes ranging from 5-1000 μL
- Low-binding sample dilution plate
- Polypropylene centrifuge tubes (e.g., 1.5 mL, 2 mL, 5 mL, 15 mL) for sample dilution or preparation of detection antibodies
- Single-channel pipette with volume adjustment range of 5-300 μL and 300-1000 μL,respectively
- Multi-channel pipette with volume adjustment range of 5-300 μL
- Suitable volumetric bottles for holding "1× Wash Buffer 1" and "1× Wash Buffer 2"
- Microplate reader capable of reading absorbance at 450 nm (required) and 650 nm
- Microplate shaker (200-500 rpm)
- Software capable of four-parameter curve fitting (can also use Microsoft Excel for plotting).