CN113136370B - Netilmicin and sisomicin monoclonal antibody hybridoma cell strain and application thereof - Google Patents
Netilmicin and sisomicin monoclonal antibody hybridoma cell strain and application thereof Download PDFInfo
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Abstract
A monoclonal antibody hybridoma cell strain of netilmicin and sisomicin and application thereof belong to the field of food safety immunodetection. The invention discloses a netilmicin and sisomicin monoclonal antibody hybridoma cell strain HLQ 3C9 which is classified and named as a monoclonal cell strain, the preservation date is 2020, 9, 27 days, and the preservation number is CGMCC No.20788. The invention mixes and emulsifies the complete antigen of netilmicin and equivalent Freund's adjuvant, and immunizes BALB/c mice through subcutaneous multipoint injection on the back of the neck. High potency, low IC 50 Fusing mouse spleen cells with mouse myeloma cells by a PEG method, and screening out fused hybrid cells; and screening cells by an indirect competitive enzyme-linked immunosorbent assay and subcloning for three times to finally obtain a monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity to both netilmicin and sisomicin, provides raw materials for immunodetection of netilmicin and sisomicin residues in food, and has practical application value.
Description
Technical Field
The invention relates to a netilmicin and sisomicin monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunodetection.
Background
Netilmicin (Netilmicin, NTM) is an aminoglycoside broad-spectrum antibiotic synthesized by using Sisomicin (SSM) as a raw material, and has similar antibacterial spectrum and clinical curative effect to gentamicin. It has the characteristic of being stable to aminoglycoside acetyltransferase AAC (3), is particularly effective against most gram-negative bacteria and many gram-positive bacteria including Staphylococcus aureus, is active against other aminoglycoside drugs such as gentamicin, sisomicin and tobramycin resistant strains, and has lower ototoxicity and nephrotoxicity than other aminoglycoside drugs. It is one of the first-line antibacterial drugs for treating serious bacterial infection, and is commonly used for respiratory system infection, skin and soft tissue infection, urinary system infection, wound infection, bone and joint infection, postpartum infection, gastrointestinal and biliary tract infection, septicemia and the like. However, like other aminoglycosides, netilmicin has a relatively narrow therapeutic index and is poorly metabolized by the body, whereas sisomicin has ototoxicity and nephrotoxicity, and is harmful to human health if used in excess or exposed to low doses for extended periods of time. Therefore, there is a need to monitor the drug in serum and urine to avoid toxicity and optimize antibiotic dosages.
In addition, netilmicin and sisomicin are also used as additives and veterinary drugs for the prevention and treatment of animal diseases in animal husbandry, and human beings exposed to these drugs for a long time through diet also have harm to health. No. 560 of Ministry of agriculture in China stipulates that the veterinary drug netilmicin sulfate is abolished, which puts requirements on the detection of netilmicin and sisomicin. Due to the polar basicity of netilmicin and sisomicin and the lack of strong uv absorbing chromophores, it is not straightforward to want direct analysis. At present, in addition to the microbiological methods recommended by the U.S. pharmacopoeia and the european pharmacopoeia, liquid chromatography-based instrumental detection methods have been developed in recent years, but these methods are highly specialized, long-lasting, and costly. With the rapid development of immunoassay technology in recent years, it becomes possible to establish a high-efficiency and rapid immunoassay method for NTM and SSM, including enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatographic test strip technology, immunomagnetic bead technology, and the like. An important prerequisite established by these methods is the need to screen out highly specific monoclonal monomers for netilmicin and sisomicin.
Disclosure of Invention
The invention aims to provide a netilmicin and sisomicin monoclonal antibody hybridoma cell strain and application thereof.
The technical scheme of the invention is as follows: a monoclonal antibody hybridoma cell strain HLQ 3C9 of netilmicin and sisomicin is preserved in China general microbiological culture Collection center (CGMCC), china academy of sciences, china, institute 3, of Nalcimicin and Sesomicin, china, nalcitonin, nakangyangju, hosthao No. 1, and is classified and named as a monoclonal cell strain, the preservation date is 2020 years, 9 months and 27 days, and the preservation number is CGMCC No.20788.
