Quantitation Methods For The Activity Determination Of Serotypes Of Botulinum Neurotoxin And Their Clinical Applications

Botulinum neurotoxin(BoNT)is the most toxic substance known to humankind,the most classical serotype classification is BoNT/A-G.BoNT can cause muscle relaxation paralysis illness in humans and animals which called botulism.Foodborne botulism,infant botulism and traumatic botulism are the most common poisoning types.Botulism incidents occur frequently at home and abroad.Due to the extreme toxicity of BoNT,the lack of accurate and reliable clinical laboratory detection methods and the timely treatment have led to poisoning deaths.In recent years,with the widespread use of BoNT in the field of medical cosmetology,botulism caused by injection of BoNT cosmetic products from informal sources or improper use is also on the rise,posing a non-negligible threat to public health and safety.In addition,BoNT is easy to be manufactured,disseminated,and abused,hence it is ranked as one of the six most threatening biological warfare agents.BoNT-related military and bioterrorist attacks can cause great panic in the international community.Therefore,it is of great medical and military importance to establish a rapid and accurate method for serotype differentiation and activity determination of BoNT.Currently,a variety of BoNT detection methods have been developed,among which the mouse bioassay,immunological assay and PCR method are more widely used.However,these assays have certain limitations,such as they are unable to determine the toxin serotype and activity simultaneously,the susceptibility to false positives of complex matrix samples,and the low sensitivity of the methods.In recent years,mass spectrometry-based detection methods have become a new trend,however,there is still a shortage of LC-MS/MS(MRM)-based methods for BoNT detection.And most of these methods have not been applied and validated in clinical samples,which makes it difficult to provide timely and reliable basis for clinical diagnosis and treatment to botulism.In addition,there is still a lack of rapid detection methods for BoNT.Therefore,the establishment of a highly sensitive method for the differentiation and activity determination of multiple serotypes of BoNT in clinical samples remains a priority and difficult area for research.To address the above difficulties,this thesis focuses on the development of a LCMS/MS(MRM)method which based on the specific endopeptidase activity of seven serotypes of BoNT.Furthermore,for serotypes A,B and E those commonly cause human botulism,an antibody simultaneously specific recognizing the three serotypes was used to easily enrich toxins from complex biological matrices,which achieved the highly sensitive identification,serotype differentiation and activity determination of BoNT/A/B/E in complex matrices.On this basis,a stable isotope dilution LC-MS/MS method for simultaneously detection of BoNT/A-G was developed and applied to detection clinical samples from foodborne botulism cases in China in the past three years,which provided timely and effective information for clinical diagnosis and treatment.Furthermore,a highly sensitive and rapid detection method for BoNT and active ricin,respectively,based on SERS-immunoassay strategy was also established.The thesis is divided into five chapters.The first chapter is a preface.We firstly outlined the generation,structure,serotype classification and the toxic action process of BoNT,and the specific enzymatic cleavage activity of BoNT was especially introduced.Secondly,the clinical presentation characteristics,differential diagnosis status,and clinical treatment of botulism were discussed.In addition,the medical use of BoNT was introduced,and BoNT-related military and bioterrorist attack events were also presented.Then,the current status of analysis and detection methods for BoNT were outlined in focus.Finally,we put forward the basis and the main research contents of this thesis.In the second chapter,an Endopep-MS method was constructed for simultaneously detection of BoNT/A/B/E in complex matrix,which based on the specific enzymatic cleavage activity of BoNT.Synthetic substrate peptides were used to mimic the target proteins of each serotype of BoNT in vivo,and the product peptides formed by enzymatic cleavage of the substrate at the respective specific sites of BoNT were detected by LC-MS/MS(MRM).Firstly,the peptide substrate of BoNT/A was screened,the LC-MS/MS(MRM)methods and enzymatic cleavage reaction buffer were optimized,which achieved highly efficient