Research paperImproved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies
Introduction
Cytokines and chemotactic cytokines (chemokines) are soluble proteins that have structural similarities and overlapping functions. When secreted during the course of an inflammatory response they interact in a complex network and function as effector molecules for cells of the immune system. During acute and chronic inflammation the dynamic equilibrium of cytokines and chemokines changes. The resulting inflammatory processes can either be systemic or restricted to a local site (Luster, 1998, Davidson and Diamond, 2001, Godessart and Kunkel, 2001, O'Shea et al., 2002). Within the inflamed microenvironment many mediators are secreted that interact directly with the surrounding tissue. The produced cytokines regulate the production of other inflammatory mediators whereas secreted chemokines function as regulatory molecules that attract and direct differentiation of new, potentially inflammatory, cells to the site of inflammation (O'Garra and Murphy, 1994, Luster, 1998, Luther and Cyster, 2001).
During the last years many different therapies have been developed that interfere in the cytokine and chemokine pathways and consequently modulate the inflammatory process. Patient categories that qualify for these forms of therapy are autoimmune diseases such as rheumatoid arthritis (RA). Immune intervention based on blocking the biological effects of tumor necrosis factor alpha (TNFα) and Interleukin 1 alpha (IL1α) is very effective for the treatment of a variety of human diseases, most notable RA and Inflammatory Bowel Disease (Elliott et al., 1994, Fleishmann, 2002).
A variety of techniques are available for detecting cytokines and chemokines, most notably the enzyme-linked immunosorbent assays (ELISA). However this technique has limitations, such as usage of large sample volumes and it is time consuming to obtain a complete protein profile. Recent applications for simultaneous detection of proteins in body fluids has resulted in particle based multiplex immunoassays (MIA). The Bio-Plex system employing the Luminex multi-analyte profiling technology (x-MAP™), allows individual and multiplex analysis of up to a hundred different mediators in a single well containing a sample volume of 50 μl (Vignali, 2000, Kellar et al., 2001, de Jager et al., 2003). However in chronic inflammatory conditions, heterophilic antibodies such as rheumatoid factor (RF) or other auto-antibodies are present (De Rycke et al., 2003, Tiittanen et al., 2004). In addition naturally occurring heterophilic antibodies can be found in up to 40% of the normal population and can either be IgG, IgM, IgA or IgE isotype (Kricka, 1999).
Detection of molecules in plasma or other complex biological fluids containing human antibodies, using a sandwich antibody system, poses problems especially when heterophilic antibodies are present. The antibody pairs used in a MIA are either monoclonal, polyclonal or a combination of both. Mouse and rat are common sources of monoclonal antibodies, whereas polyclonal antibodies are usually produced in rabbit, sheep and goat. Heterophilic- and auto-antibodies react with immunoglobulins from different mammals and thus can crosslink capture and detection antibodies resulting in either false positives or a blockade of the signal and thus critically interfere with immunoassay procedures (Kricka, 1999, Hennig et al., 2000, Kellar et al., 2001, Martins et al., 2004).
In this report we describe the development of a novel method to remove heterophilic antibodies from biological samples using protein-L. In contrast to protein-A and protein-G, protein-L has a high affinity for IgM and to a lesser extent for IgA and IgG (Akerstrom and Bjorck, 1989). In addition we describe the validation of an MIA for the quantification of 30 soluble proteins in plasma and synovial fluid containing heterophilic antibodies. These samples were derived from patients with chronic inflammatory conditions and were used as a model to demonstrate the power of this novel technique when interfering immunoglobulins are removed with protein-L.
Section snippets
Patient material
Heparinized blood samples were collected from patients with rheumatoid arthritis (n = 9) and healthy controls (HC; n = 10). Furthermore, heparinized synovial fluid (SF) samples were obtained from patients with RA (n = 10) and osteoarthritis (OA; n = 5, Table 1). All samples were stored frozen at − 80 °C until analysis. Written informed consent was obtained from the patient during visits to the outpatient clinic.
Before use all samples were centrifuged through a polypropylene centrifuge tube containing a
Immunoglobulin depletion
A multiplex immunoassay for 30 proteins uses 60 different antibodies (coating and developing reagents from mice, rat, chicken, rabbit, sheep and goat). As a consequence the potential interference of heterophillic (auto-) antibodies has to be avoided since this can induce false positive results. This is of particular importance for samples in a complex matrix and those with a high total protein content such as plasma and SF. Three different methods for the removal of immunoglobulins were tested
Discussion
We have described a validation of an MIA for the detection of 30 proteins in complex human body fluids together with a novel method for removal of antibodies from matrices that can interfere with an immunoassay. Previously we and others have validated the x-MAP™ technology for the detection of proteins using either commercial kits or own in house developed assays (Kellar et al., 2001, Prabhakar et al., 2002, de Jager et al., 2003, Prabhakar et al., 2004, Khan et al., 2004).
Since a large number
Acknowledgments
The authors wish to thank Huib de Jong and Joel van Roon (Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht) for providing the patient material. W. de Jager and B.J. Prakken are financially supported by the Dutch Rheumatoid Arthritis Foundation (Nationaal Reumafonds).
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