Protein A ELISA

General Information

Staphylococcal Protein A (SpA) is an immunoglobulin (IgG) binding protein found in the bacterial cell wall of Staphylococcus aureus. SpA binds to most mammalian IgG and can be used for detecting or purifying such antibodies. Affinity chromatography on SpA-columns is widely used for the purifying monoclonal and polyclonal antibodies. SpA may sometimes leak from the column and contaminate the preparation. Immunoglobulins reacts with SpA in vivo and may cause anaphylactic reactions. Contamination with SpA may also cause false results in immunological assays. Thus it is important that the antibody preparation be free from SpA before being used.

Interaction between SpA and IgG

Analyses aimed at detecting the presence of SpA in samples containing IgG are plagued by two major problems which have to be solved in order to obtain reliable results.

First, the Fc-reactivity of SpA with the IgG of most animal species makes it difficult to specifically detect SpA with an immunoassay. Antibodies specific to SpA will normally bind both by their specific activity and the general affinity between SpA and IgG (Fc). In this kit the problem is solved by using chicken anti-SpA IgG. Chicken antibodies are one of few immunoglobulins that do not have Fc-reactivity to SpA.
 

Second, in samples containing mammalian IgG the immunological active epitopes of SpA are normally blocked by the non-specific binding of IgG. To overcome this problem it has been suggested that the analysis of SpA be performed at low pH, at which a certain dissociation occur between SpA and the IgG. However, high-affinity antibodies (e.g. human IgG and certain mouse monoclonals) however, will even remain bound to SpA below pH 3. Unfortunately, the specific immunological detection of SpA is very difficult at such a low pH. The analytical performance of assays conducted at low pH is, consequently, very poor. We have chosen to establish an assay system that can be tailored to the specific purification needs of each one of our customers. The standard references are made up with known amounts of IgG. The IgG solution added to the standard references should be of the same isotype as the IgG of the sample. To dissociate SpA and IgG, the samples and standard references are boiled for 4 minutes. In other words, the assay is standardized using the type of IgG present as in the sample. Errors resulting from the blocking of antigenic epitopes can therefore be eliminated.

The effect of sample preparation on recovery of SpA. SpA easily binds to glass or plastic materials. However, in presence of 0,05% Tween 20 such binding is inhibited. It is absolutely necessary that the sample be eluted into a buffer containing Tween 20 (see test procedure) in order to recover all SpA from the sample matrix and prevent the walls of the sample tube from being coated with SpA. The neutralization buffer in this kit contains Tween 20 and it must be added to the sample tubes before adding the sample. The absence of Tween 20 may falsely lower the concentration of SpA.