Tool for new kinetics-based diagnostics of autoimmune diseases
A multiplex label-free biosensor is developed for diagnostics of autoimmune diseases by highly sensitive measuring in human serum both critical characteristics of autoantibody: concentration and native kinetic parameters that reflect autoantibody aggressiveness to the organism’s tissues.
The biosensor is based on the spectral-correlation interferometry and image processing of a microarray glass biochip, affordable to be single-used in medical applications. Simultaneous 25-min detection and activity characterization of several autoantibodies in the same serum sample have been demonstrated for anti-thyroglobulin (anti-TG) and anti-thyroid peroxidase (anti-TPO) as models.
The biosensor offers extremely high sensitivity: limits of detection in serum are 1.7 IU/mL and 6 IU/mL for anti-TPO and anti-TG, respectively. The dynamic range covers the whole range of clinically relevant concentrations of the autoantibodies up to 1000 IU/mL.
The developed method of characterization of autoantibody activity by recording the kinetics of their binding with free native antigens is based on autoantibody polyvalency.
The measurements in clinical serum samples have shown that the native kinetic parameters are independent of concentration. The proposed biosensor and method of native kinetic registration can be used to develop new criteria for comprehensive diagnostics of autoimmune diseases, based not only on traditional measurements of concentration but also on quantitative evaluation of autoantibody aggressiveness.
The developed method can be adapted to other label-free sensors such as those based on the surface plasmon resonance, optical waveguides, etc.
Neoparamoeba perurans is the causative agent of amoebic gill disease (AGD). Two loop-mediated isothermal amplification (LAMP) assays targeting the parasite 18S rRNA and the Atlantic salmon EF1α, used as internal control, were designed. The N.
perurans LAMP assay did not amplify close relatives N. pemaquidensis and N. branchiphila, or the host DNA. This assay detected 106 copies of the parasite 18S rRNA gene under 13 min and 103 copies under 35 min. Five “fast-and-dirty” DNA extraction methods were compared with a reference method and further validated by TaqMan™ qPCR. Of those, the QuickExtract buffer was selected for field tests.
Seventy-one non-lethal gill swabs were analysed from AGD-clinically infected Atlantic salmon. The pathogen was detected under 23 min in fish of gill score >2 and under 39 min for lower gill scores. About 1.6% of the tests were invalid (no amplification of the internal control). 100% of positives were obtained from swabs taken from fish showing gill score ˃3, but only ~50% of positives for lower gill scores.
The present LAMP assay could be implemented as a point-of-care test for the on-site identification of N. perurans; however, further work is required to improve its performance for lower scores.