Competitive Enzyme Immunoassay Kit for Quantitative Analysis of Furazolidone metabolite (AOZ)
- 1. Background
Nitrofurans are synthetic broad-spectrum antibiotics, which are frequently employed in animal production for its excellent antibacterial and pharmacokinetic properties. They had also been used as growth promoters in pig, poultry and aquatic production. In long term studies with lab animals indicated that the parent drugs and their metabolites showed carcinogenic and mutagenic characteristics. This has led to a prohibition of nitrofurans for the treatment of animals used for food production. The nitrofuran drugs furaltadone, nitrofurantoin and nitrofurazone were banned from use in food animal production in the EU in 1993, and the use of furazolidone was prohibited in 1995.
The analysis of nitrofuran drugs residue needs to be based on the detection of the tissue bound metabolites of the nitrofuran parent drugs. Since the parent drugs are very rapidly metabolized, and the tissue bound nitrofuran metabolites will retain for a long time, these metabolites are used as the target in the detection of the abuse of nitrofurans, which include Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ), Nitrofurantoin metabolite (AHD) and Nitrofurazone metabolite (SEM).
AOZ-residues are determined most commonly by LC-MS or LC-MS/MS. Enzyme immunoassays, compared with chromatographic methods, show considerable advantages regarding sensitivity, detection limit, technical equipment and time requirement. (Time cost: 45min)
- 2. Test Principle
This ELISA kit is designed to detect AOZ based on the principle of indirect-competitive enzyme immunoassay. The microtiter wells are coated with capture BSA-linked antigen. AOZ in sample competes with the antigen coated on the microtiter plate for the antibody added. After the addition of enzyme conjugate, chromogenic substrate is used and the signal is measured by a spectrophotometer. The absorption is inversely proportional to the AOZ concentration in the sample.
- 3. Applications
This kit can be used in quantitative and qualitative analysis of AOZ residue in anima tissues (muscle, liver etc), honey.
- 4. Cross-reactions
Furazolidone metabolite (AOZ)……………………..100%
Furaltadone metabolite (AMOZ)……………………<0.1%
Nitrofurantoin metabolite (AHD)……………………<0.1%
Nitrofurazone metabolite (SEM)…………………...…<0.1%
Furazolidone…………………………………….…..…16.3%
Furaltadone…………………………………………….…<1%
Nitrofurantoin…………………………………….…….…<1%
Nitrofurazone…………………………………………..…<1%
- 5. Materials Required
5.1 Equipments
----Microtiter plate spectrophotometer (450nm/630nm)
----Rotary evaporator or nitrogen drying instruments
----Homogenizer /stomacher
----Shaker
----Vortex mixer
----Centrifuge
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Volumetric flask: 100ml
----Glass flask: 10ml
----Polystyrene centrifuge tube: 2ml, 50ml
----Micropipettes: 20ul-200ul, 100ul-1000ul,
250ul-multipipette
5.2 Reagents
----Ethyl acetate (AR)
----n-hexane (or n-heptane) (AR)
----Dipotassium hydrogen phosphate trihydrate
(K2HPO4.3H2O) (AR)
----Concentrated hydrochloric acid (HCl, AR)
-----Methanol
----Sodium hydroxide (NaOH, AR)
----Deionized water
- 6. Kit Components
l Microtiter plate coated with antigen, 96 wells
l Standard solutions(6 bottles,1ml/bottle)
0ppb,0.025ppb,0.075ppb,0.225ppb,0.675ppb,2.025ppb
l Spiking standard control : (1ml/bottle)....….100ppb
l Concentrated enzyme conjugate 1ml...…..red cap
l Enzyme conjugate diluents 10ml………..green cap
l Substrate A 7ml………..............…....…..…..white cap
l Substrate B 7ml………..............…........….…..red cap
l Stop solution 7ml……………………………yellow cap
l 20×concentrated wash solution 40ml
…………………………………….……transparent cap
l 2×concentrated extraction solution 60ml….blue cap
l 2-Nitrobenzaldehyde 15.1mg………………white cap
- 7. Reagents Preparation
Solution 1: derivative reagent:
Add 10ml of methanol to the bottle having 2-Nitrobenzaldehyde and dissolve. (at the concentration of 10mM).
