CN102585006B - Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin - Google Patents

Abstract

The invention discloses a specific monoclonal antibody capable of recognizing neomycin, amikacin and paromomycin and an ELISA for detecting neomycin, amikacin and paromomycin Methods and kits. The monoclonal antibody of the present invention is secreted by hybridoma cell EDC/5G04, and the hybridoma cell is preserved in China Center for Type Culture Collection, and its preservation number is CCTCC NO: C201144. The enzyme-linked immunoassay method of the present invention includes the steps of preparation of immunogen, coating source, antibody, sample processing and detection, and the like. Compared with the prior art, the monoclonal antibody prepared by the invention can simultaneously recognize neomycin, amikacin and paromomycin, thereby improving the detection efficiency of the prior art; the animal tissue sample processing method is simple and the time is short. The ELISA method and the kit of the invention also have the characteristics of high detection sensitivity, good precision and good accuracy.

Description

Monoclonal antibody and ELISA methods and kits for the detection of neomycin, amikacin and paromomycin

technical field

The present invention relates to a monoclonal antibody capable of recognizing neomycin, amikacin and paromomycin, and an ELISA method and reagents for detecting neomycin, amikacin and paromomycin box.

Background technique

Aminoglycoside (AMGs) antibiotics are a class of water-soluble alkaline antibiotics that are extracted from the culture fluid of Streptomyces or Micromonospora, or semi-synthesized from natural products. Currently commonly used in veterinary clinics are: streptomycin, gentamicin, neomycin, kanamycin, amikacin and so on. It shows a strong bactericidal effect on most Gram-negative bacilli, and it also has an effect on Gram-positive bacteria. The main sensitive bacteria are Enterobacter sensitive strains, and it is effective against Streptococcus, Hepatitis, Clostridium, and Rickettsia. However, because AMGs are easy to accumulate in the renal cortex and inner ear perilymph, causing ototoxicity and nephrotoxicity, and the microbial drug-resistant strains produced by aminoglycoside inactivating enzymes are more serious, the detection of AMGs in animal-derived foods has increasingly attracted people. attention. In view of the toxic and side effects of AMGs, each country has made strict regulations on the maximum residue limits of AMGs. Enzyme-linked immunosorbent assay (ELISA) has been widely used in the field of rapid detection of AMGs because of its simplicity, rapidity, sensitivity, and specificity. However, the current ELISA methods mainly focus on single residue detection, and there are few ELISA methods that use one antibody to simultaneously detect AMGs with different structures. Therefore, the establishment of an ELISA method that can detect a variety of AMGs is of great significance for improving the rapid detection of this class of drugs.

The invention patent with the application number 200610171540.1 discloses an enzyme-linked immunosorbent assay kit for detecting neomycin, which uses the carbodiimide (EDC) method to couple neomycin and human serum albumin to obtain an immunogen; Rabbit serum albumin is used as a carrier, neomycin is used as a hapten, and neomycin coating is obtained by coupling with carboxymethylhydroxylamine-carbodiimide method. The prepared polyclonal antibody and monoclonal antibody can only recognize neomycin. Mycin, and the detection time of the sample is longer. The invention patent with the application number of 200710064347 discloses an enzyme-linked immunosorbent assay kit and method for detecting neomycin drug. The patent adopts the method of directly activating protein to couple neomycin and bovine serum albumin (BSA) To obtain the immunogen, the same method was used to couple neomycin and thyroid protein to obtain the coating agent. The monoclonal antibody prepared could also only specifically recognize neomycin, and the sample processing time and reagents were not optimized.

Contents of the invention

The first object of the present invention is to provide a monoclonal antibody capable of simultaneously recognizing neomycin, amikacin and paromomycin.

The second purpose of the present invention is to use the monoclonal antibody to establish an ELISA method that can be used for the detection of neomycin, amikacin and paromomycin.

The third object of the present invention is to provide a kit for the detection of neomycin, amikacin and paromomycin.

The fourth object of the present invention is to provide the application of the monoclonal antibody in the preparation of an ELISA kit for detecting neomycin, amikacin and paromomycin.

