I. Preparation

1, the kit

The kit components were allowed to equilibrate at room temperature for 30min before operation.

2, need to bring your own supplies and equipment

(1) enzyme standard instrument, thermostatic oscillator (or constant temperature incubator), washing machine, vortex generator, timer;

(2) high-precision pipettor and disposable suction head (0.5-10uL, 10-100uL, 30-300uL, 100-1000uL);

(3) deionized water (for preparation of lotion (1×));

(4) the blotting paper, EP tubes, disposable gloves;

3, the reagent preparation

(1) Lotion (1×) was prepared

Apply lotion (1 x 20), plus 19 deionized water made into job concentration lotion (1 x). If there are crystals formed in the wash solution (20×), it should be placed at room temperature or 37℃ water bath with gentle shaking, and diluted after the crystals are completely dissolved. Lotion (20 x) should not use up at 2-8 ℃ storage.

(2) Preparation of chromogenic solution

To color A and color liquid B volume mixing, avoid light place after blending. (note: the time is not too long, generally 10 min preparation before use. Do not use if the mixed chromogenic solution has turned blue.

(3) Preparation of standard substance

The standard was diluted to 5ng/mL with sample diluent, and then the standard was prepared by 2.5-fold dilution (each experiment used a newly prepared standard solution).

4, sample preparation

The samples were returned to room temperature and mixed before adding the samples. If users need to dilute the high concentration of standard of form a complete set of product samples or box, the box sample diluent can be used for dilution; For cell samples, it is recommended to centrifuge at 3000rpm/min for 5min before detection, and take the supernatant for detection.

2, the operation process

(1) will be resumed kit even at room temperature 30 min, from a balance to room temperature aluminum foil bag to take out the enzymes needed for test standard strip, with marker pen mark lath order (recommended for determination of complex pore), the remaining strip seal plate with film back again after sealing plate is aluminum foil bag, sealed and stored in 2-8 ℃. (Note: the slats are easy to fall off in the follow-up process, and pay attention to mark them.)

(2) Sample incubation: Add 50uL standard/blank (sample diluent)/sample to each well, then add 50uL antibody working solution, seal plate with plate sealing membrane, and then place at 25℃ in the dark for 30min. (note: you must first add standard, if you add antibodies will directly and board antigen response; Incubate without seal plate or block in the process of incomplete, lead to the reaction liquid evaporation, cause the experiment error; All light) to avoid as far as possible in the process of incubation

(3) Washing plate: After the incubation is completed, carefully remove the sealing plate membrane, discard the liquid in the hole, wash the plate 3 times (250uL/ well) with wash solution (1×), and pat the residual liquid in the sample hole dry. (Note: if the hand washing plate is used, the washing solution (1×) needs to be suspended, and the tip of the gun is best not to touch the inner wall of the hole; After each join lotion (1 x) let stand for 30 s (at the end of the incubation process under mild concussion); Shoot every time when pay attention to in the new absorbent paper or dry clean area on the paper)

(4) two resistance to incubate of enzyme mark: every hole to join enzyme mark two resistance, 100 ul/hole, a sealing plate membrane sealing plate, and then placed in the 25 ℃ avoid light let stand incubate for 30 min.

(5) Washing plate: the method is the same as step (3).

(6) Color development: the pre-configured color development solution was added to the microplate according to 100uL/ well, the plate was sealed with the plate sealing membrane, and the plate was left to incubate at 25℃ in the dark for 15min.

(7) Termination: Add termination solution, 100uL/ well, the color can be read after uniform. (Note: It is recommended to set 5-10s vibration in the reading program of the microplate reader)

Reading (8) : the enzyme label plate in the enzyme standard instrument, set the 450/630 nm wavelength for the double wavelength, read the absorbance value, determination shall be completed within 20 min after termination.

3. Data processing

1, the absorbance value calculation

The absorbance values of each standard or sample ranged from OD450nm to OD630nm.

2, however the computing of absorbance

The average absorbance value of each standard or sample (double well) divided by the average absorbance value of 0ng/mL standard and multiplied by 100% is the percentage absorbance:

Percentage of absorbance (%) = B/B0 by 100%

B: the average absorbance of the standard or sample values

B0: Mean absorbance value of 0ng/mL standard

3. Drawing and calculation of the standard curve

With standard percentage absorbance value as the ordinate Y, standard chroma value is stere of abscissa X, drawing standard curve, it is recommended to use four parameter mathematical model fitting Logistic equation:

Y=((A-D)/(1+(X/C)^B))+D?

