Preparation Of Monoclonal Antibodies Against Botulinum Neurotoxins And Development Of Methods For Detecting Botulinum Neurotoxins And Their Application

Botulinum neurotoxins (BoNTs) are usually produced by Clostridiumbotulinum in seven serotypes (A through G, with many subtypes for eachserotype), which are typical A-B type neurotoxins, BoNT/A, B, E and F causethe human botulism, easpecially the A serotype. The BoNTs are the mostpoisonous substance known, the lethal dose for human is supposed only0.06μgfor an adult. At the same time, BoNTs are easy to produce and transport,therefore BoNTs are classified by the American Centers for Disease Control(CDC) as one of the top six biothreat agents (Class A agents), especially the typeA. However, the outbreak with an unusual type should be considered to bedeliberately release. Each natural serotype of BoNTs is synthesized as an inactive single chain(150kDa) protein, and then cleaved to form a disulfide bond-linked dipeptideconsisting of a100-kDa heavy chain (HC) and a50-kDa light chain (LC). TheHC is comprised of two functional domains, the C-terminal HC receptor-bindingdomain (Hc domain,50kDa) specifically bind the presynaptic nerve terminalmembrane，and the N-terminal heavy chain translocation domain (Hn domain,50kDa) mediate the internalization of BoNTs into the neuron, little conservedHc amino acid sequences were observed between serotypes, and the receptorsbinding by each serotype are different. After the BoNTs enter into the neurons,the light chains cleave different members of the SNARE complex depending ondifferent types with the presence of zinc. Persons once intoxicated, the BoNTscould block the releasing of acetylcholine, which result in blockade ofneuromuscular transmission and cause flaccid paralysis or even death, if noimmediate and enough medical support.Recently, botulinum neurotoxin type A (BoNT/A) has been exploited andwidely used in clinical departments to treat disorders such as cervical dystonia,hemifacial spasm, focal spasticity, hyperhidrosis, ophthalmological andotolaryngeal disorders, as well as in cosmetic applications such as reducing skinwrinkles. Despite its medical applications, the risks of abuse and misuse of thistoxin should be vigilant. It was reported in2006that4adults were infected withbotulism after receiving treatment with an unlicensed highly concentratedBoNT/A preparation.There is no specific drug for prevention or treatment of botulism, therapyfor botulism depends on timely administration of passive neutralizing antibody,which will eliminate subsequent nerve damage and severity of disease. Theadministration of botulinum antitoxin requires early detection of BoNTs.Traditionally, the standard method to detect BoNT/A for suspect botulism isthe mouse bioassay, in which BoNT/A-induced botulism is determined byinjecting the suspicious samples to the mouse to elicit botulism syndrome. It is a sensitive method, with a detection limit of less than5mouse median lethal dose(LD50) per mL (<5LD50/ml). However, the mouse bioassay is time consuming(takes up to4days) and naturally presents an ethical dilemma due to the use oflaboratory animals. Methods for detecting the toxin in rapid, sensitive, and toxinserotype-specific ways are researched for successful therapy. Manyimmunological methods like the enzyme-linked immunosorbent assay (ELISA)have successfully been developed with which the time needed for detection ofthe toxins are as short as less than a day. However, the conventional ELISAcould detect the toxins as little as10–100folds LD50/ml. Some modifiedmethods based on ELISA such as enzyme-linked coagulation assay (ELCA),could detect the toxins with the limitation of1-2LD50/ml; but this methodsuffered from high cost and complicated procedures. By using affinity-purifiedhorse polyclonal antibodies against HC domain, the sensitivity of ELISA couldapproach the level similar to that of mouse bioassay. Recently otherimmunoassays have been introduced to detect the BoNTs, such asimmune-polymerase chain reaction (IPCR), electrochemiluminescence (ECL)assay and lateral flow assays. Although the sensitivity of IPCR could achieveabout105folds than that of ELISA (50fg/mL), it still suffers from shortcomingssuch as complexity of the method and its susceptibility to sample matrix’s effect.The ECL and lateral flow assay all failed to achieve the sensitivity of the mousebioassay. A drawback of immunological detection method is that it is supposedto be short of high-quality antibodies, the advancement of antibody wouldincrease the sensitivity of the immunological method such as ELISA. Therecombinant HC subunit of Clostridium botulinum neurotoxin serotype A (rAHc)that could elicit highly protective activity in mouse was newly designed as avaccine against botulism, indicating that the rAHc could be an effectiveimmunogen to develop high-quality antibodies. The ELISA usingchemiluminescence substrate was also known as chemiluminescence enzymeimmunoassay (CLEIA). It was reported that CLEIA could quantify the BoNT/A with the limit of detection (LOD) of5pg/ml,2–4folds lower than the mousebioassay (10–20pg/ml).It has reported that pre-vaccinated with toxoid or recombinant vaccines coulddraw protection against the BoNTs. However, because of rarity natureoccurrence of botulism, mass civilian or military vaccination is inappropriate.For the rapidly onset after intoxication, the post exposure vaccination isvalueless. The only useful method for pre-or post-exposure prophylaxis or fortreatment of botulism is the antitoxin or neutralizing antibody. Equine antitoxinand human botulinum immunoglobulin are currently available to treat adult andinfant botulism, respectively. However, considering the limited supply of theseantitoxin, monoclonal antibody (mAb) could provide an unlimited supply ofantitoxin not requiring human donors for plasmapheresis and provided apotential way for development of recombinant monoclonal antibody. To providean adequate number of doses at reasonable cost, such mAbs must be of highpotency of neutralization activity. It has reported the potent of singleneutralizing mAb could neutralize at most10to100times the50%lethal dose(LD50) of toxin in mice, the neutralizing potent of combining two or threemAbs could neutralize even more dose of toxin, which providing a way toproduce drugs for preventing and treating botulism.In this study, By immunizing mice with rAHc and screening, a total of56positive clones attributed to8epitop groups for BoNT/A and40positive clonesattributed to7epitop groups for BoNt/F were obtained. After confirmed byneutralizing mouse bioassay, the potent of mixed anti-rAHc mAbs couldneutralize as more as2×104times LD50toxins, and the potent of one singlemAbs, the FMU-BTA49, could neutralize more than50times LD50BoNT/A,which could be further exploited as candidate for development of recombinantmAbs.After screening the antibody pairs in a large-scale pairwise analysis, we aimedto establish a highly sensitive immunoassays which can be used for detecting BoNT/A in food matrix and quantify the clinical preparations of this toxin. Twohigh-affinity mouse monoclonal antibodies (mAbs) recognizing differentepitopes on the Hc domain of BoNT/A (AHc) were obtained. Cross-reactionwith other toxins in ELISA was established and the sensitivity was similar withthat of the mouse bioassay. Next, a CLEIA for BoNT/A was established basedon the ELISA sequentially, which could detect the BoNT/A complex at0.45pg/mL of150kDa BoNT/A, and the LOD of BoNT/A complex was confirmedfurther by testing the preparation of BoNT/A, BOTOX. The results showedthat this method could reach at0.1LD50/mL, about20–40folds more sensitivethan the mouse bioassay (<5LD50/ml,10–20pg/ml).