The Protein A ELISA Kit is a Sandwich ELISA for the highly sensitive detection and/or quantification of Protein A in fluids. The kit is designed to measure trace contamination of Protein A in antibody solutions. The antibodies used in this kit were produced by immunization with recombinant Protein A. Native and recombinant Protein A are quantified in this assay with similar potency.
Background:Protein A is a highly stable surface receptor produced by Staphylococcus aureus, which is capable of binding the Fc portion of immunoglobulins, especially IgGs, from a large number of species (Boyle and Reis, 1987). Each protein A molecule can bind 2 molecules of IgG, allowing the formation of a precipitate (Sjöholm, 1975).
Protein A may be coupled to a wide variety of reporter molecules including fluorescent dyes, enzyme markers, biotin, colloidal gold and radioactive iodine without affecting the antibody binding site. These conjugates may be used to track immunoglobulins in histochemical, Western Blot and ELISA.
Alternatively, Protein A may be immobilized onto a solid support to facilitate the purification and recovery of either polyclonal or monoclonal immunoglobulins. Immobilized protein A has recently been applied therapeutically to treat a variety of humoral diseases (Watson et al, 1989).
Protein A, BioAssay™ ELISA Kit, 96 tests, Quantitative detects samples containing Protein A or purified Ig's contaminated with Protein A.
Kit Components:Anti-Protein A Microwell Strip Plate, 8x12 stripsProtein A Stock, lyophilized Anti-Protein A HRP Conjugate Sample Diluent (20X) Wash Solution (100X) TMB SubstrateSample Recovery:Protein A immunosorbents are widely used for purification of monoclonal and polyclonal antibodies. Possible leakage of Protein A during processing may contaminate the purified antibody preparations. The detection/measurement of contaminating Protein A is complicated by Protein A-IgG binding in the sample, which inhibits the subsequent binding of the Protein A to the ELISA antibodies required for accurate quantification. A sample pretreatment boiling step for Protein A samples containing mouse IgG heat denatures the IgG. This uncouples the Pro A-IgG binding and, thus, allows for accurate Protein A measurement. Appropriate recovery of Protein A in solutions of IgG from other species has not been elucidated with this ELISA. In these cases, the investigator should demonstrate requirements for appropriate Protein A recovery.
High and low concentrations of Protein A were mixed into buffer containing mouse IgG at 1mg/ml. Samples were assayed before and after a boiling pretreatment step. Observed assay values in samples containing IgG compared to non-IgG sample values ranged from 41 to 62%. With a boiling pretreatment step, observed IgG sample assay values were 75 to 106% of non-IgG values, indicating accurate quantification of Protein A in the presence of IgG using the pretreatment step.
Test Principle:The Protein A ELISA kit is based on the binding of Protein A in samples to two antibodies, one immobilized on the microtiter wells, and the other conjugated to horseradish peroxidase (HRP) enzyme. After a washing step, chromogenic substrate is added and color is developed by the enzymatic reaction of HRP on the TMB substrate, which is directly proportional to the amount of Protein A present in the sample. Stopping Solution is added to terminate the reaction, and absorbance at 450nm is then measured using an ELISA microtiter well reader. The concentration of Protein A in samples is calculated from a curve of standards containing known concentrations of Protein A.
Storage and Stability:The microtiter well plate and all other reagents, if unopened, are stable at 2-8°C until the expiration date printed on the label. Stabilities of the working solutions are indicated under Reagent Preparation.
Sample Diluent and anti-Protein A-HRP contain Proclin 300 (0.05%, v/v). Stop Solution contains 1% sulfuric acid. Follow good laboratory practices, and avoid ingestion or contact of any reagent with skin, eyes or mucous membranes. All reagents may be disposed of down a drain with copious amounts of water.
