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Product Description
Competitive Enzyme Immunoassay Kit for
Quantitative Analysis of Erythromycin
1. Background
Erythromycin is a macrolide antibiotic, which is applied as antibacterial and anti-mycoplasma infective. Strict MRLs have been established since this drug may lead to serious side effect in certain groups.
This kit is a new product for drug residual detection based on ELISA technology, which only costs 1h in each operation and can considerably minimize operation errors and work intensity compared with instrumental analysis.
2. Test Principle
This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigen. Erythromycin 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 erythromycin reside in it, after comparing with the Standard Curve, multiplied by the dilution factor, erythromycin residue quantity in the sample can be calculated.
- Applications
This kit can be used in quantitative and qualitative analysis of erythromycin residue in tissue(muscle, liver), beef, aquatic products, honey and milk(raw milk, UHT milk), cream and ice cream.
4. Cross-reactions
Erythromycin....................…....100%
Erythromycin Thiocyanate.........….......114%
Erythromycin Ethylsuccinate............…67.4%
Tylosin..................…...….....…<0.1%
Tilmicosin........................…<0.1%
Spiramycin.......................…....<0.1%
Kitasamycin...............…..........<0.1%
5. Materials Required
5.1 Equipments
Microtiter plate spectrophotometer (450nm/630nm)
Rotary evaporator or nitrogen drying instruments
Homogenizer
Shaker
Vortex mixer
Centrifuge
Analytical balance (inductance: 0.01g)
Graduated pipette: 10ml
Rubber pipette bulb
Polystyrene Centrifuge tubes: 2ml, 50ml
Micropipettes: 20ml-200ml, 100ml-1000ml
250ul-multipipette
5.2 Reagents
----Methanol (AR)
-----n-hexane(AR)
----Ethyl acetate (AR)
----Acetonitrile (AR)
----Sodium hydroxide (NaOH,AR)
----Anhydrous sodium carbonate (Na2CO3, AR)
----Sodium bicarbonate (NaHCO3, AR)
----Sodium chloride (NaCl, AR)
----Deionized water
6. Kit Components
- Microtiter plate with 96 wells coated with antigen
- Erythromycin standard solutions. (1ml×6 bottles)
0 ppb, 0.2ppb, 0.6ppb, 1.8ppb, 5.4ppb, 16.2ppb
- Spiking standard control: 1ml, 1ppm
- Enzyme conjugate (7ml).....…......….red cap
- Antibody solution (7ml) …..........green cap
- Solution A (7ml) ...............…..white cap
- Solution B (7ml) .........…........…red cap
- Stop solution (7ml) .........…...…yellow cap
- 20×Concentrated wash solution (40ml)
.....................…transparent cap
- 2×Concentrated extraction solution (50ml)
..........................… blue cap
7. Reagents Preparation
Solution 1: 0.1M NaOH solution
Weigh 2.0g sodium hydroxide (NaOH) dissolve with deionized water to 500ml.
Solution 2:acetonitrile-0.1M NaOH solution
Weight 85ml acetonitrile add 15ml 0.1M NaOH solution (Solution 1),mix completely .
Solution 3: 2% NaCl solution
Weigh 2.0g sodium chloride (NaCl) dissolve with deionized water to 100ml.
Solution 4: methanol-2% NaCl solution
Weigh 100ml methanol add 50ml 2% Nacl solution and mix completely.
Solution 5:0.1M CB solution
Weigh 4.66g Na2CO3 and 0.5g NaHCO3 dilute with 500ml deionized water ,mix completely .
Solution 6:0.05M NaOH solution
Weigh 1.0g NaOH dilute with 500ml deionized water.
Solution 7: 0.1 M HCl solution
Weigh 4.15ml concentrated hydrochloric acid dissolve with deionized water to 500ml.
Solution 8: 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 9: 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.
8.2 Tissue(muscle, liver)
-----Homogenize the tissue sample.
-----Weigh 1.0±0.05g of homogenate into a 50ml polystyrene centrifuge tube;
-----Add 4ml of 0.05M NaOH(solution 6), shake fiercely for 10min,
-----Centrifuge: 3000g / ambient temperature / 5min;
-----Transfer 200µl of supernate into a 2ml polystyrene centrifuge tube, add 750µl of extraction solution(solution 8) and 50µl of 0.1M HCl(solution 7), vortex for 30s to mix thoroughly.
-----Take 50ml of the prepared solution for assay.
Dilution factor: 25
8.3 Beef
-----Homogenize the tissue sample.
