Development and comparison of two immunoassay formats for rapid detection of botulinum neurotoxin type A.

We have evaluated two formats of immunoassays for the rapid detection of Clostridium botulinum neurotoxin type A (BoNT/A), in assay buffer and various matrices (human serum and nasal swabs, fresh milk, sugar, flour and talcum). The two formats, a vertical-flow strip immunochromatography (ICT) and a small disposable immunoaffinity column (IAC), were selected because they are both rapid and readily usable in the field without sophisticated equipment. We utilised the same critical reagents to develop and optimise both assays, making it possible to compare the corresponding technologies on the same toxin preparations, without interference due to the properties of the antibodies. Results were interpreted using a standard statistical test (ANOVA) and showed little difference of sensitivity between matrices. Though both assays were completed in 40 min, the sensitivity of the IAC, evaluated at 0.45 pM (5 mouse LD50 units/ml), was 40 to 80 times better than that of the ICT. Furthermore, the sensitivity of the IAC assay was improved to 0.09 pM (1 mouse LD50 unit/ml) when performed on a 5-ml volume of human serum. Thus, the IAC appears to be one of the most sensitive and rapid assays for the detection of BoNT/A reported to date and, because it is also highly transportable, it is amongst the best suited for field diagnosis of BoNT/A poisoning.

Protective anti-V antibodies inhibit Pseudomonas and Yersinia translocon assembly within host membranes.

Pathogenic Yersinia species and Pseudomonas aeruginosa share a similar type III secretion/translocation system. The translocation system consists of 3 secreted proteins, YopB/PopB, YopD/PopD, and LcrV/PcrV; the latter is known to be a protective antigen. In an in vitro assay, the translocation system causes the lysis of erythrocytes infected with wild-type (wt) P. aeruginosa. wt Y. enterocolitica is not hemolytic, but a multiknockout mutant deprived of all the effectors and of YopN ( Delta HOPEMN) is hemolytic. In the presence of antibodies against PcrV and Y. pestis LcrV, the hemolytic activity of P. aeruginosa was inhibited. Similarly, the hemolytic activity of Delta HOPEMN was inhibited in the presence of anti-LcrV antibodies. The assembly of the translocon, composed of PopB/D and YopB/D proteins, was disturbed in immunoprotected erythrocyte membranes, mimicking the phenotypes of V knockout mutants. Thus, protective antibodies against the V antigens of Yersinia species and P. aeruginosa act at the level of the formation of the translocon pore in membranes of infected host cells by blocking the function of LcrV/PcrV. The hemolysis assay could be adapted for high-throughput screening of anti-infectious compounds that specifically target the type III translocon.