Elisa Test Kit for Erythromcin

• Environmental Protection: Yes
• Certification: REACH
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Model NO.: KA03302H
• HS Code: 38229000

Product Description

Product Description

 

Competitive Enzyme Immunoassay Kit for
Quantitative Analysis of Erythromcin

1. Background
Erythromcin residue in the production of biological products may lead to abnormal reactions of human beings, thus strict MRLs have been established. This kit is a rapid test product for the determination of tetracycline residues which is sensitive, accurate and time-saving. It can considerably reduce the operation errors in the assay.
2. Test Principle
This ELISA kit is designed to detect erythromcin based on the principle of "indirect-competitive" enzyme immunoassay. The microtiter wells are coated with capture BSA-linked antigen. erythromcin in the sample competes with antigen coated on the microtitre plate for the antibody. After the addition of enzyme conjugate, chromogenic substrate is used and the signal is measured by spectrophotometer. The absorption is inversely proportional to the erythromcin concentration in the sample.
3. Applications
This kit can be used in quantitative and qualitative analysis of erythromcin residue in biological samples.
4. Cross-reactions
erythromcin.......................…..…100%
erythromycin thiocyanate.........…....….…114%
ethylsuccinate ...........…..........…67.4%
Tylosin.................................................... <1%
tilmicosin...... ............................................ <1%
spiramycin ..................….........….<1%

5. Materials Required
5.1 Equipments:
Microtiter plate spectrophotometer (450nm/630nm)
polystyrene centrifuge tube:  2ml, 50ml
Micropipettes:  20ml-200ml, 100ml-1000ml
250ml-multipipette
5.2 Reagents:
Deionized water
6. Kit Components

1. Microtiter plate with 96 wells coated with coupling   antigen
2. erythromcin standard solutions×6 bottles: 1ml/bottle

0ng/ml, 0.2ng/ml,0.6ng/ml,1.8ng/ml,5.4ng/ml, 16.2ng/ml 

1. Spiking standard solution: 1ml, 1μg/ml
2. Enzyme conjugate (7ml)…..........….....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×sample diluent(50ml)  

............................ blue cap
7. Reagents Preparation:
Solution 1: Sample diluent
Dilute the 2×sample diluent with deionized water in the volume ratio of 1:1, which will be used for sample dilution. This solution can be stored at 4ºC for 1 month.
Solution 2: 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 for the users 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 effect the assay result.
8.2 Sample Preparation:
---- Dilute the test sample with the prepared sample diluent(solution 1) to get a final concentration of 0.2-16.2 ng/ml (erythromcin).
----Take 50 ml of the prepared solution 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, 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 wash solution should be brought to room temperature (20-25ºC) 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, enzyme conjugate and antibody: Add 50µl of standard solution or prepared sample to corresponding wells. Add 50µl of enzyme conjugate solution,50µl of antibody solution to each well, mix gently by shaking the plate manually and incubate for 40min 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 2) 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 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) 
9.2.8 Measure: Add 50µl of 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 addition of stop solution. )
(We can also measure by sight without stop solution in short of the ELISA reader).
10. Results
(1) Percentage absorbance
The mean values of the absorbance values obtained from the standards and the samples are divided by the absorbance value of the first standard (zero standard ) and multiplied by 100%.
   =
B --absorbance of standards or samples
B0 --absorbance of zero standard (0ng/ml)
(2) Standard Curve
---To draw a standard curve: The absobance value of standards as y-axis, semilogarithmic of the concentration of the standards (ng/ml) as x-axis.
---The erythromcin concentration of each sample (ng/ml), which can be read from the calibration curve, is multiplied by the corresponding dilution rate of each sample followed, and the actual concentration of sample is obtained.
11. Sensitivity, accuracy and precision
Linear range: 0.5-40.5ng/ml
Accuracy: 100±30%
Precision: CV of the ELISA kit all 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 be dry between steps to avoid unsuccessful repetitiveness and operate the next step immediately after tap the microwells holder.
12.3 Mix the homogenate and elute the plate adequately. 12.4 Avoid the stop solution touching skin for the 2M H2SO4.
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 Storage constitution:
Keep the ELISA kits at 2-8ºC without frozen. Avoid direct sunlight during all incubations. Covering the microtiter plates is recommended.
12.7 The reagents go bad:
Substrate solution should be abandoned if its color has changed. 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 need 20-30min after the addition of solution A and solution B; But you can prolong the incubation time ranges from 35min to 40min if the color is too light to be determined. On the contrary, shorten the incubation time properly.
12.9 The best reaction temperature is 25ºC, temperature too high or too low both will lead to the changes of sensitivity and absorbance values.
13. Storage condition and storage period
     Storage condition: 2-8ºC.
Storage period: 12 months.

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