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closesome questions
Posted by diana on 05 May 2008 at 16:45 GMT
This paper reports the development of a novel detection system with an excellent detection limit for BoNT/A1 and BoNT/A1 complex under optimal conditions. Congratulations on your very good work.
I also work in this field and always face the problem of false positives in very sensitive assays. From the paper, I could not find out (maybe I overlooked it?) whether you used preactivated BoNT/A1-complex for your recovery assays from complex matrices in figure 3 B? Also, I failed to find the blank values of your complex matrices like carrot juice or human serum without any BoNT. Are they listed somewhere and I just didn’t find them? I did the experience that some blank values of the matrices were detected positive in our assays. Do you think, that is will be a problem also in your assay?
I have one final question concerning your functional assay only detecting the activity of the light chain (the enzymatic active site) of BoNT/A1 and not of the whole protein. Isn’t it possible that the light chain may be perfectly active while the heavy chain (which mediates translocation into the neuron) is either detached or rendered non-functional in some other way such that the tested substance cannot be toxic because the toxin never enters any neurons? In this case your method would yield a false positive despite correctly detecting BoNT activity. This would not happen in the “gold standard” mouse test.
RE: some questions
iontrapper replied to diana on 08 May 2008 at 22:38 GMT
>>From the paper, I could not find out (maybe I overlooked it?) whether you used preactivated BoNT/A1-complex for your recovery assays from complex matrices in figure 3 B? Also, I failed to find the blank values of your complex matrices like carrot juice or human serum without any BoNT. Are they listed somewhere and I just didn’t find them?
Non-preactivated toxin was used in Fig. 3 B, however, our reaction buffer always contains at least 1.25 mM DTT (see methods). The sample-specific background was subtracted for each sample type used. Supplemental Figure S2 demonstrates the background for the bead-free reaction in 10% FBS (usually 6 to 8 thousand RFU and stable). FRET-mediated quenching of the fluorophore in our fluorogenic peptide (SNAPTide) is not perfect. Hence, there is always a certain degree of background fluorescence.
>>I did the experience that some blank values of the matrices were detected positive in our assays. Do you think, that is will be a problem also in your assay?
We do not know what assay you were performing and I cannot give you specific recommendations without knowing further details.
>>I have one final question concerning your functional assay only detecting the activity of the light chain (the enzymatic active site) of BoNT/A1 and not of the whole protein. Isn’t it possible that the light chain may be perfectly active while the heavy chain (which mediates translocation into the neuron) is either detached or rendered non-functional in some other way such that the tested substance cannot be toxic because the toxin never enters any neurons? In this case your method would yield a false positive despite correctly detecting BoNT activity. This would not happen in the “gold standard” mouse test.
We have not encountered the problem of selective degradation of heavy chain versus light chain in any natural sample types. The assay that we reported was developed for holotoxin and BoNT/A complex. In the existing form it does not distinguish between light chain and the holotoxin or complex, however, we have recently performed the ALISSA with antibodies specific to BoNT heavy chain epitopes the achieved the same level of sensitivity.