The monoclonal antibody of netilmicin and sisomicin is secreted and produced by the hybridoma cell strain HLQ 3C9 with the preservation number of CGMCC No.20788.
Netilmicin artificial antigen (hapten), the molecular formula is as follows:
The preparation method of the netilmicin artificial antigen comprises the steps of acetylating sisomicin to prepare triacetyl sisomicin, protecting hydroxyl by trimethylsilylation, reacting with 4-ethyl bromobutyrate, and finally performing alkaline hydrolysis, separation and purification to prepare the netilmicin artificial antigen.
The synthetic route is as follows:
the method comprises the following specific steps:
(1) The sisomicin sulfate is added into a DMF (dimethyl formamide) aqueous solution containing copper acetate, and the pH value of the solution system is adjusted by triethylamine; after the solution system is cooled, slowly dripping acetic anhydride diluted by DMF into the system under high-speed stirring, and maintaining the temperature and the pH; after the reaction, distilling the solvent out as much as possible by reduced pressure distillation, diluting the concentrated solution with water, standing at low temperature, filtering to separate solid matters, purifying the liquid again by using ion exchange resin, and freeze-drying to obtain a yellow powder intermediate product (1);
(2) Weighing an intermediate product (1), dissolving the intermediate product in dimethyl ether, adding trimethylchlorosilane, keeping magnetic stirring, and carrying out constant-temperature water bath reaction; finally, carrying out reduced pressure distillation on the solution to obtain a yellow solid intermediate product (2);
(3) Weighing the intermediate product (2), dissolving the intermediate product in acetone, and adding K 2 CO 3 And 4-ethyl bromobutyrate, keeping magnetic stirring, and reacting in a constant temperature water bath in a condensation reflux device; finally, carrying out reduced pressure distillation on the solution to obtain a yellow solid intermediate product (3);
(4) Intermediate (3) was added to NaOH and Na 2 CO 3 In the solution, keeping magnetic stirring, refluxing in oil bath at constant temperature in a condensing reflux device, cooling, and adding H 2 SO 4 Adjusting pH, vacuum filtering to collect clear solution, adding the solution into cation exchange resin, filtering, eluting with ammonia water, concentrating the eluate, dissolving with methanol, loading on silica gel column, eluting with chloroform-methanol-ammonia water; and distilling the collected eluent under reduced pressure, and freeze-drying the remained distillate to obtain the target product NTM-1.
Further, the method comprises the following specific steps:
(1) 1mM sisomicin sulfate was added to 40mL of DMF aqueous solution containing 0.2mM copper acetate; wherein, DMF: the volume ratio of water is 6; maintaining the pH value of the solution system at 8.5-10.5 by using triethylamine; after cooling the solution system to 5 ℃, slowly dropping 0.33mL of a solution containing 3.5mM acetic anhydride diluted with DMF into the system under high speed stirring, and reacting for 4h while maintaining the temperature at 0-10 ℃ and the pH at 8.5-10.5; then distilling off the solvent by reduced pressure distillation, diluting the concentrated solution to 10mL by water, standing for 4h at the temperature of 0-10 ℃, filtering to separate solid matters, purifying the liquid again by ion exchange resin, and freeze-drying to obtain a yellow powder intermediate product (1);
(2) Weighing an intermediate product (1) '0.8mM' and dissolving in 20mL dimethyl ether DME, adding 3.2mmol of trimethylchlorosilane, keeping magnetic stirring, reacting in a constant-temperature water bath at 50 ℃ in a condensation reflux device for 16h, and finally carrying out reduced pressure distillation on the solution to obtain a yellow solid intermediate product (2);
(3) Intermediate (2), weighed 0.6mM, is dissolved in 20mL of acetone and 600mg K is added 2 CO 3 And 1.0mM ethyl 4-bromobutyrate, keeping magnetic stirring, and reacting in a constant temperature water bath at 50 ℃ in a condensing reflux device for 24 hours; finally, carrying out reduced pressure distillation on the solution to obtain a yellow solid intermediate product (3);
(4) Intermediate (3) was added to 10mL of a solution containing 0.1M NaOH and 0.05M Na 2 CO 3 In the aqueous solution, keeping magnetic stirring, refluxing in a condensing reflux device at a constant temperature of 110 ℃ for 24 hours in an oil bath, then cooling to 10-15 ℃, and adding 1M H 2 SO 4 Adjusting the pH value to 6.5-7.0, performing suction filtration to collect a clear solution, and adding the solution into cation exchange resin; filtering, eluting with 7% ammonia water, concentrating, dissolving with methanol, loading on silica gel column, eluting with chloroform, methanol and ammonia water at volume ratio of 40: 20: 7, and separating; and distilling the collected eluent under reduced pressure, and freeze-drying the remained distillate to obtain the target product netilmicin artificial antigen NTM-1.
