Competitive Enzyme Immunoassay Kit for Quantitative Analysis of Tylosin

• Customization: Available
• Classification: Food Diagnostic
• Transport Package: Foam Box with Ice Bag
• Model NO.: KA04604H
• Trademark: kwinbon
• Origin: China
• HS Code: 38229000

Product Description

 

 

More details feel free to contact us

  

Product Description

Competitive Enzyme Immunoassay Kit for
Quantitative Analysis of Tylosin

1. Background
Tylosin is a macrolide antibiotic, which is mainly applied as antibacterial and anti-mycoplasma. Strict MRLs haven been established since this drug may lead to serious side effect in certain groups.
This kit is a new product based on ELISA technology, which is fast, easy, accurate and sensitive compared with common instrumental analysis and only needs 45min in one operation, it can considerably minimize operation error and work intensity.
2. Test Principle
This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigen. Tylosin residue in the sample competes with the antigen coated on the microtiter plate for the antibody. After the addition of enzyme labeled anti-antibody, TMB substrate is used to show the color. Absorbance of the sample is negatively related to the tylosin reside in it, after comparing with the Standard Curve, multiplied by the dilution factor, tylosin residue quantity in the sample can be calculated.
3. Applications
This kit can be used in quantitative and qualitative analysis of Tylosin residue in tissue, milk , honey and egg.
4. Cross-reactions
Tylosin.............................100%
Tilmicosin...........................<2%
5. Materials Required
5.1 Equipments:
Microtiter plate spectrophotometer (450nm/630nm)
Rotary evaporator or nitrogen drying instruments
Shaker
Vortex mixer
Centrifuge
Analytical balance (inductance: 0.01g)
Graduated pipette: 10ml
Rubber pipette bulb
Glass test tube : 10ml
Polystyrene centrifuge tubes: 50ml
Micropipettes: 20-200ml, 100-1000ml
250ml-multipipette
5.2 Reagents:
-----n-hexane(AR)
----Ethyl acetate (AR)
----Sodium hydroxide(AR, NaOH)
---- Sodium hydrogen carbonate(AR, NaHCO3)
----Sodium carbonate(AR, Na2CO3)
----Trichloroacetic acid(AR)
----Acetonitrile(AR)
---Deionized water
6. Kit Components

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

0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb

1. Spiking standard control: (1ml/bottle) 1ppm
2. Concentrated enzyme conjugate 1ml…..…red cap
3. Antibody solution 7ml.............green cap
4. Solution A 7ml.............…....white cap
5. Solution B 7ml..................…..red cap
6. Stop solution 7ml..............….yellow cap
7. 20×concentrated wash solution 40ml

