Medium-high resolution electrochemical genotyping of HLA-DQ2/DQ8 for detection of predisposition to coeliac disease

Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained.

Direct detection of myoglobin in whole blood using a disposable amperometric immunosensor

An amperometric immunosensor for the rapid detection of myoglobin in whole blood was developed. Due to its rapid kinetics, myoglobin is a useful biochemical marker for the early assessment of acute myocardial infarction (AMI). A one-step indirect sandwich assay was employed using a polyclonal goat anti-human cardiac myoglobin antibody with monoclonal mouse anti-myoglobin and goat anti-mouse IgG conjugated to alkaline phosphatase (AP), as the detecting antibodies. The final sensor required 30 min for incubation. The standard curve was linear between 85 and 925 ng/ml. The intra- and inter-assay coefficients of variation were below 8%. No cross-reactivity of the antibodies was found with other cardiac proteins. The overall performance of the sensor, rapid analysis time, wide working range, good precision and specificity demonstrate its potential usefulness for early assessment of AMI.