-----Weigh 2.0±0.05g of homogenate into a 50ml polystyrene centrifuge tube;
-----Add 10ml of acetonitrile-0.1M NaOH solution(solution 2), shake fiercely for 10min,
-----Centrifuge: 3000g / ambient temperature / 5min;
-----Transfer 1ml of supernate into a 10ml clear dry glass tube, dry with 50-60ºC under nitrogen stream;
-----Add 1ml of n-hexane, vortex for 30s, add 1ml of extraction solution(solution 8), vortex for 30s.
-----Centrifuge: 3000g / ambient temperature / 5min;
-----Remove the upper layer of organic phase, take 50ml of the lower layer of solution for assay.
Dilution factor: 5
8.4 Honey: Method A
-----Weigh 2.0±0.05g honey into a 50ml polystyrene centrifuge tube; add 2ml of 0.1M CB(solution 5), vortex to dissolve completely. Add 6ml of ethyl acetate, shake for 5min;
----- Centrifuge: 3000g / ambient temperature / 5min;
-----Take 3ml of the supernatant organic phase into a 10ml dry glass tube, dry with 50-60ºC nitrogen stream;
-----Dissolve with residue with 0.5ml extraction solution (solution 8), vortex for 5min to dissolve completely.
-----Take 50ml of the prepared solution for assay.
Dilution factor: 0.5
8.4 Honey: Method B
-----Weigh 1.0±0.05g honey into a 50ml polystyrene centrifuge tube;
-----Add 2ml methanol-2% NaCl solution (solution 4), vortex for 2min till dissolved;
-----Centrifuge: 3000g / ambient temperature / 5min;
-----Take 100ul transparent solution, mix with 900ul extraction solution (solution 8), vortex 30s, mix completely.
-----Take 50ml of the prepared solution for assay.
Dilution factor: 20
8.5 Milk
-----Take 1ml of milk sample into a 10ml polystyrene centrifuge tube;
-----Add 4ml of extraction solution(solution 8), vortex 1min to mix completely;
-----Centrifuge: 3000g / ambient temperature / 5min;
Avoid the upper fat layer, transfer 500ml substrate milk sample into 2ml centrifuge tube, then add 500ml extraction solution(solution 8), vortex for 1min to mix completely.
-----Take 50ml of the prepared solution for assay.
Dilution factor: 10
8.6 Ice Cream
-----Heat the ice cream and mix it completely;
-----Weigh 1±0.05g ice cream sample into 50ml polystyrene centrifuge tube;
-----Add 4ml extraction solution( see solution), then shake with shaker to mix it completely;
-----Transfer 200μl sample into 2ml polystyrene centrifuge tube, add 200 extraction solution(see solution 8), vortex for 30s to mix completely;
-----Take 50ml of the prepared solution for assay.
Dilution factor 10
8.7 Cream:
-----Weigh 1±0.05g ice cream sample into 50ml polystyrene centrifuge tube;
-----Add 1ml methanol, vortex for 2min to mix it completely; then add 3ml extraction solution( see solution 8), shake with shaker until mix completely;
-----Transfer 200ml sample solution into 2ml polystyrene centrifuge tube, add 200μl extraction solution(see solution 8), vortex for 30s to mix completely;
-----Take 50ml of the prepared solution for assay.
Dilution factor 10
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, homogenize 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: 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 50 µl of standard solution or prepared sample to corresponding wells. Add 50µl enzyme conjugate solution and 50µl antibody solution. Mix gently by shaking the plate manually and incubate for 40min at 37ºC 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 9) 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 solution A and 50µl solution B to each well. Mix gently by shaking the plate manually and incubate for 15 min at 37ºC with cover(see 12.8).
9.2.8 Measure: Add 50µl the stop solution to each well. Mix gently by shaking the plate manually 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 Erythromycin standards solution (ppb) as x-axis.
--- The erythromycin concentration of each sample (ppb), which can be read from the calibration curve, is multiplied by the corresponding dilution multiple of each sample followed, and the actual concentration of sample is obtained.
For data reduction of the ELISA kits, special software has been developed, which can be provided on request.
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.2 ppb
Detection limit
Tissue:...................…........…5ppb
Beef:...............…..............1ppb
Honey Method A:......….............0.1ppb
Honey Method B:..................….…....4ppb
Milk/ice cream/cream......…...............2ppb
Accuracy
Tissue........................….. 90±15%
Beef.............................… 90±20%
Honey Method A............….....… 90±20%
Honey Method B............….....… 90±20%
Milk/Ice cream/Cream…….....….......... 90±25%
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, 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 10-15min after adding Solution A and Solution B. And you can prolong the incubation time ranges from 15min 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 37ºC. 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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