Competitive Enzyme Immunoassay Kit Forquantitative Analysis of Matrine and Oxymatrine (MT&OMT)

• Customization: Available
• Classification: Biochemical Reagents
• Grade: AR
• Model NO.: KA15901H
• Content: Comparison
• Usage: Diagnostic Reagents
• Source: Imported Reagents
• Habit Appellation: Chemical Medicine
• Application: Industry, Scientific Research, Health, Environmental Protection, Agriculture
• Property: Organic Reagent
• Transport Package: Foam Box
• Specification: 10 Test
• Trademark: kwinbon
• Origin: China
• HS Code: 38229000
• Production Capacity: 520000000kit Per Year

Product Description

Competitive Enzyme Immunoassay Kit for
Quantitative Analysis of Matrine and Oxymatrine (MT&OMT) 
 

1. Background
Matrine and Oxymatrine (MT&OMT)  belong to the picric alkaloids, a class of plant alkaloid insecticides with poisoning effects of touch and stomach, and are relatively safe biopesticides. At the beginning of the year 2021, the EU countries have repeatedly notified that Oxymatrine was detected in honey exported from China, and the honey products were refused to enter the country. Therefore, it is necessary to monitor the content of this drug.
This kit is a new generation of drug residue detection products developed by ELISA technology, which has the advantages of fast, simple, accurate and high sensitivity compared with instrumental analysis technology, and the operation time is only 75 minutes, which can minimise the operation error and work intensity.
2. Test Principle
This product adopts the indirect competition ELISA method, the coupling antigen is pre-coated on the microtiter strip of the enzyme plate, the residual Matrine and Oxymatrine in the sample will compete with the coupling antigen pre-coated on the microtiter strip of the enzyme plate for the antibody of Matrine and Oxymatrine. After adding the enzyme conjugate, the colour will be developed with the TMB substrate, the absorbance value of the sample and its content of the residuals Matrine and Oxymatrine will correlate negatively, and then compare it with the standard curve, and then multiply the number of dilutions by the corresponding residuals, which can be derived from the corresponding residuals Matrine and Oxymatrine content.
3. Applications
This kit can be used for the quantitative and qualitative analysis of Matrine and Oxymatrine in honey.
4. Cross-reactions
Matrine.............................…100%
Oxymatrine….........................…82%
Sophocarpine.............…...........…122%
Sophoridine............................78%

5. Materials Required
5.1 Equipments:
----Microtiter plate spectrophotometer (450nm/630nm)
----Homogenizer (or stomacher)
----Shaker
----Vortex mixer
----Centrifuge
----Analytical balance (inductance: 0.01g)
----Polystyrene centrifuge tube: 2ml,10ml
----Micropipettes: 20ul-200ul,
100ul-1000ul, 250ul-multipipette
5.2 Reagents:
----Deionized water
6. Kit Components

1. Microtiter plate with 96 wells coated with antigen
2. MT&OMT standard solutions×5 bottles:  (1ml/bottle)

(1μg/L=1ppb=1μg/kg)
0ppb, 0.2ppb, 0.6ppb, 1.8ppb, 5.4ppb

1. Spiking standard solution: (1ml/bottle) 1ppm
2. Enzyme conjugate  12ml ........................red cap
3. Antibody solution  7ml .....................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 sample dilution 50ml

........................…..…blue cap
7. Reagents Preparation:
Solution 1: Sample dilution 
Dilute the 2×sample dilution with deionized water in the volume ratio of 1:1. 
Solution 2: Wash solution
Dilute the 20×concentrated wash solution with deionized water in the volume ratio of 1:19, which will be used to rinse the plate. This solution can be conserved at 4ºC for 1 month.
8. Sample Preparation
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.
(c) The derivative reagent can be conserved at 2-8ºC for a year;
(d) Keep untreated samples in freeze;
8.2 Honey sample
----Weigh 1.0±0.05g of the sample into a 10ml polystyrene tube;
----Add 2ml deionized water, Vortex for 5 minutes with a vortexer
----Take 50 μL into a 2 mL polystyrene centrifuge tube and add 450 μL of sample dilute solution (solution 1) and mix thoroughly. 
----Take 50 μL 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 brought to 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 antibody solution:  Add standard solution or sample solution 50μl to the corresponding well. Add 50ul antibody solution per well, Mix gently by shaking the plate manually and incubate for 30min 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 2) at interval of 10s for 5 times. Absorb the residual water with absorbent paper.
9.2.7 Add the enzyme conjugate: Add 100ul of the
 enzyme conjugate per well, Mix gently by shaking the plate manually and incubate for 30min 25ºC with cover. Remove and repeat wash (step 6).
9.2.8 Coloration: add 50µl solution A and 50µl solution B to each well. Mix gently by shaking the plate manually and incubate for 15min at 25ºC with cover (see 12.7).
9.2.9 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 then 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 (0ppb)
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 MT&OMT standards solution (ppb) as x-axis.
----The MT&OMT 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:
Software has been developed for data analysis, which can be provided upon request.
Dilution factor of samples...............…..30
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.2ppb
Detection limit..................….....10ppb
Accuracy:
Honey...….........................…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 using.
12.4 Don't use the kits out of date. Don't exchange the reagents of different batches, or else it will drop the sensitivity.
12.5 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.
12.6 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).
12.7 The coloration reaction needs 15min after the addition of solution A and solution B; But you can prolong the incubation time to 20min or more if the color is too light to be determined., never exceed 25min, On the contrary, shorten the incubation time properly.
12.8 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:  12months.