Sains Malaysiana 52(12)(2023): 3449-3461

http://doi.org/10.17576/jsm-2023-5212-08

Development of a Fast  Immunosorbent Assay for Site-Screening Dioxin Contamination in Vietnam

(Pembangunan Asai Imunosorben Pantas untuk Penyaringan Tapak Pelumusan Dioksin di Vietnam)

PHUONG NAM DANG^1,2, VAN HOANG NGUYEN¹, KIEN CUONG PHAM¹, THI NHUNG NGUYEN¹, LAN ANH TO¹, DUY KHANH LE¹, XUAN TRUONG NGHIEM³& KHANH HOANG VIET NGUYEN¹

¹Department of Molecular Biotechnology, Institute of New Technology,

Academy of Military Science and Technology, 17 Hoang Sam, Cau Giay, Hanoi, Vietnam

²Faculty of Biology, Hanoi National University of Education, 136 Xuan Thuy, Cau Giay, Hanoi, Vietnam

³Department of Chemistry and Environment, Vietnam-Russia Tropical Center, 63 Nguyen Van Huyen, Cau Giay, Hanoi, Vietnam

Received: 8 December 2022/Accepted: 23 November 2023

Abstract

Dioxins are a group of chemical compounds that cause environmental pollution and many harmful effects on human health. High-Resolution Gas Chromatography/High-Resolution Mass Spectrometry (HRGC/HRMS) is the standard method for determining dioxin concentrations in soil samples and provides the most accurate results. However, this method is time-consuming, costly, and requires modern equipment. Currently, competitive ELISA is a reliable method used for dioxin detection analysis, offering fast implementation time and low cost. Vietnam is a global hotspot for dioxin contamination, with a high number of dioxin samples for analysis. Therefore, it is essential to optimize this reliable, fast, and low-cost ELISA method for it to be applicable and replace the expensive and complex HRGC/HRMS method currently in use in Vietnam. This study presented optimized conditions for ELISA method using commercial antibodies to detect dioxin. The optimal dilution for the anti-dioxin antibody and the conjugated antibody is 1:2000 and 1:1000, respectively. The reconstitution buffer consists of 50% DMSO/H₂O, with the addition of 0.05% Triton X-100. The incubation time for anti-dioxin antibody incubated with dioxin is 60 min, while the incubation time for Horseradish Peroxidase (HRP) conjugated polyclonal antibody incubated with 3,3',5,5'-Tetramethylbenzidine (TMB) substrate is 10 min. The quenching time for the enzyme-substrate reaction is 5 min. The half-maximal inhibitory concentration (IC₅₀) of this method is 8500 pg/well and the limit of detection (LOD) is 2.02 pg/well. Although there is a difference between the analytical results of the two methods, the well-correlated results demonstrate the potential of the ELISA method for detecting and screening dioxin contamination before performing confirmatory analysis with HRGC/HRMS. These results serve as the basis for the development of a rapid dioxin detection kit, providing a new and efficient method for detecting and screening dioxin contamination in Vietnam.

Keywords: Dioxin; ELISA; fast detection; HRGC/HRMS

Abstrak

Dioksin ialah sekumpulan sebatian kimia yang menyebabkan pencemaran alam sekitar dan memberi kesan berbahaya kepada kesihatan manusia. HRGC/HRMS ialah kaedah piawai untuk penentuan kepekatan dioksin dalam sampel tanah dan dapat memberi keputusan yang paling tepat. Walau bagaimanapun, kaedah ini memakan masa, kos yang tinggi dan memerlukan peralatan moden. Pada masa ini, ELISA yang kompetitif ialah kaedah yang boleh dipercayai yang digunakan untuk analisis pengesanan dioksin dengan masa pelaksanaan yang cepat dan kos yang rendah. Vietnam ialah tempat yang tinggi pencemaran dioksin di dunia dengan bilangan sampel dioksin yang tinggi untuk dianalisis, oleh itu adalah perlu untuk mengoptimumkan kaedah ELISA yang boleh dipercayai, cepat dan kos rendah ini supaya dapat digunakan dan menggantikan HRGC/HRMS yang digunakan di Vietnam. Kajian ini membentangkan keadaan optimum untuk kaedah ELISA menggunakan antibodi komersial untuk mengesan dioksin. Pencairan optimum untuk antibodi anti-dioksin dan antibodi terkonjugasi masing-masing adalah 1:2000 dan 1:1000, kemudian penambahan penimbal ialah 50% DMSO/H₂O ditambah dengan 0.05% Triton X-100. Masa untuk antibodi anti-dioksin diinkubasi dengan dioksin ialah 60 min, masa untuk antibodi poliklonal terkonjugasi HRP yang diinkubasi dengan substrat TMB ialah 10 min dan masa untuk pelindapkejutan tindak balas enzim-substrat ialah 5 min. IC₅₀ kaedah ini ialah 8500 pg/well dan had pengesanan ialah 2.02 pg/well. Walaupun terdapat perbezaan antara keputusan analisis kedua-dua kaedah, keputusan berkorelasi dengan baik menunjukkan potensi kaedah ELISA dalam mengesan dan menapis pencemaran dioksin sebelum melakukan analisis pengesahan dengan HRGC/HRMS. Keputusan ini adalah asas untuk pembangunan kit pengesanan dioksin pantas, menyediakan kaedah baharu dan cekap untuk mengesan dan menyaring pencemaran dioksin di Vietnam.

Kata kunci: Dioksin; ELISA; HRGC/HRMS; pengesanan cepat

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*Corresponding author; email: hoangviet1015@gmail.com