Weighing netilmicin artificial antigen NTM-1, dissolving in N, N-dimethylformamide DMF, adding N-hydroxysuccinimide NHS, keeping magnetic stirring reaction, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, and continuously keeping room temperature for stirring reaction; after the reaction is finished, dropwise and slowly adding the obtained activating solution into a BSA solution dissolved in a carbonate buffer solution, and continuously keeping the room temperature and stirring for reaction overnight; finally, dialyzing with phosphate buffer solution to remove small molecules which do not participate in the reaction to obtain complete antigen NTM-1-EDC-BSA, and identifying by SDS polyacrylamide gel electrophoresis method.
Further, weighing 7.1mg of netilmicin artificial antigen NTM-1, dissolving in 700 μ L of N, N-dimethylformamide DMF, adding 4.6mg of N-hydroxysuccinimide NHS, reacting for 15min under magnetic stirring, adding 7.6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, and continuing to react for 4h under stirring at room temperature; after the above reaction was completed, the obtained activation solution was slowly dropped dropwise into 2mL of a carbonate buffer solution CB having pH =9.0 in which 10mg of BSA was dissolved; continuously keeping the room temperature, stirring and reacting overnight; finally, dialyzing with 0.01M phosphate buffer solution PBS solution with pH =7.4, and removing small molecules which do not participate in the reaction to obtain the netilmicin complete antigen NTM-1-EDC-BSA.
The application of the monoclonal antibody of netilmicin and sisomicin is applied to the detection of the residual quantity of netilmicin and sisomicin in food safety immunoassay.
Further, the method is particularly applied to the detection of the residual quantity of netilmicin and sisomicin in milk, muscle tissues and urine.
The preparation of the netilmicin and sisomicin monoclonal antibody hybridoma cell strain HLQ 3C9 provided by the invention comprises the following basic steps:
(1) Immunization of mice: the complete antigen NTM-1-EDC-BSA of netilmicin (2 mg/mL) was mixed with equal amount of Freund's adjuvant and emulsified, BALB/c mice were immunized by subcutaneous multiple injection at the back of the neck (except for the booster immunization). Freund's complete adjuvant is used for the first immunization, and the dosage is 100 mu g/mouse; freund's incomplete adjuvant is used for multiple booster immunizations, and the dosage is reduced by half to be 50 mu g/mouse; the spurt immunity does not use any adjuvant, and the spurt immunity is directly diluted by normal saline and injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse. The interval between the first immunization and the second boosting immunization is one month, the interval between the multiple boosting immunizations is 21 days, and the interval between the sprint immunization and the last boosting immunization is 18-21 days. The mice were tested for immune efficacy by tail blood sampling and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Selecting one mouse with the best effect for cell fusion by evaluating the titer and inhibition;
(2) Cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 4000) method, screening hybridoma cells by a selective medium (HAT medium), and performing cell culture by an HT medium. After one week of fusion, detecting positive cell holes by using an ic-ELISA method, further determining the inhibition effect of the positive cell holes by using the ic-ELISA method, subcloning the positive cell holes with better inhibition by using a limiting dilution method, and detecting, selecting and subcloning again after one week. Carrying out three times of subcloning according to the method to obtain a monoclonal hybridoma cell strain HLQ 3C9 of the NTM high-secretion specific antibody;
(3) And (3) identifying the properties of the hybridoma cell strain HLQ 3C 9: sensitivity and specificity were determined by ic-ELISA.
