0.1 Ppb Diethylstilbestrol Diagnostic ELISA Kit For Milk Egg Urine Serum Test Kit Feed 96 Wells/Kit

Diethylstilbestrol ELISA Test Kit For Milk Egg Urine Serum Feed 96 Wells/Kit

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Diethylstilbestrol(DES) in the feed, urine, liver, meat, shrimp and fish. The conjugate antigen is pre-coated on the micro-well stripes.The Diethylstilbestrol in the sample competes with the conjugate antigen pre-coated on the micro-well stripes, to interact with the antibodies againstDiethylstilbestrol. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with the content ofDiethylstilbestrol inthe sample. This value is compared to the standard curve and the content of the correspondingDiethylstilbestrol is subsequently obtained.

2. Technical specifications

Sensitivity: 0.1 ppb
Incubation Temperature: 37℃
Incubation Time: 30min—30min—15min

Detection limit:
Tissue (Shrimp, fish) 0.2ppb
Pork/liver, chicken/liver 2 ppb
Muscle tissue(Method Two) 0.1ppb
Urine 0.6 ppb
Feed 20 ppb

Cross-reactions rate:
DES100%
Dienestrol38.5%
Hexestrol8.5%
Ethinylestradiol< 0.1%
Estriol< 0.1%
Recovery rate
Urine70±10%
Feed90±10%
Tissue85±10%

3. Components

4. Materials required but not provided

1) Equipments:microplate reader, printer,homogenizer, nitrogen-drying device,votex, centrifuge, measuring pipets,balance (asensibility reciprocalof0.01 g)
2) Micropipettors: single-channel 20-200 µL ,100-1000 µL, andmulti-channel 30～300μl;
3) Reagents: NaOH,Acetonitrile (CH3CN), Acetone, deionized water,H3PO4 (85%),ethyl acetate,CHCl3

5. Sample pre-treatment

Instructions (Thefollowing pointsmust be dealt with beforethe pre-treatment)
1) Only thedisposable tips can be used for the experiments and the tips must be changed when used forabsorbingdifferent reagents;
2) Before the experiment, eachexperimental utensil must beclean and should be re-cleaned ifnecessary,in order to avoid thecontamination thatinterfereswith the experimental results.

Solution preparationbefore sample pre-treatment
1) 6 MH3PO4 : dissolve 100 mLH3PO4 (85%) in 150 mLdeionized water, mix properly
2) 1 M NaOH : dissolve 4 g NaOH in deionized water to 100 mL
3) 2 M NaOH : dissolve 8 g NaOH in deionized water to 100 mL
4) Acetonitrile- Acetone: add 80 mLAcetonitrile and 20 mL Acetone, mix evenly
5) The 5×concentrated redissolvingsolution is mixed withdeionized water at 1:4 (1 mL concentrated redissolvingsolution + 4 mL deionized water), used for the treated sample redissolving

5.1 Tissue (Chicken, duck, pork/liver, shrimp, fish)
1. Weigh 2± 0.05 gof the homogenized sample, add 6 mLAcetonitrile- Acetone, shake for 2 min, andcentrifuge at above 4000 r/min at15 ℃ for 10 min.
2. Transfer 3 mL supernatant into a new centrifuge tube, blow todry with nitrogen or air at 60 ℃.
3. Add 0.5 mLCHCl3, vortex for 20 sec, add 2 mL 2 M NaOH, vortex for 30 sec, centrifuge at above 4000 r /min for 5 min.
4. Take 1 mL supernatant, add 200 µL 6 MH3PO4, vortex for 5 sec.
5. Add 3 mLAcetonitrile (CH3CN) for extraction, shake properly for 2 min, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min, take the upper layer, blow todry withnitrogen or air at 60 ℃.
6. Dissolve dry residues in 1 mL of thedilutedredissolvingsolution.

