Streptomycin Elisa Test Kit for Honey

• Environmental Protection: Yes
• Certification: REACH
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Model NO.: KA01904H
• HS Code: 38229000

Product Description

Product Description

Competitive Enzyme Immunoassay Kit for
Quantitative Analysis of Streptomycin 

1. Background
Streptomycin is an aminoglycoside antibiotic, which is broadly applied in animal disease treatment. For it has neurotoxicity and kidney toxicity, its residue in animal-derived food is harmful to human; it is strictly controlled in use in EU, US and China. At present, ELISA is the common approach in supervision and control of streptomycin drug
This kit is a new product for drug residual detection based on ELISA technology, which only costs 45min in each operation and can considerably minimize operation errors and work intensity. 
2. Test Principle
This kit is based on direct-competitive ELISA. The microtiter wells are coated with coupling antibody. Streptomycin residue in the sample competes with the enzyme marker coated on the microtiter plate for the antibody. After the addition of enzyme conjugate, chromegenic substrate is used to show the color. Absorbance of the sample is negatively related to the streptomycin residue in it, after comparing with the Standard Curve, multiplied by the dilution factor, Streptomycin residue quantity in the sample can be calculated.
3. Applications
This kit can be used in quantitative and qualitative analysis of streptomycin residue in tissue (pork, chicken, liver), fish and shrimp, milk (raw milk, UHT milk, reconstituted milk, pasteurized milk, milk beverage), serum, milk powder (whole milk powder, skim milk powder), honey, royal jelly, etc.
4. Cross-reactions
Streptomycin..........................100.0%
Dihydrostreptomycin...............…....…106%
Streptomycin sulphate.....................67%
Neomycin.........…....................…<1%
Gentamycin.....................….....…<1%
Kanamycin...........................…<1%
Amikacin..................…............<1%
Spectinomycin.............................<1%
Apramycin...............................<1%

5. Materials Required
5.1 Equipments
----Microtiter plate spectrophotometer (450nm/630nm)
----Homogenizer
----Vortex mixer
----Centrifuge
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Polystyrene centrifuge tube: 2ml, 50ml
----Micropipettes: 20μl-200μl, 100μl-1000μl
250μl-multipipette 
5.2 Reagents
----Trichloroacetic acid (AR)
----Sodium hydroxide (AR)
----Deionized water
6. Kit Components

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

0ppb,0.05ppb,0.15ppb,0.45ppb,1.35ppb,4.05ppb

1. Spiking standard solution: (1ml/bottle) 1ppm
2. Concentrated Enzyme conjugate  1ml...….red cap
3. Enzyme conjugate diluent  10ml....... 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. Reagents Preparation
Solution 1: 1% trichloroacetic acid
Dissolve 1.0g of trichloroacetic acid with 100ml of deionized water, mix completely.
Solution 2: 1.5% trichloroacetic acid
Dissolve 1.5g of trichloroacetic acid with 100ml of deionized water, mix completely.
Solution 3: 0.1mol/L NaOH
Dissolve 0.4g of sodium hydroxide with 100ml of deionized water, mix completely.
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 the 20×concentrated wash solution with deionized water in the volume ratio of 1:19, which will be used for washing the plates. This solution can be stored at 4ºC for 1 month.
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; otherwise it will affect the assay result.
(c) Prepared sample should be used immediately, don not conserve more than 4h at room temperature.
8.2 Tissue(pork, chicken, liver)
----Weigh 1.0±0.05g of homogenized tissue sample into a 50ml polystyrene centrifuge tube, then add 2ml of 1% trichloroacetic acid(Solution 1), vortex for 5min to mix completely.
---Centrifuge for separation: 3000g / 5min / ambient temperature.
---Take 100ml of supernatant, add 30ml of 0.1mol/L NaOH(solution 3), then add 870ml of extraction solution(Solution 4), vortex for 30s to mix completely.
----Take 50ml of the prepared solution for assay.
Dilution factor...................30

8.3 Aquatic products
(1)Shrimp
----Weigh 1.0±0.05g of homogenized shrimp sample into a 50ml polystyrene centrifuge tube, then add 2ml of 1% trichloroacetic acid(Solution 1), vortex for 5min to mix completely.
---Centrifuge for separation: 3000g / 5min / ambient temperature.
---Take 100ml of supernatant, add 30ml of 0.1mol/L NaOH(solution 3), then add 870ml of extraction solution(Solution 4), vortex for 30s to mix completely.
----Take 50ml of the prepared solution for assay.
(2) Fish
----Weigh 1.0±0.05g of homogenized fish sample into a 50ml polystyrene centrifuge tube, then add 2ml of 1% trichloroacetic acid(Solution 1), vortex for 5min to mix completely.
---Centrifuge for separation: 3000g / 5min / ambient temperature.
---Take 100ml of supernatant, add 900ml of extraction solution(Solution 4), vortex for 30s to mix completely.
----Take 50ml of the prepared solution for assay.
Dilution factor...................30