The invention has the beneficial effects that: the monoclonal antibody secreted by the cell strain HLQ 3C9 has better specificity and detection sensitivity (IC for detecting netilmicin) for NTM and SSM 50 IC for sisomicin detection with value of 0.18ng/mL 50 The value is 0.22 ng/mL), can realize the detection of the residual quantity of netilmicin and sisomicin in milk, muscle tissues and urine, provides raw materials for the immunodetection of NTM and SSM residues in food, and has practical application value.
Biological material sample preservation: a monoclonal antibody hybridoma cell strain HLQ 3C9 of netilmicin and sisomicin is preserved in China general microbiological culture Collection center (CGMCC), china academy of sciences, china, institute 3, of Nalcimicin and Sesomicin, china, nalcitonin, nakangyangju, hosthao No. 1, and is classified and named as a monoclonal cell strain, the preservation date is 2020 years, 9 months and 27 days, and the preservation number is CGMCC No.20788.
Drawings
FIG. 1HLQ 3C9 monoclonal antibody inhibition standard curve for Netilmicin (NTM).
FIG. 2HLQ 3C9 monoclonal antibody standard curve for inhibition of sisomicin (SSM).
Detailed Description
The following examples are intended to be illustrative of the scope of the present invention and are not intended to be a limitation or scope of the present invention. The invention is further illustrated by the following examples.
According to the invention, a mouse is immunized by a netilmicin complete antigen, cell fusion and HAT selective culture medium culture are carried out, and cell supernatant is screened by ic-ELISA, so that a hybridoma cell strain with high specific antibody secretion for netilmicin and sisomicin is finally obtained.
Example 1 preparation of Netilmicin Artificial antigen
The synthetic route is shown in the specification; the method comprises the following specific steps:
(1) 1mM sisomicin sulfate was added to 40mL of DMF aqueous solution containing 0.2mM copper acetate; wherein, DMF: the volume ratio of water is 6; maintaining the pH value of the solution system at 8.5-10.5 by using triethylamine; after cooling the solution system to 5 ℃, slowly dropping 0.33mL of a solution containing 3.5mM acetic anhydride diluted with DMF into the system under high speed stirring, and reacting for 4h while maintaining the temperature at 0-10 ℃ and the pH at 8.5-10.5; then distilling off the solvent by reduced pressure distillation, diluting the concentrated solution to 10mL by water, standing for 4h at the temperature of 0-10 ℃, filtering to separate solid matters, purifying the liquid again by ion exchange resin, and freeze-drying to obtain a yellow powder intermediate product (1);
(2) Weighing an intermediate product (1) '0.8mM' and dissolving in 20mL dimethyl ether DME, adding 3.2mmol of trimethylchlorosilane, keeping magnetic stirring, reacting in a constant-temperature water bath at 50 ℃ in a condensation reflux device for 16h, and finally carrying out reduced pressure distillation on the solution to obtain a yellow solid intermediate product (2);
(3) 0.6mM of intermediate (2) was weighed out and dissolved in 20mL of acetone, and 600mg of K was added 2 CO 3 And 1.0mM ethyl 4-bromobutyrate, keeping magnetic stirring, and reacting in a constant temperature water bath at 50 ℃ in a condensation reflux device for 24 hours; finally, carrying out reduced pressure distillation on the solution to obtain a yellow solid intermediate product (3);
(4) Intermediate (3) was added to 10mL of a solution containing 0.1M NaOH and 0.05M Na 2 CO 3 In the aqueous solution of (2), magnetic stirring is maintainedStirring, refluxing in 110 deg.C oil bath at constant temperature for 24 hr, cooling to 10-15 deg.C, and adding 1M H 2 SO 4 Adjusting the pH value to 6.5-7.0, performing suction filtration to collect a clear solution, and adding the solution into cation exchange resin; filtering, eluting with 7% ammonia water, concentrating, dissolving with methanol, loading on silica gel column, eluting with chloroform, methanol and ammonia water at volume ratio of 40: 20: 7, and separating; and distilling the collected eluent under reduced pressure, and freeze-drying the remained distillate to obtain the target product netilmicin artificial antigen NTM-1.