Dilute as following method for different samples
1) Shrimp and fish-----directly take 50 µL water phase for detection;
2) Pork/liver, Chicken/liver-----take 50 µL water phase, add 450 µL of thedilutedredissolvingsolution, shake properly for 30s. Take 50 µL for analysis.
Fold of dilution of the sample:
Shrimp and fish----2 Pork/liver, Chicken/liver------20

5.2 Muscle tissue Method Two
1) Take2±0.05g homogenized sample, add 2ml 2M NaOH, shake for 2min;
2) Add 8mlethyl acetate, shake violently for 5min;
3)Centrifuge at above 4000 r/min at 15 ℃ for 10 min;
4) Take 4ml upper organic phase, blow todry withnitrogen or air at 60 ℃
5) Dissolve dry residues in 1 mL of thedilutedredissolvingsolution, shake for 30s.
Fold of dilution of the sample: 1

5.3 Feed
1) Weigh 2± 0.05 gof the homogenized sample, add 8 mLAcetonitrile, shake properly for 2 min, centrifuge at above 4000 r/min at15 ℃ for 10 min
2)Take 2 mL supernatant into a new centrifuge tube, blow todry withnitrogen or air at 60 ℃.
3) Add 0.5 mL CHCl3, vortex for 20 sec, add 2 mL 1 M NaOH, vortex for 30 sec, centrifuge at above 4000 r/min for 5 min.
4) Take 1 mL supernatant, add 100 µL 6 MH3PO4, vortex for 5 sec
Dilute as following method for different samples
Compound feed-- take 50 µL sample, add 950 µL of the dilutedredissolvingsolution, vortex for 30 sec, take 50 µL for analysis;
Concentrated /Premixed feed--take 25 µL sample, add 975 µL of the dilutedredissolvingsolution, vortex for 30 sec, take 50 µL for analysis.
Fold of dilution of the sample:
Compound feed ---------100 Concentrated / Premixed feed ----------200

5.4 Urine
1. Take 2 mL urine into centrifuge tube, centrifuge at above 4000 r/min at room temperature (20-25) for 10 min, stop when it is clear.
2. Transfer 1 mL clear urine into centrifuge tube, add 1 mL 1 M NaOH, shake vigorously for 5 min
3. Add 100 µL 6 MH3PO4, vortex for 30 sec
4. Add 8 mLCHCl3 for extraction, shake properly for 5 min, centrifuge at above 4000 r/min at15℃ for 10 min.
5. Remove the upper layer (water phase), take 4 mL of the lower layer, blow todry withnitrogen or air at 60 ℃.
6. Dissolve dry residues in 3 mL of thedilutedredissolvingsolution, shake for 30s.
7. Take 50 µL for analysis.
Fold of dilution of the sample: 6

6. ELISA procedures

6.1Instructions
1 Bring all reagents and micro-wellstrips to the room temperature (20-25 ℃) before use.
2 Return all reagentsto2-8 ℃ immediately after use
3 The reproducibility of theELISA analysis, to alargedegree, depends on the consistencyof platewashing.The correct operationof platewashing is the keypoint in theproceduresofELISA;
4 For theincubationatconstant temperatures, all the samples and reagents mustavoid light exposure, andeach microplate should be sealed by the covermembrane.

6.2Operation procedures
1. Take out the kit from the refrigerated environment.Take out all thenecessary reagents from the kit and placeattheroom temperature (20-25℃) for at least 30 min. Note that eachliquid reagent must be shaken to mix evenly beforeuse
2. Take the required micro-well strips and plateframes. Re-sealed the unused microplate,stored at 2-8 ℃,not frozen
3. Solution preparation:dilute the20× concentratedwashing buffer with thedeionized waterto800 mL (or just to the required volume) for use
4. Numbering:number themicro-wells according tosamples andstandardsolution; each sample andstandard solution should be performed in duplicate;record their positions.
5. Add 50 µL of the sample or standard solution to separate duplicate wells; add 50 µL of the antibody working solution into each well. Seal the microplate with the cover membrane, andincubate at 37℃for 30 min.
6. Pour the liquid out of the microwells, add 250 µL/well ofwashing buffer for 10 sec, repeat four to five times, thenflap to dry withabsorbent paper(if there are thebubbles after flapping, cut them with the clean tips).
7. Add 100 µL enzyme conjugate into every well, seal the microplate with the cover membrane,react at 37℃ for 30 min, continue as described in 6
8. Coloration: add50 µL of thesubstrate Asolution and then50 µL of theB solution into each well.Mix gently by shaking the plate manually, seal the microplate with the cover membrane andincubate at 37 ℃ for 15 min atdark for coloration.
9. Determination: add50 µL of thestop solution into each well.Mix gently by shaking the plate manually.Set thewavelength of themicroplatereader at 450 nm to determine the OD value (Recommend to read the OD value at thedual-wavelength 450/630 nmwithin 5 min)