The fifth object of the present invention is to provide the application of the kit containing the monoclonal antibody in the detection of neomycin, amikacin and paromomycin residues in animal tissues.

The present invention is realized through the following technical solutions:

A monoclonal antibody capable of recognizing neomycin, amikacin and paromomycin, which is secreted by the hybridoma cell EDC/5G04 with the preservation number CCTCC NO: C201144.

The above-mentioned hybridoma cell EDC/5G04 is preserved in the China Center for Type Culture Collection (CCTCC) located in Wuhan University, Wuhan City, Hubei Province, and its preservation number is CCTCC NO: C201144.

The immunogen used was prepared by conjugating the hapten neomycin with bovine serum albumin.

Further, the present invention provides an enzyme-linked immunoassay method for simultaneously detecting neomycin, amikacin and paromomycin, which method includes preparation of immunogen, coating agent, antibody, and sample processing and detection, etc. The steps are as follows:

(1) coupling the hapten neomycin with bovine serum albumin (BSA) to obtain an immunogen;

(2) Coupling the hapten neomycin with ovalbumin (OVA) to obtain the coating former;

(3) Using the immunogen in step (1) to immunize mice, and obtain the hybridoma cell EDC/5G04 with the preservation number CCTCC NO: C201144 through cell fusion and screening;

(4) Prepare the monoclonal antibody with the hybridoma cell EDC/5G04 whose deposit number is CCTCC NO: C201144;

(5) Coating the solid phase carrier (such as a microtiter plate) with the coating original of step (2);

(6) extracting, incubating, centrifuging and diluting the sample to be tested with phosphate buffered saline solution (PBS, 0.1M, pH 10-11) to obtain a solution of the test substance;

(7) ELISA is carried out to the analyte solution of step (6),

in:

The composition and ratio of phosphate buffer solution (0.1M, pH10～11) are: NaCl 20.0g, KH ₂ PO ₄ 0.4g, Na ₂ HPO ₄ 12H ₂ O 13.4g, KCl 0.5g, with a small amount of ultra-pure Dissolve in water, then dilute to 500mL.

In the present invention, the above-mentioned monoclonal antibody and coating agent are used as core reagents and combined with other conventional reagents to produce an enzyme-linked immunoassay kit capable of detecting neomycin, amikacin and paromomycin. The immunological method realizes the ELISA detection of neomycin, amikacin and paromomycin.

The main advantages of the present invention are:

1. The monoclonal antibody prepared by the present invention can simultaneously recognize neomycin, amikacin and paromomycin, while the existing patent documents can only recognize neomycin alone.

2. The tissue sample processing method involved in the present invention is simple, does not require expensive instruments, does not require organic reagents, has no health hazards to the operator, and only requires an ordinary centrifuge, which is suitable for operation at the grassroots level.

3. Compared with the prior art, the tissue sample processing time is short, the detection time is short, and the detection efficiency is high.

Description of drawings

Fig. 1 is the mass spectrogram of the carrier protein BSA of the present invention detected by the matrix-assisted laser desorption method.

Fig. 2 is a mass spectrogram of the matrix-assisted laser desorption detection of the immunogen of the present invention, which is used in conjunction with Fig. 1 to illustrate the coupling effect of the hapten neomycin and BSA.

Fig. 3 is the indirect competition ELISA reaction curve of monoclonal antibody of the present invention and neomycin (NEO) standard substance, and X axis is neomycin (NEO) standard solution concentration logarithmic value, and Y axis is the neomycin standard substance solution The optical density value was divided by the "zero" well optical density value (B/B0).

Detailed ways

Below by embodiment the present invention will be further described.

The preparation of embodiment 1 immunogen and coating former

1.1 Synthesis of neomycin-BSA

Weigh 10.0 mg of neomycin and 100.0 mg of BSA and dissolve them in 20 mL of PBS solution (pH 7.4), stir evenly, add 50.0 mg of EDC dissolved in 1 mL of pure water dropwise, and stir and react at room temperature for 8 hours. Finally, the reaction solution was transferred into a dialysis bag, dialyzed in PBS (pH7.4) solution at 4°C for 4 days, centrifuged and freeze-dried. As shown in Figures 1 and 2, the coupling was identified by matrix-assisted laser desorption (MALDI-TOF-MS), and stored at -20°C for future use.

