Doxycycline ELISA Test Kit

Catalog No. LSY-10051

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Doxycycline in the sample. The coupling antigens are pre-coated on the micro-well stripes. The Doxycycline in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti- Doxycycline antibody. 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 Doxycycline in it. This value is compared to the standard curve and the Doxycycline concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 0.2 ppb

Incubation Temperature: 25℃

Incubation Time: 30min—15min

Detection limit:

Tissue(method 1), Honey,Milk (method 1) about 5ppb

Tissue(method 2),Milk (method 2) about 10ppb

Serum about 2ppb

Note: ppb=ng/mLor ng/g

Cross-reaction rate:

Doxycycline  100%

Tetracycline 100%

Minocycline 100%

Chlortetracycline 180%

Oxytetracyline 40%

Recovery rate:  90±15%

3. Components

1） Micro-well strips: 12 strips with 8 removable wells each

2） 10× concentrated standard solution: 0 ppb, 2ppb, 6ppb, 18ppb, 54ppb (0.5ml/bottle)

3） 11X Concentrated Enzyme conjugate (0.7 mL)

4） Enzyme conjugate dilution (7 mL)

5） Substrate A (7 mL)

6） Substrate B (7 mL)

7） Stop solution (7 mL)

8） 20× concentrated washing buffer (30 mL)

9） 20× concentrated redissolving solution (10 mL)  

4. Materials required but not provided

1) Equipments: microplate reader (450nm, 630nm), rotary evaporator/nitrogen-drying device, homogenizer, oscillator, centrifuge (4000g and above), balance (a sensibility reciprocal of 0.01 g), measuring pipets, incubator (adjustable 25℃、37℃、60℃),timer

2) Micropipettors: single-channel 20~200 µL and 100~1000 µL, and multi-channel 30~300 µL;

3) Reagents: deionized water, HCl, N,N-Dimethylformamide(DMF) 

5. Sample pre-treatment

Instructions

The following points must be dealt with before the pre-treatment of any kind of sample:

1) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

2) Before the experiment, each experimental equipment must be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results.

Solution preparation before sample pre-treatment:

1) Dilute 20× concentrated redissolving solution with deionized water at 1:19(1 part concentrated redissolving solution + 19 parts deionized water ).  

2) Washing buffer: 1 part 20× concentrated washing buffer + 19 parts deionized water

3) 1M HCl: take 1ml concentrated HCl, add 11ml deionized water to dissolve and mix it evenly.

5.1Tissue (Method 1)

1. Take 1± 0.01 g of the homogenized tissue sample into 10 mL centrifuge tube, add 5 mL deionized water, shake with oscillator for 2min, centrifuge at above 4000g for 5 minutes.

2. Take 500ul up-layer clear liquid, add 500ul diluted redissolving solution, shake with oscillator for 2min;

3. Take 50 µL for analysis.

Fold of dilution of the sample:12  

5.2 Tissue (Method 2)

4. Take 1± 0.01 g of the homogenized tissue sample into 10 mL centrifuge tube, add 1 mL N,N-Dimethylformamide(DMF), shake with oscillator for 5min, to make sample completely dispersed, fully contact with the organic phase.

5. Centrifuge at above 4000g for 10 minutes.

6. Take 100ul up-layer clear liquid, add 900ul diluted redissolving solution, shake with oscillator for 10min;

7. Take 50 µL for analysis.

Fold of dilution of the sample:20      

5.3 Honey

1) Take 1± 0.01g honey sample into 10 mL centrifuge tube; Add 2ml deionized water, shake with oscillator fully for 1 min to dissolve; take 100ul dissolved solution, add 400ul diluted redissolving solution, oscillator for 10S to mix it evenly;

2) Take 50ul for analysis immediately.

Fold of dilution of the sample:10

5.4 Milk (method 1)

1) Thaw the collected liquid milk sample, then put at room temperature for 30min;

2) Put tips in the down-layer of milk, take 1ml sample into 2ml centrifuge tube(note: do not take the up-layer cream);

3) Add 50ul 1M HCl, shake strongly for 1min(or oscillator for 30s);

4) Centrifuge at above 4000 r/min at room temperature (20 - 25 ℃) for 10 minutes;

5) Take up-layer clear liquid 50ul into another clean centrifuge tube(note: do not take the up-layer cream), add 450ul diluted redissolving solution, shake strongly for 1min(or oscillator for 30s);

6) Take 50ul for analysis immediately.

Fold of dilution of the sample:10

5.5 Milk (method 2)

1) Take 50ul liquid sample into 1950ul diluted redissolving solution; oscillator fully for 1min evenly;

2) Take 50ul for analysis immediately.

