Strategies for upgrading analyte detection in immuno-PCR studied on identification of type A botulinum neurotoxin

Abstract

An efficient method of highly sensitive, specific determination of botulinic type A neurotoxin (which allows the detection of a toxin in a concentration from 1 pg/mL (10 fg per one PCR reaction) was created based on the technology of immuno-PCR. Solid phase consisting of paramagnetic organo–silicious particles carrying a detection complex of the toxin heavy chain with a pair of polyclonal antibodies to it was the most appropriate in the optimal scheme of the analysis. One of the antibodies bound to DNA (which is a matrix for PCR amplification) by means of biotin–neutravidin interaction. Matrix DNA was a molecular “net” generated by biotinylated DNA molecules with tetravalent neutravidin. The binding signal was detected by real time PCR (by the TaqMan method). An immuno-PCR method for determination of type A botulotoxin was developed.