1.2 Synthesis of neomycin-OVA

Weigh 20.0mg of neomycin and 200.0mg of OVA dissolved in 20mL of PBS solution (pH7.4), stir well, then slowly add 80.00mg of EDC dissolved in 1mL of pure water. The reaction was stirred at room temperature for 2 hours. Finally, the reaction solution was transferred into a dialysis bag, and dialyzed in PBS (pH7.4) solution at 4° C. for 4 days. Freeze-dry after centrifugation and store at -20°C for later use.

The preparation of embodiment 2 monoclonal antibody

Preparation of hybridoma cells: With reference to Yang Hanchun's "Animal Immunology", the immunogen neomycin-BSA prepared in Example 1 was used to immunize Balb/C mice (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention), and the immunization procedure was as follows: : For basic immunization, the immunogen was emulsified with an equal volume of complete Freund's adjuvant, and then injected subcutaneously at multiple points on the back of the mouse, and then boosted immunization once every 2 weeks, and emulsified with incomplete adjuvant. Finally, intraperitoneal injection three days before fusion (preferably resting for one month after the end of immunization) for booster immunization, doubling the amount of antigen without adding adjuvant.

At the time of fusion, a Balb/C mouse that had undergone the final booster immunization was taken, sacrificed by bleeding from the eye socket (serum was collected, it was a positive serum), and sterilized by immersing in 75% alcohol for 5 minutes. Remove the mouse spleen aseptically, separate the splenocytes, and immunize splenocytes with freshly prepared SP2/0 myeloma cells (SP2/0 myeloma cells come from our laboratory) according to 1-2×107 SP2/0 and 108 splenocytes (1:10-1:15) in a 50mL centrifuge tube, resuspend the cells with 15mL RPMI-1640 base solution, centrifuge at 1500r/min for 5 minutes, and wash the cells once. In the centrifugation gap, put the culture medium in the warm bath, the water in the warm bath, the PEG in the warm bath, etc. into the ultra-clean bench. Then take out the sterilized absorbent paper, pour out the supernatant of the centrifuge tube containing myeloma cells and immune spleen cells, turn it upside down on the absorbent paper to control the dry water droplets, tap the bottom of the tube to loosen the cells. Turn on the timer, draw 0.8mL PEG with a 1mL straw, hold the centrifuge tube containing the mixed cells, place it in the water bath for a while, slowly add the PEG to the mixed cells, stir gently while adding, and add within 1 minute. When finished, continue stirring for 30 seconds. Use a pipette to take 10mL of base solution, slowly add it to the fused cells along the wall of the tube, shake gently while adding (do not pipette), add 1mL, 2mL, 3mL, 4mL respectively within 5 minutes, and finally add the base solution to 40mL, add When finished, cover the lid and invert several times to mix the cells evenly. Centrifuge at 800r/min for 5 minutes and discard the supernatant. Take the HAT medium containing the feeder cells, use a pipette to gently stir the fused cells in the centrifuge tube, drop drop by drop near the liquid surface into the serum bottle containing the feeder cells, stir and mix well, gently shake the cells Stir it up, never pipette. Mix by inversion. Then the cells were seeded on the cell culture plate, two drops per well, and cultured in the incubator. One fusion can inoculate 4-6 96-well plates. It can also be planted less according to the needs, generally calculated according to the number of SP2/0 cells, and the inoculation amount of each well contains about 104 SP2/0 cells. Culture at 37°C in a 5% CO ₂ incubator.

The day of fusion is counted as day 0, try not to move the cell plate for the first 3 days, and keep the environment in the incubator stable. On the 3rd day, add 1 drop of HAT complete medium to each well; on the 5th day, aspirate 1/2 of the culture supernatant (100 μL) from each well, and then add 1 drop of HT complete medium; 2-3/4 of the culture supernatant was replaced with HT complete medium after 7 days.

