Elisa Test Kit for Amoxicillin

• Environmental Protection: Yes
• Certification: REACH
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Appearance: Liquid
• Model NO.: KA10101H
• Transport Package: Foam Box with Ice Bag
• Trademark: kwinbon
• Origin: China
• HS Code: 38229000
• Production Capacity: 50000PCS/Year

Product Description

Product Description

Competitive Enzyme Immunoassay Kit for 
Quantitative Analysis of Amoxicillin

1. Background
Amoxicillin, also spelled amoxycillin, is an antibiotic useful for the treatment of a number of bacterial infections. It is the first line treatment for middle ear infections. It may also be used for strep throat, pneumonia, skin infections, and urinary tract infections among others. It is taken by mouth, or less commonly by injection.
Common side effects include nausea and rash. It may also increase the risk of yeast infections and, when used in combination with clavulanic acid, diarrhea. It should not be used in those who are allergic to penicillin. While usable in those with kidney problems, the dose may need to be decreased. Its use in pregnancy and breastfeeding does not appear to be harmful.
2. Test Principle
This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigen. Amoxicillin 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 Amoxicillin in it, after comparing with the Standard Curve, multiplied by the dilution factor, Amoxicillin quantity in the sample can be calculated.
3. Applications
This kit can be used in quantitative and qualitative analysis of Amoxicillin in animal tissue (pork, chicken) , milk and egg.
4. Cross-reactions
Amoxicillin….............…....................100%
5. Materials Required
5.1 Equipments
----Microtiter plate spectrophotometer (450nm/630nm)
----Rotary evaporator or nitrogen gas drying system
----Homogenizer
----Shaker
----Vortex mixer
----Centrifuge
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Polystyrene centrifuge tubes: 2ml, 50ml
----Glass test tube: 10ml
----Volumetric flask: 100ml, 500ml
----Micropipettes: 20ul-200ul,100ul-1000ul,
250ul-multipipette
5.2 Reagents
---- Disodium hydrogen phosphate 12-hydrate
(Na₂HPO₄▪12H₂O, AR)
---- Sodium dihydrogen phosphate dehydrate
 (NaH₂PO₄, AR)
---- Methanol(AR)
----Deionized water
6. Kit Components

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

0 ppb, 1.5ppb, 4.5ppb, 13.5ppb, 40.5ppb, 121.5 ppb

1. Spiking standard solution: 1ml, 1ppm
2. Enzyme conjugate (12ml)….…..…......…...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. 2×Concentrated extraction solution (50ml)

 ...........................… blue cap
7. Reagents Preparation
Solution 1: pH=7.2  0.02 M PBS
Weigh 2.58g Na₂HPO₄▪12H₂O and 0.435g NaH₂PO₄, dilute with deionized water to 500ml.
Solution 2: 0.03 M PBS
Weigh 3.87g  Na₂HPO₄▪12H₂O and 0.654g NaH₂PO₄, dilute with deionized water to 500ml.
Solution 3: Tissue extraction solution
     Mix 10ml of methanol and 70ml of 0.03M PBS(solution 2) completely.
Solution 4: Raw Milk extraction solution
     Mix 8ml of methanol and 2ml of 0.02M PBS(solution 1) completely.
Solution 5: UHT Milk extraction solution
     Mix 3ml of methanol and 7ml of 0.02M PBS(solution 1) completely.
Solution 6: Extraction solution
Dilute the 2×concentrated extraction solution with deionized water in the volume ratio of 1:1(e.g. 10ml of 2×extraction solution + 10ml of deionized water), which will be used for sample extraction, this solution can be stored at 4ºC for 1 month.
Solution 7: Wash solution
Dilute the 20×concentrated wash solution with deionized water in the volume ratio of 1:19(e.g. 5ml of 20×wash solution + 95ml of deionized water), 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 absorbing different reagent.
(b) Make sure that all experimental instruments are clean.
8.2 Tissue (pork, chicken)
----Take 2.0±0.05g of homogenized tissue sample into a 50ml tube, then add 8ml of tissue extraction solution (solution 3), shake for 5min, and then centrifuge for separation: 3000g / ambient temperature / 5min..  
----Transfer 500µl of the supernate into a 2ml polystyrene centrifuge tube, add 500µl of extraction solution(solution 6), vortex for 30s to mix completely.
----Take 50µl per well for assay.

Dilution factor:                10

8.3 Milk
8.3.1 Raw milk
----Take 1ml of milk into a 2ml polystyrene centrifuge tube, add 1ml of raw milk extraction solution(solution 4), vortex for 30s to mix completely.
----Transfer 500μl of the solution into a 2ml polystyrene centrifuge tube, add 500μl of extraction solution(extraction 6), vortex for 30s to mix completely.
----Take 50µl per well for assay.
8.3.2 UHT milk
----Take 1ml of UHT milk into a 2ml polystyrene centrifuge tube, add 1ml of UHT milk extraction solution(solution 5), vortex for 30s to mix completely.
----Transfer 500μl of the solution into a 2ml polystyrene centrifuge tube, add 500μl of extraction solution(solution 6), vortex for 30s to mix completely.
----Take 50µl per well for assay.

Dilution factor:                4

8.4 Egg
----Homogenize the egg white and yolk with homogenizer.
----Weigh 1.0±0.05g of the homogenate into a 10ml  polystyrene centrifuge tube, add 2ml of deionized water, vortex for 1min, then centrifuge: 5min / 3000g / 20-25ºC.
----Transfer 200µl of the supernate into a 2ml polystyrene centrifuge tube, add 200µl of extraction solution(solution 6), vortex for 1min to mix completely(The prepared solution should be white or light yellow).
----Take 50µl per well for assay.

Dilution factor:                6

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: 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 and antibody solution: Add 50µl of standard solution(Kit provided) or prepared sample to corresponding wells. Add 50µl of antibody solution(Kit provided). Mix gently by rocking the plate manually and incubate for 30min at 25ºC with cover.
9.2.6 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl diluted wash solution (solution 7) 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 Add Enzyme conjugate: Add 100μl of enzyme conjugate(Kit provided) to each well, Mix gently by rocking the plate manually and incubate for 30min at 25ºC with cover. Repeat the wash step again.
9.2.8 Coloration: Add 50µl solution A(Kit provided) and 50µl solution B(Kit provided) to each well. Mix gently by rocking the plate manually and incubate for 15min at 25ºC with cover(see 12.8).
9.2.9 Measure: Add 50µl of stop solution(Kit provided) to each well. Mix gently by rocking 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 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 amoxicillin standards solution (ppb) as x-axis.
----The amoxicillin 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 evaluation of the result, special software has been developed, which can be provided on request.
11. Sensitivity, accuracy and precision
Test Sensitivity: 1.5ppb
Detection limit
Animal tissue...............…....….....15ppb
Milk........................…....….…6ppb
Egg.....................…...........9ppb
Accuracy
Tissue(chicken)......…........…......…100±20%
Tissue(pork)......….............…..…..…90±20%
Milk(raw Milk)......…...............…. 100±20%
Milk(UHT Milk)....................…...…90±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 the 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 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 needs 15min after the addition of solution A and solution B. And you can prolong the incubation time from 20min to more if the color is too light to be determined. Never exceed 25min, on the contrary, shorten the incubation time properly.
13. Storage
    Storage condition: 2-8ºC.
Storage period:  12 months
 

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