Miniaturized monolithic disks for immunoadsorption of cardiac biomarkers from serum

Abstract

Immunoadsorbers based on 2.0 x 6.0 mm i.d., epoxy-bearing, methacrylate-based monolithic disks were developed in order to target myoglobin and N-terminal pro-natriuretic peptide (NT-proBNP), two biomarkers involved in cardiovascular disease. In both cases, antibodies were successfully coupled to the polymeric disk material. The developed immunoadsorbers permitted the selective isolation of myoglobin and NT-proBNP from human serum. Myoglobin was successfully isolated and detected from serum samples at concentrations down to 250 fmol microL(-1). However, the affinity of the antibodies was not sufficient for the analysis of low-concentration clinical samples. Frontal analysis of anti-NT-proBNP disks revealed the ability of the immunoadsorber to bind up to 250 pmol NT-proBNP, which is more than sufficient for the analysis of clinical samples. Anti-NT-proBNP disks showed good stability over more than 18 months and excellent batch-to-batch reproducibility. Moreover, anti-NT-proBNP disks permitted the isolation of NT-proBNP at concentrations down to 750 amol microL(-1) in serum, corresponding to concentrations of strongly diseased patients. Using reversed-phase trapping columns, the detection of NT-proBNP eluted from immunoadsorbers by mass spectrometry was achieved for concentrations down to 7.8 fmol microL(-1).

The potential of mass spectrometry to study iron-containing proteins used in clinical diagnosis

“…The normal values of Mb in serum (3-90 ng mL −1 ) become significantly elevated after 1 h of the onset of myocardial infarction (MI) to peak in approximately 4-8 h with values from 200 to 1000 ng mL −1 [29]. Therefore, the measurement of serum Mb is applied nowadays as a biomarker of MI.…”

“…As miniaturization is a trend in clinical diagnosis, where low concentrations and volumes are commonly used, Delmotte et al [29] developed miniaturized monolithic immunoadsorbers for MI detection. Mb from serum samples was trapped into the immnoadsorber and further analyzed by IP-RP-HPLC-ESI-MS at concentrations down to 250 fmol L −1 .…”

Monolithic media for applications in affinity chromatography

“…Human serum albumin (HSA) and inter‐α inhibitor protein were selectively captured by specific antibodies that had been immobilized on the MSP in this manner 69. In addition to the direct immobilization of antibodies via the monolith's epoxy groups, immobilization was performed via streptavidin using biotinylated antibodies (for comparison, see Section 4.2.2) 70, resulting in the enrichment of myoglobin and N ‐terminal pronatriuretic peptide from serum.…”

“…The streptavidin/avidin–biotin complex is highly popular for the specific immobilization of biotinylated species due to its high association constant ( K A =10 15 M −1 ) 108. Streptavidin on monolithic supports has been successfully used for the immobilization of biotinylated DNA aptamers, which then allow a specific capturing of proteins 109, 110 (Section 4.2.3) and an immobilization of antibodies (Section 4.1.1) 70. Recently, MSPs with immobilized monomeric avidin ( K A =10 7 M −1 for the monomeric avidin–biotin complex) were prepared and used to enrich biotinylated cytochrome c and biotinylated cytochrome c peptides 111.…”

Organic monoliths for hydrophilic interaction electrochromatography/chromatography and immunoaffinity chromatography

“…They have demonstrated that the novel disks are suitable for the isolation of cardiac biomarkers from blood serum samples. Loading capacities in the high picomole range for immunoadsorbers with a bed volume as little as 22.4 μL are more than sufficient to isolate the proteins present at biologically relevant concentrations 72.…”