The cell colony to be fused grows to 1/10-1/5 of the culture well, and is screened by the established indirect ELISA method and indirect competitive ELISA method. Compared with the zero drug well, the OD value of the drug well can be suppressed as positive. According to the inhibition rate and the growth status of cell colonies, select 2 to 6 strongly positive cell wells with only 1-2 single colonies, and use the limited dilution method for cloning.

After 3-4 times of cloning, until the positive rate of cloning was 100%, the hybridoma cells secreting resistance to neomycin, amikacin and paromomycin were finally screened out. The average number of chromosomes in this cell line was 102.8 after chromosome counting. The applicant named the hybridoma cell EDC/5G04, and on June 29, 2011, it was sent to the China Type Culture Collection Center located in Wuhan University, Wuhan, Hubei Province for preservation, and its preservation number is CCTCCNO: C201144.

Preparation and identification of ascites monoclonal antibody: Several Balb/c mice were taken 7 days before inoculation, and each mouse was pretreated with 0.5ml Freund's incomplete adjuvant intraperitoneally injected. Use RPMI-1640 basal medium to suspend the cells expanded and cultured by the hybridoma cell EDC/5G04 with the preservation number CCTCC NO: C201144, adjust the number of cells to 1× ¹⁰⁶ cells/mL, and inoculate each mouse with 0.5ml intraperitoneally . Ascites was collected when the abdomen of the mice was obviously enlarged, the spirit became poor, and they were immobile when they were dying. According to the literature method (Zhu Liping, Chen Xueqing. Commonly used experimental methods in immunology. Beijing: People's Military Medical Publishing House, 2000), the monoclonal antibody was purified and obtained. The mouse Mab Isotyping Test Kit purchased from ROCKLAND Company was used to identify the subtype of the monoclonal antibody obtained in the present invention, and the result was mouse IgG1 subtype.

The establishment of embodiment 3 neomycin indirect competition ELISA detection method

3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

Carbonate buffer solution (pH9.6): Accurately weigh 1.59g of Na ₂ CO ₃ and 2.93g of NaHCO ₃ , dissolve in a small amount of ultrapure water, and dilute to 1000mL.

Washing solution (pH7.4): Accurately weigh 8.00g of NaCl, 0.20g of KH ₂ PO ₄ , 2.90g of Na ₂ HPO ₄ 12H ₂ O, 0.20g of KCl, dissolve in a small amount of ultrapure water, add 0.50mL of Tween 20, Dilute to 1000mL.

Phosphate buffer (pH7.4): Accurately weigh 8.00g of NaCl, 0.20g of KH ₂ PO ₄ , 2.90g of Na ₂ HPO ₄ ·12H ₂ O, 0.20g of KCl, dissolve in a small amount of ultrapure water, and set the volume to 1000mL.

Blocking solution: Accurately weigh 10.00 g of ovalbumin, add 1000 mL of phosphate buffer, stir and mix until the protein is completely dissolved.

Physiological saline: Accurately weigh 8.50g of NaCl, dissolve in a small amount of ultrapure water, and dilute to 1000mL.

Antibody diluent, enzyme-labeled antibody diluent and substrate solution were all provided by Wuhan Feiyuan Technology Co., Ltd.

Stop solution: Accurately measure 100mL of concentrated sulfuric acid and slowly add it dropwise to 800mL of ultrapure water.

3.2 Preliminary determination of coating original concentration and antibody working concentration

The first is to preliminarily select the combination of the coating source and the working concentration of the antibody by the method of square array titration. Use carbonate buffer to dilute the coated original neomycin-OVA to 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg/mL horizontally coated microtiter plate; EDC/5G04 monoclonal antibody Use phosphate buffer to dilute to 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000 and add vertically ELISA plate. The results of square matrix titration are shown in Table 1. The following combinations of original coating concentration and antibody working concentration were initially selected: (1, 1:8000), (1, 1:16000) and (2, 1:32000).