Solution 2: 0.1M K2HPO4 solution:
Weigh 22.8g K2HPO4.3H2O to 1L of deionized water to dissolve.
Solution 3: 1M HCl solution
Dissolve 8.3ml Concentrated hydrochloric acid with deionized water to 100ml.
Solution 4:1M NaOH solution
Dissolve 4g NaOH with 100ml deionized water.
Solution 5: extraction solution:
Dilute 2×concentrated extraction solution with deionized water in the volume ratio of 1:1. This solution can be conserved for 1month at 4℃, which will be used to extracted samples.
Solution 6: wash solution:
Dilute the 20×concentrated wash solution with deionized water in the volume ration of 1:19, which will be used to wash the plates. This diluted solution can be conserved for 1 month at 4℃.
- 8. Sample Preparations
8.1 Notice and precautions before operation:
a) Please use one-off tips in the experiment, and change the tips when absorbing different reagent.
b) Make sure that all experimental instruments are clean.
c) the derivative reagent can be conserved at 2-8℃ for three months;
d) The HCl solution can be conserved at room temperature for 3 months;
e) The NaOH solution can be conserved for 3 months at room temperature;
f) Keep untreated samples in freeze(-20℃);
g) Treated samples can be conserved for 24h at 2-8℃ in darkness .
8.2 Animal tissue and liver samples:
----Homogenize the samples with homogenizer;
----Weight 1.0±0.05g of the homogenized tissue sample into 50ml polystyrene centrifuge tube. Add 4ml deionized water, 0.5ml 1M HCl solution and 100ul derivative reagent (see solution 1). Shake it for 2min.
---- Incubate at 37℃ overnight ( about 16h);
---- Add 5ml 0.1M K2HPO4 (solution 2), 0.4ml 1M NaOH (solution 4) and 5ml ethyl acetate. Shake fiercely for 30s;
---- Centrifuge at room temperature (20-25℃) for 10min, at least 3000g;
---- Take 2.5ml of the supernatant organic phase into a 10ml clean glass tube, dry with 50-60℃ nitrogen gas or rotary evaporator;
---- Dissolve the dry leftover with 1ml n-hexane (or n- heptane), vortex for 30s, add 1ml extraction solution (solution 5), vortex 1min, mix completely.
---- Centrifuge at room temperature (20-25oC) for 5min, at least 3000g;
---- Remove the supernatant organic phase; take 50μl of the substrate water phase for assay.
8.4 Honey
----weigh 1.0±0.05g of the homogenized honey sample into a 50ml polystyrene centrifuge tube;
----Add 4ml deionized water, 0.5ml 1M HCl (solution 3) and 100μl derivative reagent (solution 1); shake completely for 2min;
----Incubate at 37℃ overnight ( about 16h);
----Add 5ml 0.1M K2HPO4 (solution 2), 0.4ml 1M NaOH (solution 4) and 5ml ethyl acetate, shake fiercely for 30s;
----Centrifuge at room temperature (20-25℃) for 10min, at least 3000g;
----Take 2.5ml of the supernatant organic phase into a 10ml clean glass tube, dry with 50-60℃ nitrogen gas or rotary evaporator;
----Dissolve the dry leftover with 1ml n-hexane (or n- heptane), vortex for 30s, add 1ml extraction solution (solution 5), vortex 1min, mix completely.
----Centrifuge at room temperature (20-25oC) for 10min, at least 3000g;
----Remove the supernatant organic phase; take 50μl of the substrate water phase for assay.
- 9. Assay process
9.1 Notice before assay
9.1.1 Make sure all reagents and microwells are all at room temperature (20-25℃).
9.1.2 Return all the rest reagents to 2-8℃ immediately after use.
9.1.3 Washing the microwells correctly is an important step in the process of assay; it is the vital factor to the reproducibility of the ELISA analysis.
