Description

Fipronil enzyme-linked immunoassay (ELISA)

Kit instruction manual

This kit is for research use only.

1 Purpose of use:
This kit is used for the quantitative detection of fipronil residues in feed, fish, shrimp and meat tissues (such as chicken, beef and pork), eggs, honey, milk, serum and urine.
2 Experimental principle
This kit adopts the competitive ELISA method. The microwell plate is coated with fipronil coupled antigen, fipronil standard or sample is added, free fipronil is pre-coated with fipronil coupled antigen on the microwell strip Compete with anti-fipronil antibody enzyme markers, develop color with TMB substrate, change the color from blue to yellow after adding the stop solution, use a microplate reader to detect at 450nm wavelength, absorbance and fipronil content in the sample Inversely proportional, the content of fipronil in the sample was calculated from the standard curve.
3 Kit composition
3.1 The pre-coated fipronil-coupled antigen detachable ELISA plate: 1 piece (12 wells × 8 strips).
3.2 Fipronil standard: 6 bottles (1ml/bottle), the contents are: 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb.
3.3 Anti-fipronil antibody enzyme conjugate: 1 bottle (6ml).
3.4 Color developing solution A: 1 bottle (6ml).
3.5 Color developing fluid B: 1 bottle (6ml).
3.6 Stop solution: 1 bottle (6ml), 2M sulfuric acid.
3.7 Sample diluent: 1 bottle (10×, 6ml), used for sample dilution.
3.8 Concentrated washing liquid: 1 bottle (20×, 20ml), used for plate washing.
3.9 One instruction sheet.
4 Materials needed but not provided
4.1 Equipment
4.1.1 Microplate reader with a wavelength of 450nm.
4.1.2 Crusher.
4.1.3 Measuring cylinder.
4.1.4 Oscillator.
4.1.5 Funnel.
4.1.6 Whatman No 1 or equivalent filter paper. 4.1.7 Micropipette.
4.2 Reagents
4.2.1 Deionized water or distilled water.
4.2.2 Methanol.
5 storage
5.1 The kit is stored at 2~8℃, do not freeze
5.2 Unused microplates should be sealed and stored dry
6 matters needing attention
6.1 Please read the instructions carefully before using the kit.
6.2 Do not use expired kits.