Table 1 EDC/5G04 monoclonal antibody array titration

3.3 Determination of optimal coating source concentration and antibody working concentration

Determination of the optimal coating concentration: the combination of coating concentration and antibody dilution selected by square matrix titration was used to draw the inhibition curve, and the concentration of neomycin standard was set to 0, 1, 3, 5, 7, 9 μg/mL, and zero The OD value and IC ₅₀ value of the drug well are shown in Table 2. The ratio of antigen to antibody is the key to affect its sensitivity. If there is an excess of antigen or antibody, the IC ₅₀ will be high. According to the IC ₅₀ value, the OD value of the zero drug well and the linear correlation coefficient of the inhibition curve, the optimal coating concentration was determined to be 1 μg/mL, and the antibody dilution was initially determined to be 1:16000.

Table 2 Optimum coating concentration optimization

Determination of the optimal antibody dilution: Coat the microtiter plate with the optimal coating concentration, and design 5 dilution gradients with the center concentration of 1:16000 as the equal difference. The OD value and IC ₅₀ value of the zero drug well are shown in Table 3. As the antibody dilution decreased, the IC ₅₀ value increased, and the OD value of the zero-drug well also increased; with the increase of the antibody dilution, the IC ₅₀ value decreased, and the OD value of the zero-drug well also decreased. Based on IC ₅₀ value, zero well OD value and linear correlation coefficient of inhibition curve, 1:20000 was selected as the optimal antibody dilution.

Table 3 Optimal Antibody Dilution Optimization

Antibody coefficient multiple (1:X) Zero drug well OD value _IC50 value (μg/L) 8000 2.623 7.69 12000 2.462 3.51 16000 2.291 3.35 20000 2.097 3.36 24000 2.064 2.50

3.4 Establishment of standard curve

Set the neomycin standard concentration to 6 concentration gradients of 0, 1, 3, 5, 7, and 9 μg/L, measure according to the indirect competition ELISA method determined above, and draw a standard curve. As shown in Figure 3, the regression equation and the linear correlation index of the indirect competitive ELISA method established by the present invention with neomycin as a standard are respectively: y=-0.683x+0.860, r=0.999, and the _IC50 value is 3.16±0.42 μg/L (n=5). The linear range is 1~9μg/L.

3.5 Specificity

Various commonly used AMGs were diluted into concentration gradients for indirect competition ELISA, and the IC ₅₀ value was calculated. Compared with the neomycin standard IC ₅₀ value, the cross-reaction rate was obtained. The results are shown in Table 4. The results showed that the cross-reaction rates of the indirect competitive ELISA method established by the present invention to neomycin, amikacin and paromomycin were 100.00%, 80.90%, and 151.25%, respectively, and had no cross-reaction with other AMGs.

(公式1)

(Formula 1)

The specificity of table 4 indirect competition ELISA detection method of the present invention

competitors _IC50 (μg/L) Cross-reactivity rate (%) Neomycin 3.16 100.00 Amikacin 3.91 80.90 Paromomycin 2.09 151.25 Neomycin ＞10000 ＜0.03 Gentamicin ＞10000 ＜0.03 Kanamycin ＞10000 ＜0.03 Streptomycin ＞10000 ＜0.03 Ribomycin ＞10000 ＜0.03 Spectinomycin ＞10000 ＜0.03 Apramycin ＞10000 ＜0.03 Tobramycin ＞10000 ＜0.03 sisomycin ＞10000 ＜0.03 Hygromycin ＞10000 ＜0.03 Kasugamycin ＞10000 ＜0.03

Embodiment 4 The assembly of neomycin, amikacin and paromomycin multi-residue detection ELISA kit of the present invention

4.1 The composition of the ELISA kit of the present invention

1) coated with a solid phase carrier (elisa plate) coated with original neomycin-OVA;

2) 6 bottles of neomycin standard solution, the concentrations are 0μg/L, 1μg/L, 3μg/L, 5μg/L, 7μg/L, 9μg/L;

3) Neomycin monoclonal antibody working solution;

4) Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody working solution;

5) Concentrated phosphate buffer: NaCl 80.0g, KH ₂ PO ₄ 2.0g, Na ₂ HPO ₄ 12H ₂ O 29.0g, KCl 2.0g, add ultrapure water to 1000mL;

6) Concentrated washing liquid: NaCl 80.0g, KH ₂ PO ₄ 2.0g, Na ₂ HPO ₄ 12H ₂ O 29.0g, KCl 2.0g, Tween 20 5mL, add ultrapure water to 1000mL;

7) Substrate solution A: provided by Wuhan Feiyuan Technology Company;

8) Substrate solution B: provided by Wuhan Feiyuan Technology Company;

9) Termination solution: 2mol/L sulfuric acid solution.