EXAMPLE 2 preparation of the Netilmicin complete antigen NTM-1-EDC-BSA
Weighing 7.1mg of netilmicin artificial antigen NTM-1, dissolving in 700 μ L of N, N-dimethylformamide DMF, adding 4.6mg of N-hydroxysuccinimide NHS, reacting for 15min under magnetic stirring, adding 7.6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, and continuing to react for 4h under stirring at room temperature; after the reaction was completed, the obtained activation solution was slowly dropped dropwise into 2mL of a carbonate buffer solution CB having pH =9.0 in which 10mg of BSA was dissolved; continuously keeping the room temperature, stirring and reacting overnight; finally, dialyzing with 0.01M phosphate buffer solution PBS solution with pH =7.4, and removing small molecules which do not participate in the reaction to obtain the netilmicin complete antigen NTM-1-EDC-BSA.
EXAMPLE 3 preparation of hybridoma cell line HLQ 3C9
(1) Mouse immunization: after mixing and emulsifying complete antigen of NTM-1-EDC-BSA with equivalent Freund's adjuvant, BALB/c mice were immunized by subcutaneous multiple injections at the back of the neck (except for the booster immunization). Complete Freund adjuvant is used for the first immunization, and the dosage is 100 mu g/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, and the thorny immunity is directly diluted by normal saline and injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse. One month is separated between the first immunization and the second boosting immunization, 21 days are separated between multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization. The mice were tested for immune effects by tail blood sampling and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). By evaluating the potency and inhibition, the best mouse was selected for cell fusion.
(2) Cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:
a. the method comprises the following steps of (1) taking eyeballs of a mouse, taking blood, killing the mouse by a cervical vertebra dislocation method, immediately placing the mouse into 75% alcohol for soaking and sterilizing for about 5min, taking out the spleen of the mouse by aseptic operation, properly grinding the mouse by using a syringe rubber head, obtaining spleen cell suspension through a 200-mesh cell screen, collecting, centrifuging (800rpm, 6 min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;
b. collecting SP2/0 cells: 7-10 days before fusion, the SP2/0 tumor cells are treated with 10% FBS (fetal bovine serum) -containing RPMI-1640 medium at 5% CO 2 Culturing in an incubator. Before fusion, the number of SP2/0 tumor cells is required to reach (1-4) x 10 7 Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;
c. the fusion process is 7min: 1min, 1mL of PEG 4000 was added to the cells dropwise from slow to fast; standing for 2 min; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4min respectively; dripping 2mL of RPMI-1640 culture medium within 1min at 5min and 6min respectively; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5min. Centrifuging (800rpm, 10min), discarding the supernatant, gently tapping the cells, adding thereto RPMI-1640 selective medium (HAT medium) containing 20% fetal bovine serum and 2%50 XHAT, adding to a 96-well cell plate at 200. Mu.L/well, and standing at 37 ℃ for 5% CO 2 Culturing in an incubator.
(3) Cell screening and cell strain establishment: half-changing the fused cells with HAT medium on day 3 after cell fusion; on day 5, the whole culture medium was changed with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum and 1% 100 × HT; cell supernatants were taken on day 7 for screening. The screening is divided into two steps: firstly, screening out positive cell holes by using an ic-ELISA method, and secondly, selecting netilmicin and sisomicin as standard substances and measuring the inhibition effect of the positive cells by using the ic-ELISA method. Cell wells with better inhibition of netilmicin and sisomicin standards were selected, subcloned by limiting dilution, and tested seven days later by the same method. And carrying out subcloning for three times according to the method to finally obtain the netilmicin and sisomicin monoclonal antibody cell strain HLQ 3C9.
