Lupus Anticoagulant: Performance of a New, Fully Automated Commercial Screening and Confirmation Assay

B. Montaruli
2005 Clinical Chemistry  
was performed 8 days after coupling (Fig. 2 in the online Data Supplement). However, on interpolation of data points from the calibration curve, the trend was reversed such that the highest signal was observed for experiment 3 and the lowest for experiment 1. In parallel studies, antigen-conjugated beads stored at 4°C gave consistent fluorescent emission within a period of 1 month, whereas antibody-conjugated beads showed diminished fluorescent emission (data not shown). We identified two
more » ... dentified two limitations of this methodology: The inherent instability of antibody-coupled beads and the occurrence of data points from test samples outside the linear portion of the semilogarithmic calibration curve. To resolve the latter issue, serum samples exhibiting fluorescent output within the plateau of the trendline should be reassayed after further dilution. Problems with long-term stability of protein-conjugated bead sets were evident when the beads were stored at 4°C. On the Luminex platform, antibody-conjugated beads were viable for approximately 3 weeks. Antigen-conjugated beads exhibited slightly greater longevity, although decoding of the fluorescent signatures was problematic after storage at 4°C beyond 1 month. The constituents of the storage buffer may have a detrimental effect on the fluorescent dyes within the microspheres. The reversal of the signal output profile suggests that antibody-bound beads were more liable to degradation than antigen-coupled bead sets within the same timescale. The more elaborate structural complexity of antibodies compared with antigens may account for the greater instability of the former. Rapid freezing and lyophilization were procedures explored as alternative methods to prolong the shelf-life of protein-coupled beads, and both approaches appeared to be feasible (17 ). This provides the possibility of developing calibration bead sets as reference materials, thus enabling Luminex assay standardization. This study illustrated the complexity of quantifying target analytes within antigen arrays. Production of purified antibodies is laborious and expensive. Methods that can be used for antibody purification, e.g., affinity chromatography, could theoretically be used to obtain material comparable to the target analyte of an antigen array. However, consistent antibody purity is paramount for quantification. Although this approach has broad application for the comparison of any IgG, it will not measure absolute concentrations of target analyte. This is largely because of the presence of factors (e.g., soluble receptors, heterophilic antibodies, serum binding proteins, hemoglobin, and lipids) in sera that can interfere with antibody-based immunoassays (18 ). Nonetheless, this method has reduced intraassay variability and enables interassay comparisons for a wide range of antigen arrays. Lupus anticoagulants (LAs) are acquired circulating anticoagulants that interfere with phospholipid (PL)-dependent coagulation tests and are frequently associated with thromboembolic disorders and obstetric complications.
doi:10.1373/clinchem.2004.042028 pmid:15914788 fatcat:l5d5ufdkmvd2pogf3adtfj7fme