Protein A ELISA Kits
Residual Protein A Testing in Therapeutic Antibody Manufacturing
Protein A affinity chromatography is commonly used in monoclonal antibody (mAb) manufacturing workflows because it enables selective antibody capture and high-purity product recovery (1).There are occasions where Protein A or its fragments leach from a purification column and remain bound to the Fc region of a therapeutic antibody. This must be avoided, particularly in instances where antibodies are manufactured for clinical uses in order to preclude any adverse effects of residual protein A on the patient.
R&D Systems has developed two sandwich ELISA kits for accurate detection and quantification of residual Protein A in therapeutic monoclonal antibody manufacturing workflows, one for natural and recombinant protein A and the other for engineered Protein A variants. These kits are designed with a simplified workflow and support easy transfer to automated platforms.
New to Protein A ELISAs? Explore our FAQs below to learn how residual Protein A is generated, why it is monitored during therapeutic antibody manufacturing, how ELISAs are used for detection and quantification, and key considerations for accurate Protein A measurement.
Calibrated to Natural or Engineered Protein A
Prevent over or under-reporting Protein A levels with an ELISA calibrated to the Protein A construct used in your workflow.
Ready-to-Use Components Including Buffers
Eliminate the hassle of preparing reagents. Our components are optimized and ready-to-use immediately.
Single-Step Sample Pretreatment Releases Residual Protein A
Simple and easy pretreatment allows for faster sample preparation. Just mix your sample with our pretreatment buffer to begin your assay.
Minimal Assay Incubations
Three incubation steps totaling three hours or less ensure quick turn-around for assay results.
Validated Assay Performance
Excellent recovery, dilution linearity, and intra-assay and inter-assay precision support reliable Protein A quantification.
Consistent Supply and Support
Unrivaled customer service and shipped immediately upon order.
Compatible Sample Types
Protein A ELISA Kits are designed for the quantification of residual Protein A in purified monoclonal antibody samples and biosimilar therapeutic antibody preparations. Since sample matrices can vary between workflows, users should evaluate sample-specific recovery and dilution performance when qualifying an assay for their application.
Step 1: Mix the therapeutic antibody sample with pretreatment buffer to dissociate residual Protein A from the antibody. Incubate for 5-30 minutes.
Step 2: Add the horseradish peroxidase (HRP)-conjugated detection antibody and the pretreated samples or standards to a Protein A antibody-coated 96-well plate. Incubate for 2 hours to allow Protein A capture and detection complex formation.
Step 3: Wash the plate to remove unbound substances, then add the substrate solution to the wells. Color develops in proportion to the amount of Protein A bound to the plate.
Step 4: Add stop solution and measure the absorbance using a microplate reader. Protein A concentrations are determined by comparing sample absorbance values to a standard curve generated from known Protein A standards.
Fully Validated for Accurate Protein A Detection
Multiple Protein A variants are used in therapeutic antibody purification workflows. To accurately quantify Protein A, a kit calibrated to a closely related variant must be used. Both Protein A ELISA kits have been validated and qualified based on the following parameters:
| Protein A ELISA Kit | Protein A Engineered Variant ELISA Kit | |
|---|---|---|
| Specificity | Natural and Recombinant Protein A constructs | Engineered Protein A constructs such as MabSelect SuRe™ |
| Calibration | E. coli-expressed recombinant Protein A | MabSelect SuRe Protein A |
| Intra-Assay Precision (CV) | 2.7-3.1% | 2.4-3.7% |
| Inter-Assay Precision (CV) | 6.0-8.3% | 3.8-8.5% |
| Recovery | 88-113% | 84-122% |
| Linearity | 104-112% | 102-113% |
| Limit of Detection | 16.1 pg/mL | 11.4 pg/mL |
| Limit of Quantification | 23.4 pg/mL | 11.7 pg/mL |
Protein A ELISA Kit Spiked Recovery
Protein A Spiked Recovery. Protein A constructs were spiked into in-house Trastuzumab, Cetuximab, and Rituximab biosimilar antibody samples. Spiked sample matrix Protein A recovery ranged from 80% to 101%, which was in the acceptable range.
