Elisa Test Kit for Fluoroquinolones

• Customization: Available
• Environmental Protection: Yes
• Certification: REACH
• Model NO.: KA00903H
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Trademark: kwinbon
• Origin: China
• HS Code: 38229000

Product Description

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 quinolones in honey.
4. Cross-reactions
Norfloxacin(NOR) …................…..…..100%
Pefloxacin...........................................................110%
Ofloxacin(OFL)....................….....100%
Lomefloxacin(LOM) .........….............90%
Difloxacin(DIF)..................…...…...120%
Levofloxacin...................................................110%
Enrofloxacin(ENR)........................130%
Ciprofloxacin(CIF) ...............…......100%
Sarafloxacin(SAR)..................….…..120%
Marbofloxacin..................................................90%
Pazufloxacin.................................80%
Flumequine(FLU)…...................…...95%
Enoxacin(ENO)...…............…........100%

Danofloxacin(DAN)......................….90%
Oxolinic acid (OXO) ............….....….…100%
Fleroxacin...............…....…...45%
Sparfloxacin.......................................................8%
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
---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
Extraction solution(50ml).................................blue cap  
7. Reagent Preparation
7.1 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 Honey
----Take 1.0±0.05g honey into a 10ml tube.
----Add 4ml deionized water, vortex for 5min to dissolve completely.
----Take 500ul dissolved sample, mix with 500ul extraction solution, and vortex for 3min.
----Take 50ul per well for assay.
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 or prepared sample to separate duplicate wells.
9.2.5 Mix enzyme and antibody solution: dilute the concentrated enzyme conjugate with antibody solution in the volume ratio of 1:10 according to your use (1 fold concentrated enzyme conjugate + 10 folds Antibody solution), mix completely(this solution must be use immediately )
9.2.6 Add enzyme and antibody mixture: add 50µl  enzyme conjugate and antibody mixture to each well immediately,  Mix gently and incubate for 30min at 25ºCwith cover.
9.2.6 Wash: remove the cover gently and pure the liquid out of the wells and wash the microwells with 250µl diluted wash solution 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).
9.2.7 Coloration: Add 50µl solution A and 50µl solution B to each well. Mix gently and incubate for 15min at 25ºCwith cover (see 12.8).
9.2.8 Measure: Add 50µl of the stop solution 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 absobance value of standards as y-axis, semilogarithmic of the concentration of the standards (ppb) as x-axis.
---The quinolones 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.
Sample dilution factor: 

Honey: 10
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.1ppb
Sample Detection Limit:
Honey...........................…...1 ppb
Accuracy:
Honey.........................…100%±20%
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.
13. Storage condition and storage period
Storage condition: 2-8ºC.
Storage period: 12 month.
 

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