The Fipronil ELISA Kit is intended for the determination of Fipronil in liquid whole egg samples.
Storage
The Fipronil ELISA should to be stored in the refrigerator (2–8°C). The solutions must be allowed to reach room temperature (20-25°C) before use. Reagents may be used until the expiration date on the box. Reseal the unused strips of the microtiter plate in the bag together with the desiccant bag provided. Do not intermix components from different lots.
Sensitivity
Liquid Whole Egg 0.0025mg/kg (2.5ppb)
General Description
Fipronil is a broad-spectrum phenylpyrazole insecticide. It is authorised in the EU for use in veterinary medicine to combat insects control ants, beetles, cockroaches, fleas, ticks, termites, mole crickets, thrips, rootworms, weevils, and other insects. It is not permitted for use on food-producing animals. Fipronil-sulfone is the primary biological metabolite of fipronil and Fipronil-desulfinyl is the primary environmental metabolite. The Maximum Residue Limit (MRL) for fipronil is expressed as the sum of fipronil and fipronil sulfone and has been set at 0.005mg/kg (5ppb) in the European Union. Fipronil is not carcinogenic. Animal studies carried out by the European Food Safety Authority (EFSA) and the World Health Organisation (WHO) reported adverse effects on the nervous system were observed after short-term exposure.
Reconstitution And Storage
Micro-pipetting equipment and pipette tips for pipetting the standards and the samples are necessary. We recommend using a multi-channel pipette or a stepping pipette for adding the conjugate, antibody, substrate and stop solutions in order to equalize the incubations periods of the solutions on the entire microtiter plate. Please do not mix any reagents from various kit lot numbers. 1. Adjust the microtiter plate and the reagents to room temperature before use. 2. Remove the number of microtiter plate strips required from the foil bag. The remaining strips are stored in the foil bag with desiccant and zip-locked closed. Store the remaining kit in the refrigerator (2-8°C). 3. The standard solutions, primary antibody solution, secondary HRP antibody, sample diluent, substrate and stop solutions are ready to use and do not require any further dilutions. 4. Dilute the wash buffer concentrate at a ratio of 1:50. If using the entire bottle (20 mL), add to 980 mL of deionized or distilled water. Once diluted the solution is stable for 30days when stored at 2-8°C. 5. The stop solution should be handled with care as it contains diluted hydrochloric acid.
Citations
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Background
Fipronil is a phenylpyrazole broad-spectrum insecticide, which is widely used to control various categories of crop and sanitary pests in vegetables, rice, tobacco, animal husbandry and public health because of its low production cost and high fungicidal efficiency. Its structure and insecticidal mechanism are different from those of other existing insecticides, making it effective against pests that are resistant to conventional pesticides. Fipronil is characterized by high activity and a wide range of applications, showing high sensitivity to pests such as Hemiptera, Coleoptera, Lepidoptera, and pests that have developed resistance to pyrethroids and carbamate insecticides. It is mainly used in animal health care to kill parasites such as fleas and lice on cats and dogs, and also has a remarkable effect on the control of hygienic pest cockroaches.
The main action of fipronil is to bind with γ-aminobutyric acid (GABA) receptor, blocking the chloride channel of the nerve membrane controlled by GABA, destroying the central nervous system, leading to the death of the nerves and muscle over-excitement, and ultimately achieving the purpose of killing insects. Fipronil is chemically active and is usually metabolized by direct or indirect hydrolysis and photolysis to produce three metabolites, including Fipronil desulfinyl, Fipronil sulfone and Fipronil sulfide. Fipronil is currently classified as moderately toxic to humans. Short-term intake of large amounts of fipronil can have adverse effects on the human nervous system, mainly manifested by vacuolisation of neurons, mild dilatation of the synaptic gap, and loosening of myelin sheaths of nerve fibres in the form of early demyelinating changes. And long-term ingestion of fipronil can lead to serious damage to the liver, thyroid, and digestive system. There are no specific drugs for the treatment of all types of harmful symptoms caused by fipronil, which are usually treated symptomatically in the clinic with anticonvulsants such as diazepam, sodium phenobarbital, or sedatives such as benzodiazepines.
Figure 1. Degradation of fipronil in the environment (Source: Singh NS, et al. 2021)
Chromatographic detection techniques are mostly used for physicochemical analysis of fipronil, including gas chromatography and liquid chromatography. The method has better sensitivity and accuracy, but the pre-treatment is complicated and time-consuming, and requires expensive and precise instrumentation as well as professional personnel to operate. The immunological assay is highly selective and sensitive, enabling the simultaneous selection of fipronil and metabolites for high-throughput determination. The method generally does not require expensive instruments, can simplify or even eliminate the need for pre-treatment methods, is inexpensive and simple to operate.
References
1. Wang X, et al. Fipronil insecticide toxicology: oxidative stress and metabolism. Crit Rev Toxicol. 2016 Nov;46(10):876-899.
2. Singh NS, et al. A comprehensive review of environmental fate and degradation of fipronil and its toxic metabolites. Environ Res. 2021 Aug;199:111316.
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Figure 1. Degradation of fipronil in the environment
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