Fold of dilution of the sample:40

5.6 Serum

1. Take 100ul sample, add 700ul diluted redissolving solution, shake with oscillator for 10s;

3. Take 50 µL for analysis immediately.

Fold of dilution of the sample:8  

6.ELISA procedures

Instructions

1. Bring all reagents and micro-well strips to the room temperature (20-25℃).

2. Return all reagents to 2-8℃immediately after use.

3 .The reproducibility of the ELISA analysis, to a large degree, depends on the consistency of plate washing. The correct operation of plate washing is the key point in the procedures of ELISA.

4. For the incubation at constant temperatures, all the samples and reagents must avoid light exposure, and each microplate should be sealed by the cover membrane.

Operation procedures

1. Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate; record their positions. The Unused Micro-well strips need to seal and store at 2~8℃to avoid metamorphism.

2. Dilute the 5 concentrated standard solution separately: take 5 pieces of 2ml centrifuge tube, mark 0、0.2、0.6、1.8、5.4ppb accordingly, add 900µL the diluted redissolving solution into each tube, then add the five 10X concentrated standard solution into above 5 tubes accordingly, 100ul/tube. The 5 diluted standard solutions will be: 0—0、0.2—2、0.6—6、1.8—18、5.4—54.

3. Enzyme conjugate preparation: take 1 part 11X Concentrated Enzyme conjugate, add 10 parts Enzyme conjugate dilution, dilute at 1:10.

4. Add 50µL of the sample or standard solution to separate duplicate wells, then add enzyme conjugate, 50 µL each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, andincubate at25 ℃ at darkfor30 minutes.

5. Pour liquid out of microwell, add 250 µL/well of washing buffer for 15-30 seconds, repeat three to four times, then flap to dry (if there are the bubbles after flapping, cut them with the clean tips).

6. Coloration: add 100 µL mixture of the substrate A and substrate B into each well (Note: mix Substrate A and Substrate B at 1:1, the mixture should be used in 10min, never use metal container or metal to stir the solution, otherwise the substrate may be invalid.). Mix gently by shaking the plate manually, andincubate at25 ℃ for15 minutes at dark for coloration.

7. Determination: add 50 µL of the stop solution into each well (The substrate color from blue to yellow, it means the stop succeeds). Set the wavelength of the microplate reader at 450 nm to determine the OD value (Recommend to read the OD value at the dual-wavelength 450/630 nm within 5 minutes).

7. Result judgment

There are two methods to judge the results; the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with the content of Doxycycline in the sample.

7.1 Qualitative determination

The concentration range (ng/mL) can be obtained from the comparison the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sample Ⅰ is 0.3, and that of the sample Ⅱ is 1.0, while those of the standard solutions are as the followings: 2.243 for 0ppb, 1.816 for 0.2ppb, 1.415 for 0.6ppb, 0.74 for 1.8ppb, 0.313 for 5.4ppb, accordingly the concentration range of the sampleⅠis 1.8 to 5.4ppb, and that of the sample Ⅱ is 0.2 to 0.6 ppb.

7.2 Quantitative determination

The mean values of the absorbance values is equivalent to the percentage of the average OD value (B) of the sample and the standard solution divided by the OD value (B0) of the first standard solution (0 standard) and subsequently multiplied 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 0 ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the Doxycycline standard solutions (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the sample from the standard curve by incorporating its absorption percentage into the standard curve. The resulting value is subsequently multiplied by the corresponding dilution fold, thus finally obtaining Doxycycline concentration in the sample.

Using the professional analyzing software of this kit will be more convenient for the accurate and rapid analysis of a large amount of samples. (Please contact us for this software)

8. Precautions

1. Read the instruction carefully before use.

2. Take out all the necessary reagents from the kit and return to room temperature (25±2℃) (note: about 1h)

3. Note that each liquid reagent must be shaken to mix evenly before use, avoid bubble.

4. The tips is disposable, do not repeat use it to avoid cross pollution.

5. Do not use kit out of date, do not mix use reagents from different lots.

6. Analysis the sample immediately after sample treatment, otherwise it may affect the result.

7. Both substrate A and substrate B is Colorless transparent liquid, if it becomes blue color or become blue color after mixing before use, it means the reagents is polluted or deteriorated.

8. Add sample quickly under the premise of accuracy, to avoid time difference affect the result.

9. The stop solution has H2SO4, avoid contacting with the skin.wash with large volume water if splash on skin or clothes. If contact eyes, go the the hospital after washing.

9. Storage and expiry date

Storage: store at 2-8 ℃, not frozen.

Expiry date: 12 months; date of production is on the box.