A chromatographic procedure of high interest for the rapid and specific purification of proteins from complex samples is immunoaffinity chromatography. For example, see the chapter in “Methods in Molecular Biology: Affinity Chromatography, Volume 147, Anuradha Subramanian, p.95ff.; Pfeiffer et al., J. Immunol. Methods 97, p.1-9; and Cuatrecasas, J. Biol. Chem. 245, p.3059ff. The methodology has been refined with respect to the application of polyclonal and monoclonal antibodies for separating components from samples over immunoaffinity chromatography columns. A significant improvement for site-specific immobilization of antibodies on chromatographic matrices that facilitate binding in the first step and eluting in the second step of antigens with higher capacity was reported by Gersten & Marchalonis, J. Immunol. Methods 127, p.215ff. and Schneider et al., J. Biol. Chem. 257, p.10766ff.
In most cases, immunoaffinity chromatography has been used for the purification of a specific protein. In fewer cases, subtraction or removal of undesired proteins has been the purpose for the use of immunoaffinity chromatography matrices, e.g. in the specific removal of viral (protein) components from a tissue or body fluid sample (Schreiber et al., Curr. Stud. Hematol. Blood Transfus. (1989) 56, 146ff.) or in the removal of clinically undesirable proteins from blood of patients (Vallar et al., J. Chromatogr. Biomed. Appl. (1995) 664, 97ff.). In this situation, recovery of a protein mixture that has no affinity for the immunoaffinity chromatography matrix is of primary interest. Flurer and Novotny (Anal. Chem. (1993) 15, 817ff.) describe an analytical-scale procedure in which immunoaffinity chromatography columns are used to subtract proteins from human plasma to generate a plasma protein profile by second-dimension RP-HPLC.
Two-dimensional gel electrophoresis (2-DGE) is a method that enables distinguishing individual proteins in a sample that contains a plurality of proteins. Such samples include cell lysates, tissue lysates, serum samples etc. Difficulties in discriminating proteins can arise when proteins have similar charge and molecular weight so that the two or more proteins comigrate and are located in the same general vicinity of the display.
Another factor contributing to poor discrimination is the disparate abundance of one or several high abundance proteins that generally comigrate on 2-D gels with other comigrating proteins of lower abundance. In an attempt to detect lower abundance proteins, sufficient protein is loaded onto the gels such that the images on the 2-D gels are stained or labeled very heavily due to high abundance proteins thereby obliterating weaker signals of nearby, unrelated proteins. This effect may be partially ameliorated by taking a series of optical measurements as the staining develops as demonstrated in WO 01/16884.
Additionally, a limited amount of sample protein may be used in the isoelectric focusing procedure to achieve acceptable separation. 2-D gels have limits on the amount of total protein they can hold and thus it is desirable to maximize the amount of protein sample of low abundance in the gel. Thus partitioning the sample before running one or more 2-DGE has been proposed in WO 00/33075.
Such confounding conditions might be overcome by using differential detection methods that might reveal the different proteins that are situated in the same vicinity on a gel. For example, a specific detection means can be employed such as using a reagent (e.g. antibodies, receptors or ligands as reporter molecules) that specifically bind to a particular protein, and perhaps minor variants thereof. Thus, there is a need for techniques, which permit measurement of lower abundance proteins in samples containing higher abundance proteins.