enzymatic cleavage of BoNT and accurate quantification of product peptides.Then,a highly specific antibody mAb 01-3 conjugated with immunomagnetic beads was used for one-step enrichment of BoNT/A/B/E,which solved the difficult problems such as the low concentration of BoNT in complex matrices,high matrix interference and complicated pre-treatment process to capture target toxin.The LOD values of BoNT/A,B and E detection in serum were 0.5 U/mL,1.5 U/mL and 1.8 U/mL,respectively.There was no interference among these three serotypes.Finally,the method was successfully applied to the analysis of suspected cosmetic botulism cases,which providing a basis for the clinical diagnosis and treatment of cosmetic related botulism.In the third chapter,a stable isotope dilution LC-MS/MS method for simultaneously detection of BoNT A-G was developed.Seven peptide substrates were employed to mimic the targets in vivo of BoNT A to G respectively,and the respective stable isotope labelled peptides of each peptide product were used as internal standards(IS).Combined with the stable isotope IS of each serotype,the LC-MS/MS(MRM)method for quantification product peptides was validated to be sensitive and accurate,which met the methodological requirements for the analysis of biological samples.By extracting MRM transitions and performing retention time matching of each product peptides of BoNT/A-G,the method can accurately differentiate seven serotypes and determine their activities with high specificity.Our research provides a simple and powerful method for detection clinical samples from human and animal botulism and their related environmental samples.In the fourth chapter,the established method was applied to the analysis of 63 clinical samples from 17 suspected cases in China from 2019 to 2022 to determine the existence of BoNTs and differentiate their serotypes.Serum,urine,vomitus,postmortem blood,gastric mucosa and food samples were comprehensively involved and analyzed.Totally 11 cases were identified as positive for BoNT.In this process,the first cases of mixed botulism of BoNT/A/B/E with predominantly type E toxin from one family were identified.The relationship between the detection results of the samples and the development of clinical symptoms was explored in detail for the four cases of mixed botulism.Results showed that BoNT concentration levels and serotypes in the clinical samples were consistent with the patients’ clinical symptomatology.Our results indicated that the serum for detection BoNT/A with a detection window up to 12 days,and for the first time revealed that urine sample is suitable for testing BoNTs with a longer detection window up to 25 days.The above findings provide an important basis for the suitable type and time of clinical sample collection.The established multiplex Endopep-MS method with high sensitivity and specificity was proved to be of great significance for timely and accurately diagnosis of botulism,especially for the mixed botulism.In the fifth chapter,a rapid SERS-based sandwich immunoassay was developed and applied for the detection of active ricin and BoNT.Immunomagnetic beads coupled with toxin-specific antibodies were used as capture elements for rapid separation and enrichment of toxins from complex matrices,and gold nanoparticles(AuNPs)decorated both with ricin-specific monoclonal antibody(mAb)14C12 and Raman reporter molecule Nile blue A(NBA)were used as both recognition elements and SERS nanoprobes.A sandwich immunocomplex "magnetic beads-biotoxins-SERS nanoprobes" based on the antigen-antibody interaction was finally formed.The presence and concentration of toxin can be indicated by the SERS signal at 591 cm-1 of the characteristic Raman shift of NBA.Firstly,we selected ricin as a target model for the strategy construction.A specific monoclonal antibody recognizing the active ricin was used as the capture antibody,which can achieve the sensitive detection of ricin,the LOD values for ricin detection in PBS and plasma were 1 ng/mL and 5 ng/mL,respectively.The established method can be used for distinguishing active ricin from inactive ricin,and it also achieved a good discrimination among ricin and its highly homologous biotoxins,such as RCA120,abrin and AAG.The strategy was then applied to the detection of BoNT,employing a high affinity nanoantibody for BoNT/A for constructing SERS nanoprobes.The LOD of BoNT/A analysis in PBS was as low as 20 U/mL.Our method could provide a new SERS-based strategy for evaluating the threat of BoNT,ricin and other biotoxins in defense and security field.