(4) Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10 6 Hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the caprylic acid-saturated ammonium sulfate method. Under the condition of meta-acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
4.1 coating: coating stock NTM-1-EDC-OVA is diluted by 3 times from 1 mu g/mL by 0.05M carbonate buffer solution with pH9.6, 100 mu L/hole, and reacted for 2h at 37 ℃;
4.2 washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;
4.3 sealing: after patting to dryness, 200. Mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. Drying for later use after washing;
4.4 sample adding: diluting antiserum from 1; after washing sufficiently, adding 1;
4.5 color development: taking out the ELISA plate, fully washing, adding 100 mu L of TMB color development liquid into each hole, and reacting for 15min at 37 ℃ in a dark place;
4.6 termination and determination: the reaction was stopped by adding 50. Mu.L of a stop solution to each well, and the OD 450 value of each well was measured by a microplate reader.
IC determination of monoclonal antibodies to Netilmicin by IC-ELISA 50 0.18ng/mL, the monoclonal antibodyIC to sisomicin 50 The concentration is 0.22ng/mL, which indicates that the kit has good sensitivity to netilmicin and sisomicin and can be used for immunoassay detection of netilmicin and sisomicin.
Solution preparation:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to fix the volume to 1000mL, and storing at 4 ℃ for later use;
phosphate Buffered Saline (PBS): 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12H 2 Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and metering the volume to 1000mL;
PBST: PBS containing 0.05% tween 20;
TMB color development liquid: solution A: na (Na) 2 HPO 4 ·12H 2 18.43g of O, 9.33g of citric acid and pure water with constant volume of 1000mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. And the volume ratio of the solution B to the solution B is 5:1 to obtain the TMB color developing solution which is mixed at present.
Claims (4)
1. A monoclonal antibody hybridoma cell strain HLQ 3C9 of netilmicin and sisomicin is preserved in China general microbiological culture Collection center (CGMCC), china academy of sciences, china, institute 3, of Nalcimicin and Sesomicin, china, nalcitonin, nakangyangju, hosthao No. 1, and is classified and named as a monoclonal cell strain, the preservation date is 2020 years, 9 months and 27 days, and the preservation number is CGMCC No.20788.
2. A netilmicin and sisomicin monoclonal antibody, characterized in that: the hybridoma cell strain HLQ 3C9 with the preservation number of CGMCC No.20788 as defined in claim 1.
3. The use of the netilmicin and sisomicin monoclonal antibodies of claim 2, wherein: the method is applied to detection of the residual quantity of netilmicin and sisomicin in food safety immunoassay.
4. The use of netilmicin and sisomicin monoclonal antibodies as claimed in claim 3, wherein: the method is particularly applied to detection of the residual quantity of netilmicin and sisomicin in milk, muscle tissues and urine.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0044441A1 (en) * | 1980-07-17 | 1982-01-27 | Miles Laboratories, Inc. | Monoclonal antibodies to drugs, methods for their production and somatic cell hybridomas secreting them |
| EP0242589A2 (en) * | 1986-03-18 | 1987-10-28 | Research Corporation | Detection of haptens in immunoassay techniques |
| CN102776151A (en) * | 2011-11-29 | 2012-11-14 | 塔里木大学 | Monoclonal antibody for detecting aminoglycoside antibiotics and kit thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0044441A1 (en) * | 1980-07-17 | 1982-01-27 | Miles Laboratories, Inc. | Monoclonal antibodies to drugs, methods for their production and somatic cell hybridomas secreting them |
| EP0242589A2 (en) * | 1986-03-18 | 1987-10-28 | Research Corporation | Detection of haptens in immunoassay techniques |
| CN102776151A (en) * | 2011-11-29 | 2012-11-14 | 塔里木大学 | Monoclonal antibody for detecting aminoglycoside antibiotics and kit thereof |
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