Microarray Immunoassay Tests for Simultaneous Detection of Five Botulinum Toxins by Phosphorescence Analysis (PHOSPHAN)

Combination of multiplex microarray immunoassay tests and luminescent nanoparticle tags is considered as a promising approach to the development of highly sensitive, specific, and rapid methods of causative agent detection. Objective of this study was to develop and compare the sensitivity of the tests for simultaneous detection of five botulinic toxins (A,B,C,E,F) applying multiplex phosphorescence analysis (PHOSPHAN) using standard (Pt coproporphyrin tag-based) and modified (europium containing nanoparticles) systems of phosphorescent signal registration. Materials and methods. PHOSPHAN assay was performed in standard 96 well microplate. The toxoids added to the wells interacted with monospecific and polyvalent immunoglobulins printed as tiny spots on the bottom of each well, and with a mixture of the same antibodies conjugated to biotin. Analyzed anatoxin concentration range – 0.005 to 100 ng/ml. The reaction was manifested by streptavidin conjugated to either Pt coproporphyrin, or the luminescent nanoparticles. The luminescence of both tags was recorded in time-resolved mode by Biochip Analyzer. The limit of detection corresponded to a minimum toxoid concentration, at which the P/N ratio was equal or exceeded 2, while the number of such samples (in a series of 10-30 experiments) was no less than 50%. Results and conclusions. Both multiplex tests provided for simultaneous group-specific detection of five botulinum toxins with the option of type-specific (A, B, E) identification. No cross-reactivity was revealed. The use of phospho￾rescent nanoparticles allowed for the increase in detection sensitivity by an order of magnitude, up to 10 pg/ml. The tests developed could be recommended for specific detection and identification of botulinum toxins in clinical, environmental, and food samples. 

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