Volume 13, Issue 2 (2023)
JMBS 2023, 13(2): 133-146 |
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Samiei Abianeh H, Nazarian S, Amani J, Hojjati razgi A S, Rahmani M R. Detection of Clostridium botulinum toxin type B by Sandwich ELISA method. JMBS 2023; 13 (2) :133-146
URL: http://biot.modares.ac.ir/article-22-60352-en.html
URL: http://biot.modares.ac.ir/article-22-60352-en.html
Hossein Samiei Abianeh1 
, Shahram Nazarian * 2, Jafar Amani3 , Amir Sajjad Hojjati razgi1 , Mohammad reza Rahmani1
, Shahram Nazarian * 2, Jafar Amani3 , Amir Sajjad Hojjati razgi1 , Mohammad reza Rahmani1
1- Biological Sciences Dept., Imam Hosein University, Tehran, Iran
2- Biological Sciences Dept., Imam Hosein University, Tehran, Iran , nazarian56@gmail.com
3- Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
2- Biological Sciences Dept., Imam Hosein University, Tehran, Iran , nazarian56@gmail.com
3- Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
Abstract: (1002 Views)
Background and Aim: Toxins produced by Clostridium botulinum are among the deadliest compounds known that cause botulism. Currently, the detection of BoNTs in food using bioassays on laboratory mice is a very sensitive method with a detection range of 7 to 20 pg.mL-1. However, bioassay for mice is time consuming. This method is fast, highly specific, and sensitive to experiments on mice. The aim of this study was to use the modified Sandwich ELISA method to detect BoNT/B toxin.
Materials and Methods: Recombinant 370 amino acid protein was expressed from the carboxyl terminus of the binding moiety of BoNT / B toxin with a molecular weight of 45 kDa as antigen and purified by Ni-NTA affinity chromatography. IgG antibodies were isolated from mouse and rabbit sera byG protein affinity chromatography. The sensitivity and specificity of the method designed to detect recombinant BoNT/B-HcC antigen and botulinum toxin type B were evaluated.
Results: Purified rat and rabbit antibody concentrations were 3 and 4.5 mg / ml serum, respectively. The minimum concentrations of detectable protein were determined by indirect ELISA with purified mouse and rabbit antibodies at 475 and 118 pg. By optimizing the sandwich ELISA method, at least 30 ng of recombinant BoNT/B-HcC antigen and146 pg of highly specific BoNT/B were detected.
Conclusion: sandwich ELISA method can be used for accurate and sensitive identification of Clostridium botulinum toxin type B. It is necessary to evaluate the effectiveness of this method in the future to detect botulinum toxin in environmental and food samples.
Materials and Methods: Recombinant 370 amino acid protein was expressed from the carboxyl terminus of the binding moiety of BoNT / B toxin with a molecular weight of 45 kDa as antigen and purified by Ni-NTA affinity chromatography. IgG antibodies were isolated from mouse and rabbit sera byG protein affinity chromatography. The sensitivity and specificity of the method designed to detect recombinant BoNT/B-HcC antigen and botulinum toxin type B were evaluated.
Results: Purified rat and rabbit antibody concentrations were 3 and 4.5 mg / ml serum, respectively. The minimum concentrations of detectable protein were determined by indirect ELISA with purified mouse and rabbit antibodies at 475 and 118 pg. By optimizing the sandwich ELISA method, at least 30 ng of recombinant BoNT/B-HcC antigen and146 pg of highly specific BoNT/B were detected.
Conclusion: sandwich ELISA method can be used for accurate and sensitive identification of Clostridium botulinum toxin type B. It is necessary to evaluate the effectiveness of this method in the future to detect botulinum toxin in environmental and food samples.
Article Type: Original Research |
Subject:
Microbial biotechnology
Received: 2022/03/19 | Accepted: 2022/08/24 | Published: 2023/07/10
Received: 2022/03/19 | Accepted: 2022/08/24 | Published: 2023/07/10
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