Fluoroquinolones Residue Elisa Kit for Egg

• Environmental Protection: Yes
• Certification: REACH
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Appearance: Liquid
• Model NO.: KA00906H
• Transport Package: Foam Box with Ice Bag
• Specification: 96WElls
• Trademark: KWINBON
• Origin: China
• HS Code: 38229000
• Production Capacity: 50000PCS/Year

Product Description

 

 

Competitive Enzyme Immunoassay Kit for
Quantitative Analysis of Fluoroquinolones

 

 

1. Background
Quinolones (QNS) is a synthetic class of antibiotics, which all act by inhibition of DNA gyrase, abolishing its activity by interfering with the DNA-rejoining reaction. Since gyrase is an essential enzyme in prokaryotes but is not found in eukaryotes, bacteria are ideal targets for these antibiotics. In veterinary practice, they are administrated by subcutaneous injection to cattle, intramuscularly to pigs, and orally to cattle, pigs, and chicken for the treatment of infections of the respiratory and alimentary tract.
2. Test Principle
This ELISA kit is designed to detect quinolones based on the principle of competitive enzyme immunoassay. The microtiter wells are coated with capture BSA-linked antigen. Quinolones in the sample competes with antigen coated on the microtitre plate for the antibody. After the addition of enzyme conjugate, chromogenic substrate is used and the signal is measured by spectrophotometer. The absorption is inversely proportional to the quinolones concentration in the sample.
3. Applications
This kit is used for the quantitative and qualitative analysis of fluoroquinolones in tissue(pork ,duck,check,fish,shrimp) milk , milk powder and egg.
4. Cross-reactions
Norfloxacin(NOR) …..................…..…..100%
Difloxacin(DIF)..................…........197%
Enrofloxacin(ENR)........................160%
Flumequine(FLU)…...................…...155%
Sarafloxacin(SAR)..................….…..175%
Danofloxacin(DAN)......................….120%
Pefloxacin(PEF)...…...........…........…...205%
Ciprofloxacin(CIF) ........................…120%
Enoxacin(ENO)...…............…........….65%
Ofloxacin(OFL)....................….......70%
Levofloxacin(LVX).....................…...7%
Oxolinic acid (OXO) ............….....…...…13%
Lomefloxacin(LOM) .........…..........…...8%
Marbofloxacin(MAR)...............…....…...4%
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, 400ml, 500ml
---Glass test tube: 10ml
---Polystyrene centrifuge tube: 2ml, 15ml, 50ml
---Micropipettes: 20ul-200ul,
100ul-1000ul, 250ul-multipipette
5.2 Reagents
---Anhydrous acetonitrile(AR)
---n-hexane(AR)
---Concentrated hydrochloric acid(HCl, AR)
---Deionized water
6. Kit Components

1. Microtiter plate coated with antigen, 96wells.
2. Standard solutions×6 bottles: (1ml/bottle)

0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb

1. Spiking standard solution: 1ml, 100ppb
2. Concentrated enzyme conjugate (1ml)….....red cap
3. Antibody solution (7ml).........…....green cap
4. Substrate solution A (7ml).............white cap
5. Substrate solution B (7ml)............…red cap
6. Stop solution (7ml)...............…yellow cap
7. 20×Concentrated wash solution(40ml)

 .....................…transparent cap

1. 2×Concentrated Extraction solution(50ml) 

