Protein A ELISA Kits

Protein A ELISA Kits

Use Protein A ELISA kits in a therapeutic antibody manufacturing workflow

Protein A affinity chromatography is commonly used in monoclonal antibody (mAb) manufacturing workflows because of its high binding affinity and the purity of the product obtained with it (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 ELISA kits for reliable supply and accurate detection of Protein A residuals in the manufacturing of therapeutic monoclonal antibodies, one for natural and recombinant protein A and the other for Protein A engineered variants. These kits have been designed with a simplified procedure, accurate quantification, and allow for easy transfer to automated platforms.

View Protein A ELISA Kits

 

R&D Systems Protein A ELISA Advantages

Protein A ELISA calibration graph     Protein A ELISA Kit buffer     Protein A ELISA pretreatment step     Protein A ELISA Incubation time     Protein A ELISA trouble shooting    

Calibrated to Natural or
Engineered Protein A

 

    Ready-to-use Components
Including Buffers

 
    Single-step Sample
Pretreatment

 
    Minimal Assay
Incubations

 
   

Consistent Supply
and Technical Support

 

   
Prevent over or
under-reporting
Protein A levels
with an ELISA
calibrated to the
Protein A construct
used in your workflow.
    Eliminate the hassle
of preparing reagents.
Our components are
optimized and ready
to use immediately.

 
   

Simple and easy
pretreatment allows
for faster sample
preparation. Just mix
your sample with
our pretreatment buffer
to begin your assay.

    3 incubation steps
total 3 hours or less
ensuring quick
turn-around for
assay results.

 
    Unrivaled customer
service and shipped
immediately upon order.



 
   

 

Fully Validated for Accurate Protein A Detection

Multiple Protein A variants are used in the manufacturing workflow. 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 Recombinant Protein A  Engineered Protein A Variants
Calibration E. coli expressed recombinant Protein A  MabSelect SuReTM
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% 102-112%
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 ELISA Kit Linearity of Dilution
Protein A ELISA spike recovery data   Protein A ELISA kit linearity of dilution data
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 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 Protocol

Protein A ELISA Protocol

Pretreatment releases Protein A from the therapeutic antibody sample followed by Protein A quantification via sandwich ELISA. Click on image to view larger.

 

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.

 

References

  1. Ramos-de-la-Pena, A.M. et al. (2019) Journal of Separation Science. 42: 1816
  2. Rigi, G. et al (2019) Biotechnology and Applied Biochemistry. 66:454
  3. Mazigi, O. et al (2019) Protein Engineering and Selection. 32: 359

    MabSelect SuRe™ is a trademark of GE Healthcare.