The percentage absorbance value of the sample was substituted into the standard curve, the corresponding concentration value of the sample was read out, and the actual concentration of kanamycin in the sample was multiplied by its corresponding dilution.

Kanamycin amino glucoside is a kind of antibiotics, used in the treatment of animal diseases, strain screening, and cell and gene therapy drug in the preparation of raw materials, since it has nerve toxicity and kidney toxicity, damage the eighth cranial nerve, cause the vestibular and cochlear damage. Residues in animal food and biological drugs can affect human health and even cause allergic reactions. European and American countries and our country require its limited use.

? ? ? ? Kanamycin quantitatively detected by indirect competitive ELISA method, coupling in the process of the package is on the microporous article kanamycin antigen and the drug residues in the sample card competition combined with enzyme labeled kanamycin resistance of monoclonal antibody, then join enzyme mark 2, again by adding TMB chromogenic substrates, enzyme standard instrument were used to detect absorbance value, Absorbance values with the sample content of kanamycin in negative correlation. The operating time of this kit is only about one hour, and the linear range is 0.05ng/ ml-5ng /mL.

? ? ? ? In the linear verification of kanamycin raw material, it is recommended to dilute kanamycin raw material to the linear range of the kit before determination, and dilute to at least 5 concentrations according to a certain proportion. The detection concentration is linearly related to the dilution.

? ? ? ? Specificity should be validated against other substances present in the process. It is recommended to use high concentration and low concentration quality control samples, and add increasing concentrations of related interfering substances for specificity investigation. Did not join the analyte matrix should be measured at the same time. Quality control accuracy should be within a certain range, and did not join the analyte measured value of the matrix should be lower than the quantitative limit.

Product Features:

Strong specificity

with penicillin, streptomycin, gentamicin sulfate and ammonia benzyl no cross reaction.

High sensitivity: kit sensitivity of 0.05 ng/mL.

Convenient and efficient

use the two-step method, the results in a 1 hour or so.

Excellent performance

standard curve and high accuracy, good repeatability.

Product performance index:

Detection limit: < 0.05 ng/mL

Loq: 0.05 ng/mL

The linear range was 0.05-5 ng/mL

Accuracy (recovery rate) : 70%-130%

Accuracy (measurement deviation) : ≤15%

Difference in repetitive (batch) : ≤15%

Specificity:

1. The optimal reaction temperature for this reagent kit is 25 ℃. If the temperature is too high or too low, it will cause changes in the detection absorbance and sensitivity& Nbsp3. All components should be thoroughly mixed before use, and the standard sample needs to be briefly centrifuged for 5 seconds to concentrate all the liquid on the tube wall and lid at the bottom of the tube; Immediately return all reagents to 2-8 ℃ after use& Nbsp<4. The reagent kit must be used within its validity period, and corresponding standard curves must be prepared for each experiment. It is not recommended to use different batches of related reagents in mixed batches& Nbsp
2. When adding liquid to the microplate, be careful not to touch the bottom of the microplate to prevent damage to the coating layer. Timely replace the sampling tank and suction head between different samples and steps to avoid cross contamination& Nbsp
3. When the Flat noodles is dried after washing, pay attention to prevent the Flat noodles from falling off, and the sealing film should not be reused& Nbsp
4. During color rendering, high concentrations may produce black flocculent matter, which is a normal phenomenon and does not affect the final reading result to a slight extent& Nbsp
5. When reading, pay attention to checking whether the detection wavelength and fitting equation selection are correct& Nbsp
6. Only by strictly following the operating methods in the manual and using all the reagents matched with this kit can the best detection effect be guaranteed& Nbsp11. Our company is only responsible for the reagent kit itself and is not responsible for sample consumption caused by the use of the reagent kit. Users are advised to fully consider the possible usage of the sample and reserve sufficient samples before use& Nbsp
7. The termination solution in this reagent kit is an acid solution, and special attention should be paid during operation
8. Operators should wear personal protective equipment, such as laboratory clothing, gloves, masks, and goggles.