.....................…......blue cap
7. Reagent Preparation
Solution 1: 0.15M HCl
Dilute the 5ml of concentrated hydrochloric acid with 400ml of deionized water, mix completely.
Solution 2: 3%Trichloroacetic acid solution
       Weight 3.0g Trichloroacetic acid ,dilute with 100ml deionized water ,dissolved completely.
Solution 3:  2M sulfuric acid solution solution
     Weight 11.1ml 98.3%concentrated sulfuric acid,Add in deionized water at constant volume to 100ml,mix completely.
Solution 4: Sample buffer
      Mix 10ml of 0.15M HCl(Solution 1) with 90ml of Anhydrous acetonitrile completely.
Solution 5: Extraction solution
Dilute the 2×concentrated extraction solution with deionized water (1:1), which will be used to dilute samples.
Solution 6: Wash solution
Dilute the 20×concentrated wash solution with deionized water(1:19), which will be used to wash the plate. The wash solution can be conserved for a month at 4ºC.
8. Sample Preparation
8.1 Notice and precautions before operation
(a) Please use one-off tips in the process of experiment, and change the tips when absorb different reagent.
(b) Please check up whether all experimental tools are clean and to purify if necessary, which as against interfering with the results.
(c) Store the untreated sample in freeze.
(d) The treated sample can be stored for 24 hours, at 2-8ºC in dark except for the serum.
(e) The enzyme conjugate should be prepared freshly before use.

8.2 Tissue(Chicken, pork, duck, fish, shrimp etc.)
----Homogenize the sample with homogenizer.
----Weigh 2.0g±0.05 of the homogenate into a 50ml polystyrene centrifuge tube, add 8ml of sample buffer(solution 4), shake for 3min, then centrifuge: 3000g,5 min, ambient temperature.
----Transfer 2ml of the supernate organic phase into a clean and dry test tube, and then evaporate to dry with water bath under 50-60ºC nitrogen stream.
----Dissolve the residue with 1ml of n-hexane, vortex for 2min to dissolve, then add 1ml of extraction solution (solution 5), and then vortex for 30s.
----Centrifuge: 3000g, for 5min, ambient temperature.
----Remove the supernatant organic phase, take 50ul of the substrate solution per well for assay.

Dilution factor:                2

8.2 Milk
----weight 1ml of milk sample into 10ml Polystyrene centrifugal tube ,add 1ml 3% Trichloroacetic acid solution (Soluton 2),vertex 1min,mix completely, Centrifuge: 3000g, for 5min, ambient temperature.
----Avoid the upper fat layer and remove 100 ml of supernatant to 2ml Polystyrene centrifugal tube, Add 900ul extraction solution(solution 5), vortex for 1min to mix completely.
----Take 50µl per well for assay.

Dilution Factor:                      20

8.3 Milk powder
----weight 1±0.05g milk powder sample into 10ml Polystyrene centrifugal tube ,add 5ml deionized water,vertex 2min,mix completely .
-----Take 1ml milk powder solution to 2ml polystyrene centrifugal tube,Add 20ul 2M sulfuric acid solution solution(Soluton 3),vertex 2min,mix completely, Centrifuge: 3000g, for 5min, ambient temperature.
----Avoid the upper fat layer and remove 100 ml of supernatant to 2ml Polystyrene centrifugal tube, Add 900ul extraction solution(solution 5), vortex for 2min to mix completely.
----Take 50µl per well for assay.