4.2 Preparation of ELISA plate

Dilute neomycin-OVA to 1 μg/mL with coating solution, add 100 μL to each well, and coat at 4 °C for 12-16 hours; pour off the coating solution, add 250 μL washing solution to each well, wash 3 times, pat dry, Then add 250 μL of blocking solution to each well, and incubate at 37°C for 2 hours; pour off the liquid in the well, wash with the washing solution 3 times, and pat dry; place the microplate upside down in a 37°C incubator, and let it dry for 30 minutes. After drying, put it into an aluminum foil bag together with a desiccant, and seal it with a vacuum packaging machine.

Example 5 The assay procedure of the ELISA kit of the present invention

5.1 Preparation of reagents

1) Washing solution: Dilute the washing solution provided in the kit 10 times with ultrapure water before use.

2) Substrate mixed solution: According to the required amount each time, mix the prepared substrate solution A and substrate solution B according to the volume of 1:100, and prepare and use immediately.

3) The composition and ratio of phosphate buffer solution (0.1M, pH10~11) are: NaCl 20.0g, KH ₂ PO ₄ 0.4g, Na ₂ HPO ₄ ·12H ₂ O 13.4g, KCl 0.5g, with a small amount of Dissolve in ultrapure water, then dilute to 500mL.

5.2 Pretreatment of tissue samples

Pretreatment of porcine muscle tissue: Weigh 1.00±0.01g of homogeneous muscle sample into a 50mL centrifuge tube, add 10mL of phosphate buffer solution (0.1M, pH10-11), vortex and mix well for 1-2 minutes, and store at 60°C After incubating for 30 minutes, take it out, cool it to room temperature and shake well, centrifuge at room temperature for 10 minutes at 4000r/min, take the supernatant, dilute it with 4 times the volume of phosphate buffer solution (0.1M, pH10～11), and take 50μL for sample .

Note: The dilution factor for pig muscle in this method is 50.

5.3 Determination steps

1) Adding samples: Add 50 μL of neomycin series concentration standard solution or sample solution to the microwells of the microplate, then add 50 μL of neomycin monoclonal antibody working solution, place in a wet box, and incubate at 37°C for 30 minutes;

2) Washing: Pour out the liquid in the wells, add 250 μL of washing solution to each well, let stand for 30 seconds, wash 3 times and pat dry;

3) Add enzyme-labeled secondary antibody: add 100 μL of enzyme-labeled secondary antibody working solution to each well, and incubate in a humid box at 37°C for 30 minutes;

4) Washing: Pour out the liquid in the wells, add 250 μL of washing solution to each well, let stand for 30 seconds, wash 3 times and pat dry;

5) Add substrate: add 100 μL of substrate mixture to each well, and incubate in a humid chamber at 37°C for 15 minutes;

6) Add stop solution: add 50 μL of stop solution to each well;

7) Determination: Measure the optical density value (OD value) of each well at 450 nm with a microplate reader.

5.4 Result Judgment

The OD value of the measured standard divided by the "zero" hole OD value (B/B ₀ ) was taken as the ordinate, and the logarithm value of neomycin concentration was taken as the abscissa to draw a standard curve, and linear regression was performed to give a regression equation. Calculate the inhibition rate of the sample according to formula 1, substitute the inhibition rate into the regression equation, calculate the measured concentration, and multiply it by the corresponding dilution factor, which is the residual concentration of neomycin, amikacin and paromomycin in the sample.

