General Information

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

Interaction between Protein A and IgG

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

Second, in samples containing mammalian IgG the immunological active epitopes of protein A are normally blocked by the non-specific binding of IgG. To overcome this problem it has been suggested that the analysis of protein A be performed at low pH, at which a certain dissociation occur between protein A and the IgG. However, high-affinity antibodies (e.g. human IgG and certain mouse monoclonals) however, will even remain bound to protein A below pH 3. Unfortunately, the specific immunological detection of protein A 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 protein A 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 protein A. Protein A 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 protein A from the sample matrix and prevent the walls of the sample tube from being coated with protein A. 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 protein A.

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