Acetylcholinesterase (AChE) polyclonal antibody from hybrid catfish (C. macrocephalus × C. gariepinus): Specification, sensitivity and cross reactivity
| Autor: | Amnuay Wattanakornsiri, Krittima Saowakoon, Jakkaphun Nanuam, Phochit Nanthanawat, Nanthika Khongchareonporn, Chutima Thanomsit, Witchuda Prasatkaew |
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| Rok vydání: | 2020 |
| Předmět: |
010504 meteorology & atmospheric sciences
Physiology Health Toxicology and Mutagenesis Glycine Dot blot 010501 environmental sciences Cross Reactions Toxicology medicine.disease_cause 01 natural sciences Biochemistry Cross-reactivity Antibodies Snakehead chemistry.chemical_compound Western blot Antibody Specificity medicine Animals 0105 earth and related environmental sciences biology medicine.diagnostic_test Chemistry Herbicides fungi Cell Biology General Medicine biology.organism_classification Molecular biology Acetylcholinesterase Perciformes Polyclonal antibodies biology.protein Antibody Catfish |
| Zdroj: | Comparative biochemistry and physiology. Toxicologypharmacology : CBP. 237 |
| ISSN: | 1532-0456 |
| Popis: | AChE (acetylcholinesterase) is generally classified as a specific biomarker of pesticide exposure. The aim of this study was to produce AChE polyclonal antibody from hybrid catfish that were exposed to commercial glyphosate. The hybrid catfish was exposed to glyphosate (0.75 mL/L) for 24 h. After that, the fish brain was dissected, AChE was extracted and purified by hydroxyapatite column chromatography and eluted with 0.2 M potassium phosphate buffer pH 6.8. This protocol gave 70% yield. Then, the brain extract was characterized using 10% SDS-PAGE and Western blot probed with commercial polyclonal antibody specific to AChE (PAb-AChE). The protein, 71 kDa, was then used as an antigen to immunize mice for antibody production. The polyclonal antibody (PAb) was characterized using dot blot, Western blot and immunohistochemistry for immunolocalization of AChE in hybrid catfish exposed to glyphosate. We found that the appropriate dilution of antibody for both dot blot and Western blot was 1:3500, and 1:2500 for immunohistochemistry. Cross reactivity testing showed that PAb-AChE can be used with AChE from striped snakehead fish at the same dilution as used with AChE from hybrid catfish. It was concluded that PAb specific to hybrid catfish AChE from this work was highly specific and sensitive, and can cross-react with striped snakehead fish AChE. Thus, this polyclonal antibody may be used in monitoring glyphosate exposure in hybrid catfish and striped snakehead fish. |
| Databáze: | OpenAIRE |
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