Protein A ELISA Kit Linearity of Dilution
Protein A ELISA Linearity of Dilution. Protein A constructs were spiked at a high concentration into in-house biosimilar antibody samples and diluted to produce samples with values within the dynamic range of the assay. The linearity is between 97%-112%.
Protein A ELISA Kit Precision
Precision describes the reproducibility of assay results within a single assay run and across multiple assay runs. Intra-assay precision evaluates consistency among replicate measurements performed within the same assay, while inter-assay precision evaluates reproducibility across different assay runs, operators, and reagent lots.
Protein A ELISA Kit (Catalog # BPPA00)
| Intra-Assay Precision | Inter-Assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 157 | 507 | 1519 | 148 | 458 | 1442 |
| Standard Deviation | 4.90 | 13.6 | 42.3 | 12.2 | 28.9 | 86.8 |
| CV% | 3.1 | 2.7 | 2.8 | 8.3 | 6.3 | 6.0 |
Protein A ELISA Kit for Engineered Variant (Catalog # BPPAEV0)
| Intra-Assay Precision | Inter-Assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (pg/mL) | 70.0 | 227 | 725 | 77.7 | 239 | 753 |
| Standard Deviation | 1.70 | 6.54 | 26.6 | 6.58 | 10.3 | 28.4 |
| CV% | 2.4 | 2.9 | 3.7 | 8.5 | 4.3 | 3.8 |
R&D Systems Protein A ELISA Kits Demonstrate Excellent Precision. The Protein A ELISA Kit showed intra-assay coefficients of variation (CVs) ranging from 2.7-3.1% and inter-assay CVs ranging from 6.0-8.3%. The Protein A Engineered Variant ELISA Kit demonstrated intra-assay CVs ranging from 2.4-3.7% and inter-assay CVs ranging from 3.8-8.5%. These results indicate highly reproducible Protein A quantification within individual assay runs and across multiple assays.
Protein A ELISA Kit Sensitivity
Assay sensitivity is commonly described using the limit of detection (LoD) and lower limit of quantification (LLoQ). The LoD represents the lowest concentration of Protein A that can be distinguished from background signal, while the LLoQ represents the lowest concentration that can be quantified with acceptable accuracy and precision.
The Protein A ELISA Kit demonstrated an LoD of 16.1 pg/mL and an LLoQ of 23.4 pg/mL. The Protein A ELISA Kit for Engineered Variant demonstrated an LoD of 11.4 pg/mL and an LLoQ of 11.7 pg/mL, supporting sensitive detection and quantification of residual Protein A.
Common Applications for Protein A ELISA Kits
Protein A ELISA Kits support residual Protein A monitoring throughout biopharmaceutical development and manufacturing workflows, from purification process optimization through process validation and routine manufacturing testing.
- Purification process optimization
- Protein A clearance studies
- Process validation
- Quality control testing
What is Protein A?
Protein A, also known as staphylococcus aureus protein A (SpA) is a 40-60 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus (2). The carboxyl terminal end of Protein A is anchored to the staphylococcus cell wall by the transpeptidase enzyme sortase. It is encoded by the spa gene and consists of five homologous 6.7 kDA immunoglobulin domains (E, D, A, B, and C) joined by conserved linkers of six to nine residues (3). Protein A has been shown to stimulate inflammatory responses by interacting with several proteins including tumor necrosis factor alpha receptors, Willebrand factor, and C1qR.
Protein A is most known for interacting with the Fc region of the antibody heavy chain. Although it binds all mouse immunoglobulin (IgG) subtypes (IgG1, IgG2a, IgG2b and IgG3), in humans, it binds to IgG1, IgG2 and IgG4 but not IgG3 (3). Protein A fails to bind IgG3 because of a single histidine to arginine change at amino acid 435 (3). Protein A reacts with Ig from a variety of other species including rhesus monkey, guinea pig, pig, cow, and goat.
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Residual Protein A Testing and Protein A ELISA FAQs
Residual Protein A testing is an important part of therapeutic antibody manufacturing because Protein A used during affinity chromatography can carry through the purification process as a process-related impurity. The following FAQs explain why residual Protein A is monitored, how Protein A ELISAs are used, and the factors that can influence accurate Protein A detection and quantification.