8.4 Milk (raw milk, UHT milk, reconstituted milk, pasteurized milk, milk beverage), serum
----Add 30ml of milk or serum sample into a 2ml polystyrene centrifuge tube,
----Add 870ml of extraction solution(Solution 4), vortex for 30s to mix completely.
----Take 50ml of the prepared solution for assay.
Dilution factor...................30

8.5 milk powder
----Weigh 1.0±0.05g of milk powder sample into a 50ml polystyrene centrifuge tube, then add 10ml of deionized water. vortex for 5min to mix completely.
---Take 100ml of supernatant, add 900ml of extraction solution(Solution 4), vortex for 30s to mix completely.
----Take 50ml of the prepared solution for assay.
Dilution factor...................100

8.6Honey
----Weigh 1.0±0.05g honey sample into a 50ml polystyrene centrifuge tube, then add 4ml of deionized water, vortex for 5min till the sample is completely dissolved.
---Take 200ml of clean solution, add 600ml of extraction solution(solution 4), vortex for 30s to mix completely.
----Take 50ml of the prepared solution for assay.

Dilution factor...................20

8.5 Royal jelly
----Weigh 1.0±0.05g of the royal jelly sample into a 50ml polystyrene centrifuge tube.
----Add 3ml of 1.5% trichloroacetic acid(solution 2), vortex for 5min, and then centrifuge at room temperature (20-25ºC) for 5min, at 3000g;
----Take 100ml of the supernatant layer(avoid the floating object), and mix with 110ml of 0.1mol/L NaOH (solution 3), adjust the pH to 6-8, then add 790ml of extraction solution(solution 4), vortex for 1min,  mix completely.
----Take 50ml of the prepared solution for assay.
Note: This method may have 2ppb background effect, which will not affect the determination of the sample according to MRLs.

Dilution factor...................30

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 concentrated wash solution and concentrated extraction solution should be rewarmed before use;
9.2.4 Number: Number every microwell position and all standards and samples should be run in duplicate. Record the standards and samples positions.
9.2.5 Add standard solution/sample: Add 50µl of standard solution or prepared sample to corresponding wells.
9.2.6 Dilute the concentrated enzyme conjugate: Mix the concentrated enzyme conjugate and enzyme conjugate diluent in the volume ratio of 1:10(e.g. 0.5ml of concentrated enzyme conjugate + 5ml of enzyme conjugate diluent ), mix completely, (this mixture can't be conserved, use immediately).
9.2.7 Add the diluted enzyme conjugate (9.2.6): Add 50µl of the diluted enzyme conjugate to each well, shake the plate manually to mix and then incubate for 30min at 25ºC with cover.
9.2.8 Wash: Remove the cover gently and pour 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).
9.2.9 Coloration: Add 50µl of solution A and 50µl of solution B to each well. Mix gently by shaking the plate manually and incubate for 15 min at 25ºC with cover (see 12.8)
9.2.10 Measure: Add 50µl of the stop solution 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. 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 of standard (or sample)
B₀ --absorbance of 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 streptomycin standards solution (ppb) as x-axis.
----The streptomycin 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 analysis, which can be provided on request. 
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.05ppb
Detection limit
Tissue.....................….…....…1.5ppb
Fish and shrimp...............….…....…1.5ppb
Milk,serum...........................…1.5ppb
Milk powder ...................….......5ppb Honey.....................….….......1ppb
Royal jelly.............................…1.5ppb
Accuracy
Tissue/fish and shrimp/serum /Honey / Royal jelly.............................................................90±15%
Milk/milk powder................................................…100±30%
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 the 0.5M H2SO4 solution.
12.5 Don't use the kits out of date. Don't exchange the reagents of different batches, for it will drop the sensitivity.
12.6 Keep the ELISA kits at 2-8ºC,do not freeze. Seal rest microwell plates Avoid straight sunlight for the standard sample and the colorless chromogenic reagent are sensitive to light.
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 needs 15 min after adding Solution A and Solution B. And you can prolong the incubation time 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 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: 12months.

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