Dilution Factor:                         50
8.4 Egg
---weight 1.0±0.05g Homogeneous sample material to 10ml polystyrene centrifugal tube.
-----Add 5ml deionized water ,Use an oscillator to dissolve completely.
----Take 100ul sample ,add 400ul extraction solution(solution 5),vertex 1min until mix completely .
---Take 50ul for assay.
Dilution Factor:                         30
9. Assay Steps
9.1 Notice before assay
9.1.1 Keep all reagents and microwells at room temperature (20-25ºC) for more than 30min until rewarm.
9.1.2 Return all the rest reagents to 2-8ºC immediately after used.
9.1.3 Wash the microwells correctly is an important point in the process of assay, it is a vital factor for the reproducibility of the ELISA analysis
9.1.4 Cover the microwells during incubation.
9.2 Assay process
9.2.1 Bring all reagents to room temperature (20-25ºC) for more than 30min, and shake gently before use.
9.2.2 Get microwells needed out, and return the rest into the zip-lock bag and stored at 2-8ºC immediately.
9.2.3 Number: insert a sufficient number of wells into the microwell holder for all standards and samples to be run in duplicate. Record the position of standards and samples.
9.2.4 Add sample / standard: add 50µl of each standard solution(Kit component) or prepared sample to separate duplicate wells.
9.2.5 Mix the antibody and enzyme conjugate: Mix the antibody(Kit component) and enzyme conjugate(Kit component) in the volume ratio of 10:1 according to your use (e.g. 3ml of antibody + 0.3ml of enzyme conjugate), mix completely(The mixture can not be preserved, use immediately).
9.2.6 Add the mixture of antibody and enzyme conjugate: Add 50µl of the antibody-enzyme conjugate mixture to each well immediately, Mix gently and incubate for 30min at 25ºCwith cover.
9.2.7 Wash: Remove the cover gently and pure the liquid out of the wells and wash the microwells with 250µl of diluted wash solution(solution 6) at intervals of 10s. Tap the microwells holder upside down vigorously against absorbent paper to ensure complete removal of liquid from the wells (repeat 4-5 times).
9.2.8 Coloration: Add 50µl of solution A(Kit component) and 50µl of solution B(Kit component)  to each well. Mix gently and incubate for 15min at 25ºCwith cover (see 12.8).
9.2.9 Measure: Add 50µl of the stop solution(Kit component)  to each well. Mix gently and measure the absorbance at 450nm against an air blank (It's suggested measure with the dual wavelength of 450 / 630nm, Read the result within 5min after adding stop solution)
10. Results
10.1 Percentage absorbance
The mean values of the absorbance values obtained from the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied by 100%.
   =
B --absorbance of standards or samples
B₀ --absorbance of zero standard
10.2 Standard Curve
---To draw a standard curve: The absorbance value of standards as y-axis, semilogarithmic of the concentration of the standards (ppb) as x-axis.
---The fluoroquinolones concentration of each sample (ppb), which can be read from the calibration curve, is multiplied by the corresponding dilution rate of each sample followed, and the actual concentration of sample is obtained.
Notice: Special software has been developed for result calculation, which can be provided on request.
11. Sensitivity, accuracy and precision
11.1 Test Sensitivity: 0.1ppb
11.2 Detection limit
Norfloxacin(NOR)…...................…..…0.2ppb
Difloxacin(DIF)........................…0.2ppb
Enrofloxacin(ENR)......................0.2ppb
Flumequine(FLU)…...................….0.2ppb
Sarafloxacin(SAR).....................….0.2ppb
Danofloxacin(DAN)...............…......0.2ppb
Pefloxacin(PEF)...…...........…......…..…..0.2ppb
Ciprofloxacin(CIF) ......................…0.3ppb
Enoxacin(ENO)...…....................…0.3ppb
Ofloxacin(OFL)..................…...…...0.3ppb
Levofloxacin...........................0.3ppb
Oxolinic acid (OXO) ....................…0.6ppb
11.3 Sample Detection Limit
Tissue...........................…..0.3ppb
Milk................................…3ppb
Milk powder............................…6ppb
Egg..............................................................................3ppb
11.4 Accuracy
Tissue............….......…....…100%±30%
Milk......................…....…95%±25%
Milk powder..................…....…95%±25%
Egg...................................................................100%±30%
11.5 Precision
CV of the ELISA kit all less than 10%.
12. 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 (20-25ºC).
12.2 Do not allow microwells to be dry between steps to avoid unsuccessful repetitiveness and operate the next step immediately after tap the microwells holder.
12.3 Mix the homogenate and elute the plate adequately. 12.4 Avoid the stop solution touching skin for it is 2M     H₂SO₄.
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 Keep the ELISA kits at 2-8ºC. Avoid direct 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 need 10-15min after the addition of solution A and solution B; But you can prolong the incubation time ranges to 20min if the color is too light to be determined. Never exceed 25min. On the contrary, shorten the incubation time properly.
12.9 The best reaction temperature is 25ºC, temperature too high or too low both will lead to the changes of sensitivity and absorbance values.