6.3 Before using the kit, return the reagents to room temperature (25±2°C). It is recommended to return to the temperature for at least 2 hours.
6.4 The standard product contains fipronil, so special care should be taken when using it, and gloves should be worn during operation.
6.5 The stop solution contains sulfuric acid, which prevents skin burns and corrosion of clothing during use.
6.6 Tips for different standards and samples cannot be mixed, otherwise the test results will be affected.
6.7 The reagents in the kits of different batch numbers must not be mixed; the tips used for different standards and samples must not be mixed, otherwise it will affect the experimental results.
6.8 The sample diluent in this kit must be used when diluting the sample, otherwise it will affect the experimental results
6.9 Foaming should be avoided when mixing reagents.
7 Preparation of working fluid
7.1 Fipronil standard solution: 0ppb, 0.1ppb, 0.3 ppb, 0.9ppb, 2.7 ppb, 8.1ppb
7.2 Concentrated washing liquid: Dilute 1:20 (1+19) with distilled water for use
7.3 Sample diluent: Dilute 1:10 (1+9) with distilled water for use
7.3 Chromogenic agent: reserved, avoid direct light
7.4 Reaction stop solution: ready for use
8 Sample processing procedures (samples should be operated strictly in accordance with the instructions during the extraction process, and the extraction process should be accurately diluted, otherwise the results will be inaccurate, the samples should be stored in a cool and dark place and refrigerated)
8.1 Take 10g of crushed sample and add 20ml of 70% methanol solution
8.2 Vibrate vigorously for 3 minutes
8.3 Filter with Whatman No 1 filter paper
8.4 Take 25µl of the processed sample and add 25µl of sample diluent to the reaction well (the sample dilution factor is 2)
9 Enzyme immunoassay steps
9.1 Experimental instructions
9.1.1 Please fully restore all reagents outside the box to room temperature (25±2°C) before the experiment starts for about 2 hours. After warming to room temperature (25±2℃), take out the microporous strips, reseal the excess microporous strips, and store them immediately at 2~8℃
Note: Make sure that the temperature return is sufficient, otherwise the accuracy and accuracy of the detection will be affected.
9.1.2 Please put the reagent back to 2~8℃ for storage immediately after use
9.1.3 Please do not change the analysis program
9.1.4 Please use an accurate micropipette
9.1.5 Once the operation starts, please do not interrupt any program
9.1.6 The reproducibility of ELISA results greatly depends on the operating procedures, please operate strictly in accordance with the requirements
9.1.7 In order to avoid cross-contamination, each standard and sample should be filled with different tips
9.1.8 Do not let the pipette tip touch the solution or the inner surface of the microwell when adding samples
9.2 Analysis steps
9.2.1 Numbering in advance, marking the position of B0, standards and samples, it is recommended to carry out double-hole detection
9.2.2 Take the required number of micropores (micropore strips can be removed), reseal the excess strips and immediately put them back at 2~8℃ for storage
9.2.3 Sample dilution solution (10×), concentrated washing solution (20×) are diluted into working solution (diluted with distilled water or deionized water)
9.2.4 Add 50µl 0.0ppb standard solution to well B0
9.2.5 Add 50μl of standard solution to each standard well
9.2.6 Add 50µl of sample solution to each sample well
9.2.7 Add 50 µl of anti-fipronil antibody enzyme conjugate to all wells
9.2.8 Gently shake the reaction plate for a few seconds.
9.337℃ warm bath for 30 minutes (tapping the reaction plate from time to time during warm bathing can reduce the double hole error)
9.3.1 Shake off the liquid in the wells, wash the microplate 5 times with lotion, and tap on absorbent paper for the last time to completely remove the liquid in the wells.

9.4 Reaction
9.4.1 After the washing procedure is completed, immediately use a micropipette to add 50µl of chromogenic solution A and then 50µl of chromogenic solution B to each microwell; shake the reaction plate slightly to mix it thoroughly
9.4.237℃ warm bath for 10min
9.4.3 Add 50µl stop solution to each well and mix well
9.4.4 Detect the absorbance at 450nm and read the result within 5min.
10 Result calculation
10.1 Quantitative analysis
10.1.1 The average value (B) of the absorbance value of each concentration standard solution and sample obtained is divided by the absorbance value (B0) of the first standard (0 standard) and multiplied by 100%, which is the percent absorbance value.
B—average absorbance value of standard solution or sample solution B0—average absorbance value of 0ppb standard solution
10.1.2 Use the logarithmic value of fipronil concentration as the X-axis and the percent absorbance value as the Y-axis to draw a standard curve. According to the percent absorbance value of the sample, the abscissa of the corresponding point can be obtained from the curve, which is the logarithm of the fipronil concentration, and the antilog is the fipronil concentration C (ppb) in the measurement solution
10.1.3 Since the sample has been pre-diluted, the concentration of the sample obtained from the standard curve must be multiplied by its dilution factor.
10.2 Semi-quantitative determination
10.1.1 Visual semi-quantitative determination: First, select an appropriate standard solution and run it with the sample, and judge whether the sample concentration value is less than or greater than the standard value based on the color comparison between the sample and the standard.
10.1.2 Semi-quantitative measurement of the instrument: First select an appropriate standard solution and run it with the sample, and judge whether the sample concentration value is less than or greater than the standard value based on the comparison of the absorbance values ​​of the sample and the standard.
11 specificity
Substance Cross-reaction fipronil 100%

12 Kit parameters
The lower limit of detection of this kit is 0.05ppb. The best absorbance value of B0 should be greater than 1.0
The error within the kit absorbance plate is less than 8%, and the error between the plates is less than 15%. The recovery rate of tissue sample extraction method provided in this manual is greater than 80%. 13
The standard curve range provided by the kit is 0.1ppb~8.1ppb.

14 Analysis limitations
The samples tested positive by this kit should be confirmed by another method such as HPLC or GC/MS.