(Formula 1)

The sensitivity, precision and accuracy of embodiment 6 kit of the present invention

6.1 Sensitivity of the kit of the present invention

和标准差(SD)，根据公式

计算出组织中的最低检测限。本发明的IC₅₀值为3.16±0.42μg/L，新霉素在猪肌肉中的最低检测限为15.90μg/L。The _IC50 value of the standard curve and the lowest tissue detection limit (LOD) are used as the sensitivity index of the detection kit of the present invention. Dilute the neomycin standard to 6 concentrations of 0 μg/L, 1 μg/L, 3 μg/L, 5 μg/L, 7 μg/L, and 9 μg/L, with 5 replicate wells for each concentration, and repeat the determination for 5 times according to the indirect competitive ELISA method times, the average value of the _IC50 determined for 5 times was taken. LOD is determined by the following steps: measure the OD value of muscle tissue of 20 blank pigs, calculate the corresponding neomycin concentration according to the regression equation of the standard curve, and then calculate the average value of the neomycin concentration

and standard deviation (SD), according to the formula

Calculate the lowest limit of detection in the tissue. The IC ₅₀ value of the present invention is 3.16±0.42 μg/L, and the lowest detection limit of neomycin in pig muscle is 15.90 μg/L.

6.2 The precision of the kit of the present invention

Dilute the neomycin standard to 6 concentrations of 0 μg/L, 1 μg/L, 3 μg/L, 5 μg/L, 7 μg/L, and 9 μg/L, with 5 replicates for each concentration, and repeat the determination 5 times according to the indirect competition ELISA method, Use the regression equation of the standard curve to calculate the measured values of the neomycin standard solutions at various concentrations, and calculate the coefficient of variation within and between plates. The results are shown in Table 5.

The precision of table 5 kit of the present invention

6.3 Accuracy of the kit of the present invention

Add neomycin standard solution to 1 g of homogenized pig muscle tissue to make the final concentrations 200 μg/kg, 100 μg/kg and 50 μg/kg; add amikacin standard solution to make the final concentrations 200 μg/kg kg, 100 μg/kg and 50 μg/kg; paromomycin standard solution was added to make the final concentrations respectively 200 μg/kg, 100 μg/kg and 50 μg/kg; each concentration was repeated 5 times, and the determination was repeated 3 times. Determine the concentration of neomycin, amikacin and paromomycin in the added tissue, calculate the recovery rate according to the following formula, and assess the accuracy of the kit; calculate the intra-assay and inter-assay coefficient of variation, and assess the repeatability of the kit sex. Accuracy and repeatability results are shown in Table 6, Table 7 and Table 8, indicating that the kit has reliable accuracy, small intra-assay and inter-assay coefficients of variation, and good repeatability.

Neomycin added recovery and coefficient of variation in table 6 pig muscle

Table 7 Paromomycin added recovery and coefficient of variation in pig muscle

Amikacin addition recovery and coefficient of variation in table 8 pig muscle

Claims (7)

1. A monoclonal antibody that can recognize neomycin, amikacin and paromomycin is characterized in that it is secreted by the hybridoma cell EDC/5G04 with the preservation number CCTCC NO:C201144. 2. The hybridoma cell EDC/5G04 described in claim 1 is preserved in China Center for Type Culture Collection, and its preservation number is CCTCC NO:C201144. 3. A kit comprising the monoclonal antibody of claim 1. 4. The test kit according to claim 3, which is an enzyme-linked immunosorbent assay kit for detecting neomycin, amikacin and paromomycin. 5. An enzyme-linked immunosorbent method for detecting neomycin, amikacin and paromomycin, comprising the preparation of coating former, antibody and sample processing and detection, its steps are as follows: (1) Coupling the hapten neomycin and ovalbumin to obtain the coating; (2) Prepare monoclonal antibody with the hybridoma cell EDC/5G04 with the preservation number CCTCC NO: C201144; (3) Coating the solid phase carrier with the coating material of step (1); (4) Extract, incubate, centrifuge and dilute the sample to be tested with a 0.1M, pH 10-11 phosphate buffer solution to obtain a solution of the test substance; (5) Perform enzyme-linked immunoassay on the analyte solution in step (4). 6. the application of monoclonal antibody described in claim 1 in the enzyme-linked immunoassay kit of preparation detection neomycin, amikacin and paromomycin. 7. The application of the kit according to claim 3 or 4 in the detection of neomycin, amikacin and paromomycin residues in animal tissues.

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