Article

Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies

Utrecht University, Utrecht, Utrecht, Netherlands
Journal of Immunological Methods (Impact Factor: 2.01). 06/2005; 300(1-2):124-35. DOI: 10.1016/j.jim.2005.03.009
Source: PubMed

ABSTRACT Cytokines, chemokines and soluble adhesion molecules interact in a complex network within the immune system. Fingerprinting of these proteins may allow the use of these proteins as biomarkers for identification of disease, disease subtyping and monitoring therapeutic interventions. We developed a multiplex immunoassay (MIA) for the detection of 30 proteins in a variety of human body fluids such as plasma and synovial fluid (SF). The measurement of these proteins is hampered by the presence of human (auto-) antibodies, which can cause non-specific binding. We have validated a novel approach for the removal of interfering immunoglobulins using pre-absorption with protein-L. Interfering (auto-) antibodies, such as rheumatoid factor (RF), were removed using three methods; polyethylene glycol (PEG) precipitation, pre-absorption with human gamma-globulin or pre-absorption with protein-L. A significant decrease of RF was observed after a 2 h incubation with protein-L. RF IgM levels were reduced by 89% whereas total IgM, IgG and IgA levels were reduced by 60%. Residual immunoglobulins were blocked with rodent serum and did not interfere with the multiplex immunoassay. Comparing the MIA with a conventional enzyme-linked immunosorbent assay (ELISA) using a panel of spiked plasma samples resulted in correlation coefficients for all mediators between R2 = 0.88 and R2 = 0.99. Intra-assay variance was less than 10% whereas inter-assay variance ranged between 6% and 16%. Pathological samples with heterophilic antibodies hamper immunoassays such as ELISA and MIA. We show that pre-absorption with protein-L is a powerful tool for removal of interfering immunoglobulins from human bodily fluids to be used in immunoassays for studying changes in protein patterns.

1 Bookmark
 · 
226 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The role of non-diagnostic features in the pathophysiology of autism spectrum disorders (ASDs) is unclear. Increasing evidence suggests immune system alterations in ASD may be implicated in the severity of behavioral impairment and other developmental outcomes. The primary objective of this meta-analysis was to investigate if there is a characteristic abnormal cytokine profile in ASD compared with healthy controls (HCs). We identified relevant studies following a search of MEDLINE, EMBASE, PsycINFO, Web of Knowledge and Scopus. A meta-analysis was performed on studies comparing plasma and serum concentrations of cytokines in unmedicated participants with ASD and HCs. Results were reported according to PRISMA statement. Seventeen studies with a total sample size of 743 participants with ASD and 592 HC were included in the analysis. Nineteen cytokines were assessed. Concentrations of interleukin (IL)-1beta (P<0.001), IL-6 (P=0.03), IL-8 (P=0.04), interferon-gamma (P=0.02), eotaxin (P=0.01) and monocyte chemotactic protein-1 (P<0.05) were significantly higher in the participants with ASD compared with the HC group, while concentrations of transforming growth factor-β1 were significantly lower (P<0.001). There were no significant differences between ASD participants and controls for the other 12 cytokines analyzed. The findings of our meta-analysis identified significantly altered concentrations of cytokines in ASD compared to HCs, strengthening evidence of an abnormal cytokine profile in ASD where inflammatory signals dominate.Molecular Psychiatry advance online publication, 17 June 2014; doi:10.1038/mp.2014.59.
    Molecular Psychiatry 06/2014; DOI:10.1038/mp.2014.59 · 15.15 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: New discoveries about the pathophysiology changed the concept that all forms of osteoarthritis are alike; this lead to the delineation of different phenotypes such as age, trauma or obese related forms. We aim to compare soluble mediator profiles in primary knee and posttraumatic wrist osteoarthritis. Based on the general faster progression rate of wrist osteoarthritis, we hypothesize a more inflammatory profile.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: IntroductionThis study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.Methods Samples were obtained from donors without joint pathology (n¿=¿39), with focal defects (n¿=¿65) and osteoarthritis (n¿=¿61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1¿, IL-1ß, IL-1RA, IL-4, IL-6, IL-6R¿, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)¿, Interferon (IFN)¿, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).ResultsIn synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFN¿ and OSM levels were higher than in donors without joint pathology (P ¿0.001). IL-13, IFN¿ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ¿0.001). IL-1¿, IL-6, TNF¿ and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.Conclusions Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.

Full-text

Download
18 Downloads
Available from
Oct 23, 2014