Colistin (CLS) Elisa Kit for Chicken and Milk

• Customization: Available
• Environmental Protection: Yes
• Certification: REACH
• Model NO.: KB05202H
• 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 Colistin (CLS)

1. Background
Colistin is a polypeptide antibiotic, which is commonly applied in curing sensitive organism infection and promoting growth of pup and poultry. It is employed as medicine feed additives and inhibitor of Gram-negative bacteria such as bacillus pyocyaneus and Escherichia coli. Colistin is excreted by kidney, and it can cause kidney epithclium denaturalization.
The common approach to detect colistin is microbiology inhibition assay. Due tothe big molar weight, strong polarity and thermal instability, it is inappropriate to detect colistin with GC, thus the sensitivity and detection limit is restricted. Enzyme immunoassay is more precise and sensitive, and simpler operating, and can considerably minimized work intensity.
2. Test Principle
This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigen. Colistin residue in the sample competes with the antigen coated on the microtiter plate for the antibody. After the addition of enzyme conjugate, TMB substrate is used to show the color. Absorbance of the sample is negatively related to the colistin reside in it, after comparing with the Standard Curve, multiplied by the Dilution factor, colistin residue quantity in the sample can be calculated.
3. Applications
This kit can be used in quantitative and qualitative analysis of colistin in chicken and milk.
4. Cross-reactions
Colistin.............................…100%
Bacitracin zinc...............….........…< 1%
5. Materials Required
5.1 Equipments
----Microtiter plate spectrophotometer (450nm/630nm)
----Homogenizer
----Vortex mixer
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Volumetric flask: 500ml
----Polystyrene centrifuge tubes: 2ml, 50ml
----Glass centrifuge tubes: 10ml
----Micropipettes: 20ml-200ml. 100ml-1000ml
250ml-multipipette
Reagents
----Methanol (AR)
----Concentrated sulfuric acid (H₂SO₄, AR)
----Sodium hydroxide (NaOH, AR)
----Concentrated hydrochloric acid (HCl, AR)
----Sodium chloride (NaCl, AR)
----Deionized water
6. Kit Components

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

0ppb, 2.5ppb, 7.5ppb, 22.5ppb, 67.5ppb, 202.5ppb

1. Spiking standard solution : (1ml/bottle)  10ppm
2. Enzyme conjugate 7ml ..........….….…red cap
3. Solution A 7ml ............…......…white cap
4. Solution B 7ml .........…..........….red cap
5. Stop solution 7ml ...................yellow cap
6. 20×concentrated wash solution 40ml

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

1. Extraction solution 60ml......….......…blue cap

7. Reagents Preparation
Solution 1: Tissue extraction solution
Dilute 8.3ml of concentrated hydrochloric acid with deionized water to 500ml, then add 50g of sodium chloride, mix completely.
Solution 2: 1M NaOH
Dissolve 20g of sodium hydroxide with deionized water and dilute to 500ml.
Solution 3: 2M H₂SO₄
Dilute 10ml of 98.3% H₂SO₄ with deionized water to 90ml.
Solution 4: Extraction solution
Dilute the 2×concentrated extraction solution with deionized water in the volume ratio of 1:1, which will be used for sample extraction; this solution can be stored at 4ºC for 1 month.
Solution 5: 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 experimental instruments are clean.
8.2 Chicken samples
----Homogenize sample with homogenizer.
----Take 2.0g±0.05g homogenized sample into a 50ml polystyrene centrifuge tube, add 1.8ml of tissue extraction solution(solution 1) and 1.2ml of methanol, vortex for 2min, and then centrifuge for separation: 3000g / ambient temperature / 5min.
----Take 200µl of supernate, add 30μl of 1M NaOH (solution 2), then add 600µl of extraction solution (solution 4), vortex for 20s to mix completely.
----Take 50µl of the prepared solution for assay.

Dilution factor................10

8.3 Milk
----Take 1ml of milk into a 2ml polystyrene centrifuge tube, add 20μl of 2M H₂SO₄(solution 3) and 300μl of methanol, vortex for 30s to mix completely.
----Centrifuge for separation: 3000g / ambient temperature / 5min.
----Take 100μl of supernate (avoid the floating substance), add 900μl of extraction solution(solution 4), vortex for 20s.
----Take 50µl of the prepared solution for assay.

Dilution factor................13

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 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 (20-25ºC) 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/sample and enzyme conjugate: Add 50µl of standard solution(kit component) or prepared sample to corresponding wells. Add 50µl of enzyme conjugate(kit component). Mix gently by shaking the plate manually and incubate for 30min at 25ºC with 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 5) 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).
9.2.7 Coloration: Add 50µl of solution A(kit component) and 50µl of solution B(kit component) to each well. Mix gently by rocking the plate manually and incubate for 15min at 25ºC with cover.(see 12.8)
9.2.8 Measure: Add 50µl of stop solution(kit component) to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm (It's suggested measure with the dual-wavelength of 450/630nm and read the result within 5min after addition of stop solution).
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 colistin standards solution (ppb) as x-axis.
----The colistin 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 data analysis, which can be provided on request.
11. Sensitivity, accuracy and precision
Sensitivity: 2.5ppb
Detection Limit:
Chicken...…........................…..30ppb
Milk......…........................….30ppb
Accuracy:
Chicken...….......................…85±20%
Milk......…..........................85±15%
Precision:
C.V. 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 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 Keep the ELISA kits at 2-8ºC, do not freeze. Seal rest microwell plates. Avoid direct sunlight during all the incubations. Covering the microtiter plates is recommended.
12.7 Substrate solution should be abandoned if it turns colors. The reagents may 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 needs 10-15min after the addition of solution A and solution B. And 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
   Storage condition: 2-8ºC.
Storage period: 12 months.

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