title: Expression, purification and development of neutralizing antibodies from synthetic BoNT/B LC and its application in detection of botulinum toxin serotype B.
abstract: Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) neurotoxins (BoNTs). The mouse bioassay is the gold standard for the detection of botulinum neurotoxins, however it requires at least 3-4 days for completion. Most of the studies were carried out in botulinum toxin A and less on type B. Attempts have been made to develop an ELISA based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. In the present study, the synthetic BoNT/B LC gene was constructed using PCR overlapping primers, cloned in a pET28a+ vector and expressed in E. coli BL21DE3. The maximum yield of recombinant proteins was optimized after 16 hrs of post induction at 21°C and purified the recombinant protein in soluble form. Antibodies were raised in Mice and Rabbit. The IgG antibody titer in the case of Mice was 1: 1,024,000 and Rabbit was 1: 512,000 with alum as adjuvant via intramascular route. The biological activity of the recombinant protein was confirmed by in-vitro studies using PC12 cells by the synaptobrevin cleavage, the rBoNT/B LC protein showed the maximum blockage of acetylcholine release at a concentration of 150nM rBoNT/B LC in comparison to the control cells. When the cells were incubated with rBoNT/B LC neutralized by the antisera raised against it, the acetylcholine release was equivalent to the control. IgG specific to rBoNT/B LC was purified from raised antibodies. The results showed that the developed antibody against rBoNT/B LC protein were able to detect botulinum toxin type B approximately up to 1 ng/ml. These developed high titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.
authors.affiliation: Biotechnology Division, Defence Research and Development Establishment, Gwalior, Madhya Pradesh, India. ponmariappans@yahoo.com
authors.forename: S
authors.initials: S
authors.lastname: Ponmariappan
authors.forename: Swati
authors.initials: S
authors.lastname: Jain
authors.forename: Richa
authors.initials: R
authors.lastname: Sijoria
authors.forename: Arvind
authors.initials: A
authors.lastname: Tomar
authors.forename: Om
authors.initials: O
authors.lastname: Kumar
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000276:Adjuvants, Immunologic / Q000378:metabolism; D000536:Aluminum Hydroxide / Q000276:immunology; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D001905:Botulinum Toxins / Q000032:analysis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D019274:Botulinum Toxins, Type A; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression; D005813:Genes, Synthetic / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D057230:Limit of Detection; D051379:Mice; D016716:PC12 Cells; D010957:Plasmids / Q000235:genetics; D050683:R-SNARE Proteins / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013696:Temperature
chemical_list: D000276:Adjuvants, Immunologic; D057134:Antibodies, Neutralizing; D004274:DNA, Recombinant; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; C096323:rimabotulinumtoxinB; D007544:Isopropyl Thiogalactoside; D000536:Aluminum Hydroxide; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D000109:Acetylcholine