A one-step purification of membrane proteins using a high efficiency immunomatrix.

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.

Multi‐component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome

The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed. Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined.

Tutorial review. Oriented immobilization of antibodies and its applications in immunoassays and immunosensors

Oncornaviruses transform cells either directly through a virus-coded oncogene1 or, if they lack such a gene, indirectly by promoter insertion2–4 into the cellular genome and activation of a cellular gene. Both transformation mechanisms ultimately result in abnormally high expression of normal genes. Recently, the activated cellular gene in certain lymphomas4 has been shown to be homologous to the oncogene of an acute avian leukaemia virus, MC29. In MC29 the oncogene is fused to the viral structural gene, gag. The product of this fused gene is a protein of molecular weight 110,000 (110 K)5–7, designated p110gag–myc. We have characterized this protein by using monoclonal antibodies against p19, the N-terminal portion of the gag–myc fusion or 110 K protein and purified it 3,700-fold by immune affinity column chromatography. Immunofluorescence studies and cell fractionation of MC29-transformed fibroblasts indicate that the 110 K protein is predominantly located in the nucleus. Moreover, the purified protein binds to double-stranded DNA. These properties may be related to the role of the protein in transformation.

Nuclear localization and DNA binding of the transforming gene product of avian myelocytomatosis virus

An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor

A rapid, novel method for the solid-phase derivatization of IgG antibodies for immune-affinity chromatography

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A rapid, novel method for the solid-phase derivatization of IgG antibodies for immune-affinity chromatography