1, ready to work

1. Kit preparation

Place the kit even at room temperature balance start operation after 30 min.

2, need to bring their own consumables and equipment

(1) Microplate reader, constant temperature oscillator (or constant temperature incubator), washing machine, vortex oscillator, timer;

(2) high precision pipetting, and disposable suction (0.5-10 (including L, 10-100 (including L, including 30-300 L, 100-1000 (including L);

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

(4) absorbent paper, EP tube, disposable gloves.

3. Reagent preparation

(1) Preparation of lotion (1×)

One part of the wash solution (20×) was taken and 19 parts of deionized water was added to make the working concentration wash solution (1×). 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) the color of liquid preparation

An equal volume of chromogenic solution A and B was mixed and placed in the dark after mixing. (note: the time is not too long, generally 10 min preparation before use. Such as color liquid mixture has turned blue, please do not use).

(3) Preparation of standard substance

The standard dilution with sample diluent to 10 ng/mL, then use 2.5 times than dilution method standard (each experiment using new preparation of 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 50μL standard/blank (sample diluent)/sample to each well, then add 50μL antibody working solution, seal plate with plate sealing membrane, and then place at 37℃ in the dark for 30min.

(Note: the standard must be added first, if the antibody is added first, it will directly react with the antigen on the plate; Incubate without seal plate or block in the process of incomplete, lead to the reaction liquid evaporation, cause the experiment error; Avoid light exposure during all incubations.)

(3) Washing plate: After the incubation is completed, remove the sealing plate membrane carefully, discard the liquid in the hole, wash the plate 3 times (250μL/ 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 addition of lotion (1×), it was left for 30s and slightly shaken. When patting dry, pay attention to each new absorbent paper or patting dry on a clean area of the paper)

(4) two resistance to incubate of enzyme mark: every hole to join enzyme mark two resistance, including 100 L/hole, a sealing plate membrane sealing plate, and then at 37 ℃ for incubate 30 min.

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

(6) color: preconfigured color liquid press, 100 (including L/holes in enzyme label plate, seal plate membrane sealing plate, with 37 ℃ avoid light let stand incubate for 15 min.

(7) termination: join terminated liquid, 100 (including L/hole, reading after color evenly.

(Note: It is recommended to set 5-10s vibration in the reading program of the microplate reader)

(8) Reading: Put the microplate into the microplate reader, set the wavelength to double wavelength 450/630nm, and read the absorbance value. The determination should be completed within 20min after termination.

3. Data processing

1. Calculation of absorbance value

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

2. Calculation of percentage 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: Mean absorbance value of standards or samples

B0:0 ng/mL the average absorbance of the standard value

3. Drawing and calculation of the standard curve

The standard curve was drawn with the percentage absorbance value of the standard as the ordinate Y and the concentration value as the abscissa X. It is recommended to use the four-parameter Logistic mathematical model to fit the 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 gentamicin in the sample was multiplied by its corresponding dilution.

1. The four-parameter Logistic mathematical model is recommended for the fitting equation. ② Using the recommended double wavelength correction method as much as possible, using 630nm wavelength for correction, OD450-OD630 is the corrected OD value, which can be directly used for calculation, or according to the data quality, and then blank correction. If there is no dual-wavelength microplate reader, after reading OD450 data, the data quality should be judged before blank correction.

Gentamicin, a basic compound extracted from the fermentation broth of monospore genus of actinomycetes, is a commonly used aminoglycoside antibiotic and is widely used in the preparation of culture medium. It is mainly used to treat bacterial infections in clinic. Gentamicin has neurotoxicity, nephrotoxicity and ototoxicity. Its residues in animal food and biological drugs will affect human health and even cause allergic reactions. European and American countries and China require its limited use

? The gentamicin quantitative detection kit adopts indirect competitive ELISA method. The pre coated gentamicin antigen on the micro well strip competes with the residual gentamicin in the sample to bind enzyme labeled gentamicin monoclonal antibody. Then the enzyme labeled secondary antibody is added, and then the TMB substrate is added to develop color. The absorbance value is detected by a microplate reader. The absorbance value is negatively correlated with the gentamicin content in the sample. The operation time of this kit is only about one hour, and the linear range is 0.1ng/ml-10ng/ml

? When doing the linearity verification of gentamicin raw material, it is recommended to dilute the gentamicin raw material to the linear range of the kit and then determine it. Dilute it to at least 5 concentrations according to a certain proportion. The detection concentration is linearly related to the dilution multiple&Nbsp

? Specificity should be verified for other substances present in the process. It is suggested to use high concentration and low concentration quality control samples and add increasing concentrations of relevant interfering substances for specificity investigation. The matrix without analytes should also be measured at the same time. The accuracy of the quality control sample should be within a certain range, and the measured value of the matrix without analytes should be lower than the lower limit of quantification.

Product Features:

Strong Specificity

No cross reaction with penicillin, streptomycin sulfate, kanamycin and ampicil

High Sensitivity: The sensitivity of the kit is 0.1ng/ml

Convenient and Efficient

Using two-step method, the result can be obtained in about 1 hour

Excellent Performance

good linearity of standard curve, high accuracy, Good repeatability

Product Performance Index:

detection limit: <0.1 ng/ml

limit of quantification: 0.1 ng/ml

linear range: 0.1-10 ng / ml

accuracy (spiked recovery): 70% - 130%?

accuracy (measurement deviation): ≤ 15%

repeatability (intra batch difference): ≤ 15%

Specificity:

1. All components in the reagent kit must be restored to room temperature (20-25 ℃) before use& Nbsp
2. 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<3. 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<4. 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 flocs, which is a normal phenomenon and does not affect the final reading result to a slight extent& Nbsp8. Only by strictly following the operating methods in the manual and using all the reagents matched with this reagent kit can the best detection effect be guaranteed& Nbsp10. 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
5. The termination solution in this reagent kit is an acid solution, and special attention should be paid during operation& Nbsp
6. Operators should wear personal protective equipment, such as laboratory clothing, gloves, masks, and goggles.