..................…..…transparent cap

1. 4×concentrated extraction solution 50ml

............................blue cap
7. Reagents Preparation:
Solution 1: 0.1M NaOH solution
Weigh 0.4g NaOH and add deionized water  to 100ml and mix it completely.
Solution 2: 1M NaOH solution
Weigh 4.0g NaOH and add deionized water  to 100ml and mix it completely.
Solution 3: carbonate buffer
Weigh 4.664g Na2CO3  and 0.504g NaHCO3, add deionized water  to 500ml and mix completely.
Solution 4: 3% trichloroacetic acid solution
Weigh 3.0g trichloroacetic and add deionized water to 100ml ,mix compeletly.
Solution 5: sample extraction
Take 82ml anhydrous acetonitrile, add 18ml 0.1M NaOH solution(solution 1), mix completely.
Solution 6: extraction solution for tissue
Dilute 4×Concentrated extraction solution with deionized water in the volume ration of 3:5(3 folds 4xconcentrated extraction+5 folds deionized water), which will be used to dilute tissue samples. This solution can be stored for 1 month at 4ºC.
Solution 7: extraction solution for milk sample
Dilute 4×Concentrated extraction solution with deionized water in the volume ration of 1:4(1 folds 4xconcentrated extraction+4 folds deionized water), which will be used to dilute the milk samples. This solution can be stored for 1 month at 4ºC.
Solution 8: extraction solution for honey sample
Dilute 4×Concentrated extraction solution with deionized water in the volume ration of 3:37(3 folds 4xconcentrated extraction+37 folds deionized water), which will be used to dilute honey samples. This solution can be stored for 1 month at 4ºC.
Solution 9: extraction solution for egg
Dilute 4×Concentrated extraction solution with deionized water in the volume ration of 1:1(1 folds 4xconcentrated extraction+1 folds deionized water), which will be used to dilute egg samples. This solution can be stored for 1 month at 4ºC.
Solution 10: wash solution
Dilute 20×Concentrated wash solution with deionized water in the volume ratio of 1: 19, which will be used to wash the plates. This diluted solution can be stored for 1 month at 4ºC.
8. Sample Preparations
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) Make sure that all instruments are clean.
8.2 Tissue
----Homogenize the tissue with homogenizer;
----Weigh 1.0±0.05g tissue into a 50ml polystyrene centrifuge tube; add 1ml carbonate buffer (solution 3), then add 4ml ethyl acetate, vortex for 1min to dissolve it.
----Centrifuge at room temperature (20-25ºC) for 5min , at least 3000g.
----Take 2ml of the supernatant phase into a 10ml glass tube, dry with 50-60ºC nitrogen gas stream;
----Dissolve the dry leftover with 200μl n-hexane, add 1ml extraction solution for tissue(solution 6), shake it with shaker for 1min;
----Centrifuge at room temperature (20-25ºC) for 5min , at least 3000g.
----Remove the supernatant phase, take 50μl of the substrate solution for assay.
8.3 Milk:
----Take 1ml milk sample into a 10ml polystyrene centrifuge tube, add 1ml 3% trichloroacetic acid solution
(solution 4), shake for 1min with shaker to dissolve it;
----Centrifuge at room temperature (20-25ºC) for 5min ,
at least 3000g;
----Avoid the supernatant fat layer, take 100μl the
middle clear phase, add 900μl extraction solution for
milk(solution 7) and 7μl 1M NaOH solution(solution 2),
shake it for 30s with shaker to dissolve it;
----Take 50μl of the prepared solution for assay.
8.4 Honey
----Take 1.0±0.05g honey sample into a 50ml polystyrene centrifuge tube, add 4ml sample extraction solution(solution 5), shake it with shaker for 1min to dissolve it;
----Centrifuge at room temperature (20-25ºC) for 5min ,
at least 3000g.
----Take 2ml of the supernatant phase into a 10ml
glass tube, dry with 50-60ºC nitrogen gas stream;
----Dissolve the dry leftover with 500μl extraction
solution for honey sample(solution 8), shake it with
shaker for 2min to dissolve it;
----Take 50μl of the prepared solution for assay.
8.5 Egg sample
---- Homogenize the egg with homogenizer to mix the white and yolk completely;
----Take 1.0±0.05g egg sample into a 10ml polystyrene centrifuge tube, add 3ml sample extraction solution(solution 5), shake it with shaker for 1min to dissolve it;
----Centrifuge at room temperature (20-25ºC) for 5min , at least 3000g.
----Take 1ml of the supernatant organic phase into a 10ml glass tube, dry with 50-60ºC nitrogen gas stream;
----Dissolve the dry leftover with 1ml n-hexane, shake it for 1min, then add 1ml extraction solution for egg sample(solution 9) ,shake it with shaker for 15s to dissolve it;
----Centrifuge at room temperature (20-25ºC) for 5min , at least 3000g.
---- Remove the supernatant phase, take 50μl of the substrate solution for assay.
9. Assay process
9.1 Notice before assay:
9.1.1 Make sure all reagents and microwells are all at room temperature (20-25ºC).
9.1.2 Return all the rest reagents to 2-8ºC immediately after used.
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 (20-25ºC) for more than 30min, shake gently before use.
9.2.2 Get the microwells needed out and return the rest into the zip-lock bag at 2-8ºC immediately.
9.2.3 The diluted wash 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 Add standard solution/sample: Add 50 µl of standard solution or prepared sample to corresponding wells.
9.2.5Dilution of enzyme conjugate: 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).Notice: this mixture can
not be stored, please use it immediately.
9.2.6 Add antibody and enzyme conjugate mixture:
add 50µl antibody and enzyme conjugate 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 rinse the microwells with 250µl diluted wash solution (solution 10) at interval of 10s for 4-5times. Absorb the residual water with absorbent paper (the rest air bubble can be eliminated with unused tip).
9.2.8 Coloration: Add 50µl solution A and 50µl solution
B to each well. Mix gently and incubate for 15min at
25ºC with cover.
9.2.9 Measure: Add 50µl 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. We can also measure
by sight without stop solution if there is no ELISA
reader).
10. Results
10.1 Percentage 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 (%) = -- ×100%
                 B0
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 tylosin standards solution (ppb) as x-axis.
---The tylosin 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:
For data reduction of the ELISA kits, special software has been developed, which can be ordered on request.
Dilution factor of samples:
Tissue: 2
Honey: 1
Milk: 20
Egg: 3
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.5ppb
Detection limit:
Tissue............................1.5ppb
Honey............................…1ppb
Milk...............…..........…..…10ppb
Egg...............................3ppb
Accuracy:
Tissue........................…100±20%
Honey sample.....................90±20%
Milk........................….... 100±20%
Egg........................….... 100±20%
Precision:
Variation coefficient of the ELISA kit is 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 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 2M 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 Keep the ELISA kits at 2-8ºC,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 turn bad 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 from 20min to 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 25ºC. Higher or lower temperature will lead to the changes of sensitivity and absorbance values.
13. Storage condition and storage period
     Storage condition: 2-8ºC.
Storage period: 12 months.
 

 

Detailed Photos

 

Company Profile