The Establishment Of An Easy-to-use Method For The Detection Of Botulinum Neurotoxin Type A

Botulinum neurotoxin(BoNT)is produced by Clostridium botulinum in an anaerobic environment,and is subtyped from A to G according to their antigenicity.BoNT/A is the most toxic substances among all biological toxins and chemical poisons,and the clinical symptoms caused by it is far more severer than the BoNT/B and BoNT/E.Bo NT/A product has been used in the treatment of neuromuscular block function such as blepharospasm,hemifacial spasm,children with cerebral palsy,cosmetic wrinkles,and lose weight,the treatment of autonomic nervous system dysfunction such as hyperhidrosis and gastric achalasia,and the treatment of sensory nerve function such as migraine and chronic pain.With the development of economy and society,people's demand for beauty is increasing.In recent years,more and more people use BoNT to lose weight and to be more beautiful and younger.At present,there are some BoNT products named Botox(America Allergen company),Dysport(UK Ipsen Ltd),Myobloc(America Elan Inc),BTXA(Hengli,China Lanzhou Institute of Biological Products)and so on.In China,there are only two BoNT products,including Botox and BTXA,are approved to treat some diseases,lose weight and to be more beautiful and younger.Botox's can be used to treat cervical dystonia,severe primary axillary hyperhidrosis,neurogenic detrusor hyperactivity,overactive bladder,strabismus,blepharospasm,chronic migraine,and upper limb spasm.BTXA can be used to treat eyelid spasticity and hemifacial spasm of adult patients,strabismus especially caused by acute paralysis,endocrine myopathy and patients over 12 years which cannot be corrected or due to poor surgical results,temporary improve moderate or severe eyebrow pattern of adults under 65 years old due to frown.However,in the use of such botulinum toxin products,there have been some cases of poisoning such as varying degrees of eyelid spasm,blurred vision,ptosis,drinking water cough,dysphagia,dysarthria,dysphonia,muscle weakness,even difficulty breathing which are mainly caused by the over injection and/or the improper dispersion of them.As a commercial biological agents,the study of BoNT is particularly important.Biological products should generally illustrate in quality and activity.The construction of activity should be measured by the biological activity contained in one unit of biological product.The aim of this study was to develop an easy-to-use lateral flow test strip,which was used to detect the activity of BoNT based on the digestion kinetic of metal zinc and immunoreaction principles.The principle of detection is that the biotin at the amino terminus of the peptide separate from the carboxy-terminal His-Tag after incubation with BoNT/A-spiked serum sample for a certain time duration,resulting in that gold nanoparticle-labelled streptavidin-substrate peptide complexes could not be captured on the test zone of LFTS,thus,LFTS presented a different coloration from BoNT/A-negative result.First,we cultured the Clostridium botulinum type A and B.The Clostridium botulinum type A was inoculated to TPYG liquid medium,and incubated for 48 h in an anaerobic incubator at 37?.The fresh culture medium was transfered into TPOM medium for toxic culture.The released toxin was got with the H2SO4 dropped into the medium.And then the nucleic acid was removed with(NH4)2SO4.And finally BoNT/A was purified by Sephacryl-200 column and DEAE-Sepharose.The purified BoNT/A was quantified and the biological activity of toxins was determined by mouse lethality assay.The Clostridium botulinum type B culture,purification,quantification and biological activity were identified with botulinum toxin type A,but the step of purification had not been present.The purified BoNT/A and B were applied to simulate spiked clinical serum samples.Then,the sequence of BoNT/A substrate was designed as follows: N-terminal biotinylated-TRIDEANQRATKM-6×His-Tag.The substrate peptide consists of three parts: the N-terminal biotin label,the BoNT/A specific substrate peptide sequence and the His-tag.Bo NT/A specific cleavage sites is between Q and R.When BoNT/A acts on the peptide as a endopeptidase,it can break down the peptide chain,so that the peptides become to two different biomarkers with different color patterns on colloidal gold lateral flow test strip.The anti-His monoclonal antibodies and the anti-streptavidin antibody were dispensed respectively as test line and control line,the colloidal gold was conjugated with streptavidin as the probe according to the producer's instruction.We optimized the concentration of test line,the minimum dose of substrate peptide,and the incubation time of substrate peptides and toxins in vitro.According to the optimized results,the concentration of test line was 1 mg/m L;the dose of the specific substrate peptide was 1 ng/?L;the incubation time of toxins and substrate peptides in vitro was 12 h.In addition,we evaluated the specificity and sensitivity of the LFTS with blank serum,BoNT/A-spiked and BoNT/B-spiked serum.According to the experimental results,the minimum detection limit of the LFTS can reach 1.3 LD50/mL(about 20 pg/mL),the sensitivity is consistent with the mouse lethality assay of gold standard method,but the detection time reduced from 4 days to 12 h.The method established in this study consumes significantly less sample and reagents,which will reduce assay cost and simplify disposal and is more suitable for use in the remote medical institutions and remote areas with limited resources.Our LFTS assay combines the very best of LFTS and endopeptidase activity assays.The former offers a familiar and easy-to-use laboratory assay platform.The latter provides the specificity to BoNT/A and enzyme-based signal amplification processes.In China,the majority of the cases caused by botulism were from less-developed rural areas.It is difficult to diagnose and treat of botulism in the regions where traffic were inconvenience and professional equipment were poor.The establishment of the detection method can effectively alleviate the misdiagnosis and missed diagnosis of botulism,improve the diagnosis and treatment rate.And by designing the toxin-specific substrate peptide,the regimen can also be extended for the detection of other serotypes of BoNT.In addition,BoNT is currently used by some international terror and extremist organizations to make panic and is listed as one of the most likely to use bioterrorism.Our existing means of detection are time-consuming,costly and require specialized professionals to carry out in well-equipped professional laboratories.It is critical to design and develop a fast,sensitive and easy-to-use detection method for the diagnosis and identification of botulism in China,especially when infant botulism and food safety is uncertain.