Protein A affinity chromatography is a purification technique commonly used during monoclonal antibody manufacturing. Protein A binds with high affinity to the Fc region of many IgG molecules, allowing antibodies to be selectively captured from complex mixtures. Following purification, downstream processes and analytical testing are used to confirm the removal of process-related impurities, including residual Protein A.
Residual Protein A refers to Protein A and Protein A fragments that remain in a therapeutic antibody sample following Protein A affinity chromatography purification. Because Protein A is used to capture and purify antibodies through Fc binding, small amounts can occasionally leach from the chromatography resin and persist through downstream purification steps. Residual Protein A is considered a process-related impurity and is routinely monitored during biopharmaceutical development and manufacturing.
Protein A leaching occurs when Protein A molecules or fragments detach from the affinity chromatography resin during antibody purification. Factors that can contribute to leaching include repeated column use, cleaning and sanitization procedures, chemical or mechanical stress on the resin, and the specific Protein A ligand and chromatography conditions being used.
Protein A is widely used during monoclonal antibody purification, but any Protein A remaining in the final product is considered a process-related impurity. Because residual Protein A is not part of the intended therapeutic product, downstream purification processes are designed to reduce its levels to acceptable limits. Analytical testing is then used to verify adequate removal, helping support product quality and patient safety.
Residual Protein A testing is performed to verify that Protein A used during affinity chromatography has been effectively removed from purified therapeutic antibody preparations. As a process-related impurity, residual Protein A is routinely monitored to assess the effectiveness of downstream purification steps and support product quality throughout biopharmaceutical development and manufacturing.
Residual Protein A is commonly measured using highly sensitive immunoassays such as ELISAs. These assays use Protein A-specific antibodies to detect and quantify trace amounts of residual Protein A that may remain in purified therapeutic antibody samples following affinity chromatography. Because ELISAs provide sensitive, specific, and quantitative measurement of low Protein A concentrations, they are widely used for residual Protein A analysis in process development, process validation, and manufacturing workflows.
Protein A concentrations are determined by comparing sample absorbance values to a standard curve generated from known Protein A standards. The standard curve establishes the relationship between absorbance and Protein A concentration, allowing unknown sample concentrations to be calculated and quantified.
Total time to results depends on the assay workflow and sample preparation requirements. The R&D Systems Protein A ELISA protocol includes a brief pretreatment step followed by a 2-hour incubation and assay development steps, providing an efficient workflow for residual Protein A quantification in approximately 2.5 hours.
Detecting residual Protein A in monoclonal antibody samples can be challenging because Protein A is typically present at very low concentrations relative to the therapeutic antibody, requiring highly sensitive analytical methods. In addition, Protein A binds strongly to the Fc region of many antibodies, which can mask epitopes or interfere with detection if the Protein A is not effectively released from the antibody prior to analysis. Assay performance may also be influenced by the specific Protein A construct used during purification, as engineered Protein A variants can differ from native Protein A in their structure and antibody-binding characteristics. Together, these factors can complicate detection and highlight the importance of selecting an assay appropriate for the sample being analyzed.
Many modern affinity chromatography resins use engineered Protein A ligands that have been optimized for properties such as improved binding capacity, stability, or alkaline resistance. Because these engineered variants can differ from native Protein A in their amino acid sequence, domain structure, and antibody-binding characteristics, assays developed for one Protein A construct may not accurately detect, recover, or quantify another. Using an assay that has been calibrated and validated for the specific Protein A variant used in the purification process can help improve measurement accuracy and reduce the risk of under- or overestimating residual Protein A levels.
Inaccurate Protein A measurements can lead to incorrect conclusions about residual Protein A levels in purified antibody samples. Underestimation may result in residual Protein A levels being underreported or not detected, while overestimation can trigger unnecessary investigations, additional purification efforts, or rejection of otherwise acceptable material. Reliable quantification is therefore important for making informed process development and manufacturing decisions.
References
- Ramos-de-la-Pena, A.M. et al. (2019) Journal of Separation Science. 42: 1816.
- Rigi, G. et al (2019) Biotechnology and Applied Biochemistry. 66:454.
- Mazigi, O. et al (2019) Protein Engineering and Selection. 32: 359.
MabSelect SuRe™ is a trademark of GE Healthcare.