9.1.4 Avoid the light and cover the microwells during incubation.
9.2 Assay Steps
9.2.1 Take all reagents out at room temperature (20-25℃) for more than 30min, homogenize before use.
9.2.2 Get the microwells needed out and return the rest into the zip-lock bag at 2-8℃ immediately.
9.2.3 The concentrated wash solution and concentrated extraction solution should be rewarmed to be at room temperature before use.
9.2.4 Number: Numbered every microwell positions and all standards and samples should be run in duplicate. Record the standards and samples positions.
9.2.5 Dilution of concentrated antibody solution: dilute the concentrated enzyme solution with enzyme conjugate diluents in the volume ratio of 1:10(1 fold concentrated enzyme solution: 10 folds enzyme conjugate diluents).
9.2.6 Add standard solution / sample and enzyme conjugate solution: add 50µl of standard solution or prepared sample to corresponding wells, add 50µl enzyme conjugate solution. Mix gently by shaking the plate manually and incubate for 30min at 25℃ with cover.
9.2.7 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl diluted wash solution (solution 6) at interval of 10s for 4-5 times. Absorb the residual water with absorbent paper (the rest air bubble can be eliminated with unused tip).
9.2.8 Coloration: Add 50µl substrate solution A and 50ul substrate solution B to each well. Mix gently by rocking the plate manually and incubate for 15min at 25℃ with cover(see 12.8).
9.2.11 Measure: Add 50µl the stop solution to each well. Mix gently by rocking the plate manually and measure the absorbance at 450nm (It suggested measure with the dual-wavelength of 450/630nm. Read the result within 5min after addition of stop solution. )
10 Results
10.1Percentage absorbance
The mean values of the absorbance values obtained for the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied by 100%. The zero standard is thus made equal to 100% and the absorbance values are quoted in percentages.
=
B ——absorbance standard (or sample)
B0 ——absorbance zero standard
10.2 Standard Curve
----To draw a standard curve: Take the absorbance value of standards as y-axis, semi logarithmic of the concentration of the AOZ standard solution (ppb) as x-axis.
----The AOZ concentration of each sample (ppb), which can be read from the calibration curve, is multiplied by the corresponding dilution factor of each sample followed, and the actual concentration of sample is obtained.
Please notice:
Special software has been developed for all data reduction, which can be provided on request.
Dilution factor………………………………………..……2
10. Sensitivity, accuracy and precision
Sensitivity: 0.025ppb
Detection limit:
Tissue (muscle, liver)…………………………0.1ppb
Honey--------------------------------------------------0.1ppb
Accuracy:
Animal tissue (muscle and liver)……...…………100±20%
Honey………………………………..………….... 100±20%
Precision: CV of the ELISA kit is less than 10%.
11. Notice
12.1 The mean values of the absorbance values obtained for the standards and the samples will be reduced if the reagents and samples have not been regulated to room temperature ( 20-25℃).
12.2 Do not allow microwells to dry between steps to avoid unsuccessful reproducibility and operate the next step immediately after tap the microwells holder.
12.3 Shake each reagent gently before use.
12.4 Keep your skin away from the stop solution for it is the 0.5M H2SO4 solution.
12.5 Don’t use the kits out of date. Don’t exchange the reagents of different batches, or else it will drop the sensitivity.
12.6 Storage condition: Keep the ELISA kits at 2-8℃,do not freeze. Seal rest microwell plates, Avoid straight sunlight during all incubations. Covering the microtiter plates is recommended.
12.7 Substrate solution should be abandoned if it turns colors. The reagents may be deteriorated if the absorbance value (450/630nm) of the zero standard is less than 0.5 (A450nm<0.5).
12.8 The coloration reaction needs 15min after the addition of substrate solution; You can prolong the incubation time to 20min or more if the color is too light to be determined., never exceed 25min.On the contrary, shorten the incubation time properly.
12.9 The optimal reaction temperature is 25℃. Higher or lower temperature will lead to the changes of sensitivity and absorbance values.
12. Storage condition and storage period
Storage condition: 2-8℃.
Storage period: 12months.
