Competitive Enzyme Immunoassay Kit for Quantitative Analysis of
Sulfanilamide (SAs) Residue Detection (7-in-1)
1. Background
SAs are broadly applied bacteriophages, which are very important in controlling and curing animal disease. These drugs have very serious side effects and will lead to SAs resistance of some bacilus if they exist in human body for a long period. They also have potential carcinogenicity. Doc.235 of Ministry of Agriculture prescribes that the residue limit of SAs is 100mg/kg.
This kit is a new generation product for drug residue detection based on ELISA technology. It is fast, simple, accurate and sensitive. And it requires only 1.5 hours in one operation, which considerably minimizes work intensity and operation error. This kit is documented in Doc.558 of MOA in Oct.2005.
2. Test Principle
This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigen. Drug residue including sulfamonomethoxine, sulfadiazine, sulfamethoxazole and sulfathiazole in the sample competes with the antigen coated on the microtiter plate for the antibody. After the addition of enzyme labeled anti-antibody, TMB substrate is used to show the color. Absorbance of the sample is negatively related to the SAs residing in it, after comparing with the Standard Curve, multiplied by the dilution factor, SAs residue quantity in the sample can be calculated.
3. Applications
This kit can be used in quantitative and qualitative analysis of SAs residue (SMM, SD/SDZ, SMZ, ST, PST, Sulfamethizole and Sulfapyridine) in honey.
4. Cross-reactions
Sulfadiazine(SD or SDZ)................…100.0%
Sulfamonomethoxine (SMM)..........…..… …97.6%
sulfamethoxazole (SMZ) ...............…169.0%
sulfathiazole (ST)......................188.0%
Phthalylsulfathiazole (PST)......…........…62.8%
Sulfamethizole ........................103%
Sulfapyridine.................….......…51.9%
Sulfamethazine ........................ <1%
Sulfadimethoxine .......................…<1%
Sulfaquinoxaline ........................<1%
Sulfamerazine.........…...............................…<1%
5. Materials Required
5.1 Equipments
Microtiter plate spectrophotometer (450nm/630nm)
Rotary evaporator or nitrogen drying instruments
Shaker
Vortex mixer
Centrifuge
Analytical balance (inductance: 0.01g)
Graduated pipette: 10ml
Rubber pipette bulb
Volumetric flask: 500ml,
Glass test rube: 10ml
Polystyrene Centrifuge tubes:2ml, 50ml
Micropipettes: 20ml-200ml, 100ml-1000ml
250ml -multiple
5.2 Reagents
---- Acetonitrile (AR)
---- Ethyl acetate (AR)
----Disodium hydrogen phosphate 12-hydrate (Na2HPO4·12H2O , AR)
---- Sodium dihydrogen phosphate dehydrate
(NaH2PO4·2H2O , AR)
---- Sodium hydroxide (NaOH, AR)
---- Concentrated hydrochloric acid(HCl, AR)
---- Deionized water
6. Kit Components
Microtiter plate with 96 wells coated with antigen
Standard solutions(6 bottles×0.5ml/bottle)
0ppb, 2ppb, 4ppb, 8ppb, 16ppb, 32ppb
Spiking standard control:(1ml/bottle) 1ppm
Enzyme conjugate 12ml............…... red cap
Antibody solution 10ml ...........….…..green cap
Solution A 7ml........................white cap
Solution B 7ml ......…............... red cap
Stop solution 7ml ...........…....…yellow cap
20×Concentrated wash solution 40ml
.....................…..…..transparent cap
2XExtraction solution 50ml...…......… blue cap
7. Reagents Preparation
Solution 1: 1 M hydrochloric acid solution
Measure 41.5ml concentrated hydrochloric acid and dilute to 500ml with deionized water.
Solution 2: 1 M sodium hydroxide solution
Dissolve 20.0g sodium hydroxide with deionized water and dilute to 500 ml.
Solution 3: Phosphate buffer solution (pH=6)
Weigh 1.75g disodium hydrogen phosphate 12-hydrate, 10.52g sodium dihydrogen phosphate dehydrate, dilute to 500ml with deionized water.
Solution 4: Extraction solution
Dilute the 2×concentrated extraction solution with deionized water in the volume ration of 1:1, which will be used for sample extraction. This diluted solution can be conserved for 1 month at 4ºC.
Solution 5: Wash solution
Dilute the 20×concentrated wash solution with deionized water in the volume ration of 1:19, which will be used to wash the plates. This diluted solution can be conserved for 1 month at 4ºC.
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.
(c) Samples that are treated with other methods can be stored at 4ºC for 24 hours.
8.2 Honey
Weigh 1.0±0.05g honey sample to a 50ml polystyrene centrifuge tube.
Add 1ml 1M hydrochloric acid solution (see solution 1), shake with until the honey completely dissolved.
Keep the tube in 37ºC incubator for 30min.
Add 1ml 1M sodium hydroxide solution(see solution 2) and 1ml phosphate buffer solution (see solution 3),shake for 1min,and then add 2ml acetonitrile and 6ml ethyl acetate, shake for 1min with shaker, centrifuge for 5min , 3000g, at ambient temperature.
Take the supernatant organic phase 4ml to a 10ml clean glass test tube, dry it with 50-60ºC water bath under nitrogen flow.
Add 0.5ml extraction solution solution (see solution 4), vortex for 1min to dissolve the dry leftover;
Take 20ml of the prepared solution for assay.
Dilution factor: 1
9. Assay process
9.1 Notice before assay:
9.1.1 Make sure all reagents and micro wells 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 repetitiveness 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: Add 20 µl of standard solution or prepared sample to corresponding wells. Add 80µl antibody solution. 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 (see 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).
9.2.7 Enzyme conjugate: Add Enzyme conjugate 100ml to each well, Mix gently by rocking the plate manually and incubate for 20min at 25ºC with cover. Repeat the wash step again.
9.2.8 Coloration: Add 50µl solution A and 50µl solution B 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 the stop solution to each well. Mix gently by rocking 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 ELIASA instrument)
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 SAs standards solution (ppb) as x-axis.
----The SAs concentration of each sample (ppb), 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.
Please notice:
Special software has been developed for all data analysis, which can be provided on request.
11. Sensitivity, accuracy and precision
Test Sensitivity: 2ppb
Detection limit: 2ppb
Accuracy: 80±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 repetitiveness and operate the next step immediately after tap the microwells holder.
12.3. Homogenize each reagent before using.
12.4. Keep your skin away from the stop solution for it is the2M 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 Storage condition:
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 Indications for the reagents going bad:
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 need 10-15min after the addition of solution A and solution B; But you can prolong the incubation time ranges from 20min to more if the color is too light to be determined., never exceed 30min,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 condition and storage period
Storage condition: 2-8ºC.
Storage period: 12 months
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