7. Result judgment

There are two methods to judgethe results; the first one is therough judgment, while the second is thequantitative determination.Note that the OD value of the samplehas anegative correlation with the content ofDiethylstilbestrol (DES).
7.1Qualitative determination
Theconcentration range (ng/mL) can be obtained from the comparison theaverageOD value
of the sample with that of the standard solutin.Assuming that the OD value of the sampleⅠ is 0.3, and that of the sampleⅡ is1.0, while those of the standard solutions are as the followings:2.243 for 0 ppb,1.816 for0.1ppb, 1.415 for0.3 ppb,0.74 for0.9ppb, 0.313 for2.7 ppb and0.155 for8.1 ppb, accordingly the concentration range of the sampleⅠis2.7 to8.1ppb, and that of the sampleⅡ is 0.3to 0.9ppb. (multiplied bythe corresponding dilution fold)

7.2 Quantitative determination
The mean values of the absorbance values is equivalentto the percentage of the average OD value (B) of the sample and the standard solutiondivided by the OD value (B0) of thefirststandardsolution(0 standard) andsubsequentlymultiplied by 100%, that is,

B—the average (double wells) OD value of the sample or the standard solution
B0—the average OD value of the 0ng/mL standardsolution
Draw thestandard curvewith theabsorption percentages of the standard solutions and thesemilogarithm values of the Diethylstilbestrol(DES) standard solutions(ng/mL) asY-and X-axis, respectively.Read thecorrespondingconcentration of the sample from thestandard curve byincorporating itsabsorption percentageinto the standard curve.The resulting value issubsequently multiplied bythe corresponding dilution fold, thusfinally obtaining theDiethylstilbestrol (DES)concentrationin the sample.
Using theprofessionalanalyzingsoftware of thiskit will bemore convenient for theaccurate and rapid analysisof a large amount ofsamples. (Please contact us for thissoftware)

8. Precautions

1. Theroom temperature below25℃ orthetemperature of the reagents andthe samples being notreturned to the room temperature (20-25 ℃) will lead to a lowerstandard OD value
2. Dryness of the microplate in the washingprocess will be accompaniedbythe situationsincluding the non-linearstandardcurves and theundesirable reproducibility; So continue to next step immediately after washing
3. Mix evenly, otherwise there willbe theundesirable reproducibility
4. The stop solution is the2 M sulfuric acid solution,avoid contacting with the skin;
5. Do not usethe kit exceeding itsexpiry date.The use of diluted or adulteratedreagents from the kits will lead to the changes inthesensitivity and thedetecting OD values. Do not exchangethereagents from the kits ofdifferent lot numbers touse
6. Put the unused microplate into anauto-sealing bag to re-seal it.Thestandard substance andthecolorless color former is light sensitive, and thus they cannot be directly exposed to the light
7. Discard thecoloration solution with anycolor that indicates thedegeneration of this solution.The detecting value of the0 standard solution of less than 0.5 (A450 nm< 0.5 ) indicates itsdegeneration
8. The optimum reaction temperatureis 37℃,and too high or low temperatures will result inthe changes in thedetectingsensitivity andOD values.
 

9. Storage andexpiry date

Storage: store at2-8 ℃, not frozen.
Expiry date: 12 months; date of production is on box.
Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.

Q1: When will it be shipped?
A1: We will ship the goods for you as soon as possible within 7 working days after receiving the payment. (In case of external factors such as the epidemic, the delivery may be delayed)

Q2: Does it support OEM/ODM?
A2: It can be supported, but the specific quantity needs to be more than 100,000 pieces, which is convenient for customized products.

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A3: We have nationally certified ISO9001 and ISO13485. Our production process conforms to standard procedures to ensure optimum product quality.

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