CN105131121B - Detect monoclonal antibody, ELISA method and the kit of Furaxone metabolite - Google Patents

Abstract

The invention belongs to wild animal resources and technical field of immunoassay, and in particular to a kind of monoclonal antibody of detection Furaxone metabolite, ELISA method and kit.The invention mainly comprises the synthesis of haptens, immunogene and coating antigen, the preparation of monoclonal antibody and the foundation of ELISA method.The kit of the present invention is mainly made of the monoclonal antibody of anti-Furaxone metabolite AOZ, the sheep anti-mouse igg of horseradish peroxidase-labeled, the ELISA Plate that is coated with the coating antigen combined with AOZ monoclonal antibody specificities.The enzyme linked immunological kit sensitivity of the present invention is good, cost-effective, the residue detection of Furaxone metabolite AOZ suitable for aquatic products.

Description

Detect monoclonal antibody, ELISA method and the kit of Furaxone metabolite

Technical field

The invention belongs to wild animal resources technical fields, and in particular to a kind of monoclonal of detection Furaxone metabolite Antibody (monoclonal antibody), ELISA (enzyme linked immunological) methods and kit.

Background technology

Furazolidone belongs to Nitrofuran metabolites, and because of its rear difficult absorption for oral administration, concentration is high in intestines and blood level is low, therefore Treatment suitable for various enteric infections.Such drug has certain toxicity, large dosage or long-term continuous use to livestock and poultry, easily occurs Toxic reaction shows as apocleisis, diarrhea, gastrointestinal bleeding, peripheral neuritis, excitement, convulsions, paralysis etc., can draw when being poisoned serious Play animal dead.Furazolidone is metabolized rapidly in vivo, metabolite 3- amino -2- oxazolidones (AOZ) energy and protein In conjunction with the formation protein conjugates more more stable than active compound compound.These metabolins can be under mildly acidic conditions from protein In release, therefore when the mankind eaten contain the remaining food of Nitrofuran antibiotics, these metabolins can be in people It is released from protein under the acid condition of class gastric juice and is absorbed by the body and causes damages to human health.European Union in Nineteen ninety-five forbids using Nitrofuran antibiotics in edible animal, and China, which has also been promulgated in 2002, is forbidden to use nitro furan The ban of class of muttering antibiotic.

For ensure animal derived food safety and export abroad trade development, establish it is accurate and reliable, high sensitivity Qualitative-and-quantitative method is very necessary.Majority state is by detecting original shape medicine to the monitoring of furazolidone when initial The residual of object, due to the difficulty of the unstability and detection monitoring of original shape drug, researcher takes up to study and searching can The residual marker being detected instead of original shape drug.2000, European Commission, which proposes, will will develop two distinct types of sieve Choosing experiment improves detection and the monitoring capacity in the residual laboratory of each medicine of European Union with a kind of confirmatory test.Then, Enzyme-linked Immunosorbent Assay The birth of (ELISA) method of experiment, and high-throughput with it, highly sensitive, low detection limit, plurality of advantages easy to operate, consuming is few It is generalized the screening operation of residue detection.

2004, Cooper etc. designed AOZ haptens, first plant of polyclonal antibody was obtained, using Salmonella method Specific detection is carried out to derivative, lowest detection is limited to 0.1 μ g/kg, 0.4 μ g/kg of quantitative limit.2005, Diblikova etc. Mouse is immunized with identical haptens, obtains AOZ monoclonal antibody specifics, for the remaining detections of AOZ in animal tissue, inspection Survey is limited to 0.3 μ g/kg, has high correlation to the testing result of AOZ with LC-MS/MS.It is 2008, often superfine to fish, pig, chicken Tissue uses benzaldehyde as derivative medium, and AOZ, which is derived, becomes N- benzylidene -3- amido-2-oxazolidinones, with indirect ELISA method is detected, and doing standard curve with phosphate buffer carries out regression analysis, is obtained tissue detection limit and is respectively less than 0.5μg/kg。

The instrumental method of furazolidone residual detection is ripe day by day, and it is sensitive that the application of LC-MS/MS substantially increases detection Degree, usually as confirmation means.Specific binding reaction of the ELISA kit based on antigen-antibody, has conventional physics and chemistry The features such as analytical technology is unrivaled highly selective and highly sensitive is very suitable for the analysis of matrix complexity, trace residue, is One of ideal residual screening assays, are the high-throughput screening methods that current countries in the world are carried out and implemented.

Invention content

It is an object of the invention to overcome the deficiencies of existing technologies, a kind of Dan Ke that can identify Furaxone metabolite is provided Grand antibody establishes a kind of ELISA (enzyme linked immunologicals of energy Furaxone metabolite (AOZ) residue detection using the monoclonal antibody Method) and kit and application.

The invention is realized by the following technical scheme：

Applicant by prepare, the monoclonal antibody of Furaxone metabolite (AOZ) can be identified by obtaining one kind, it be by Preserving number is CCTCC NO：Secreted by the hybridoma cell strain SC20150701A of C2015129, which is ordered Entitled hybridoma cell strain SC20150701A delivered the Chinese Typical Representative culture of the Chinese Wuhan Wuhan Universitys on July 20th, 2015 Object collection (CCTCC) preservation, preserving number are CCTCC NO：C2015129.

Further, the present invention proposes a kind of enzyme linked immunological side being suitable for Furaxone metabolite (AOZ) residue detection Method (ELISA), this method include the preparation of haptens, immunogene, coating antigen and antibody and the steps such as the processing of sample and detection Suddenly, it is of the invention be as follows it is described：

(1) Furaxone metabolite (AOZ) is subjected to derivative and haptens CNPAOZ and CPAOZ is prepared；

(2) haptens CNPAOZ and hemocyanin (KLH is purchased from sigma companies) coupling are obtained into immunogene；

(3) haptens CPAOZ and bovine serum albumin(BSA) (BSA) coupling are obtained into coating antigen；

(4) utilizing step, (2) the immunogene prepares and can secrete the miscellaneous of the monoclonal antibody for identifying Furaxone metabolite Hand over tumor cell strain SC20150701A；

(5) the coating primordial covering solid phase carrier (that is, ELISA Plate) of step (3) is used；

(6) obtain determinand after being handled before sample to be checked and carry out enzyme linked immunosorbent detection.

Based on above-mentioned detection method, it is CCTCC NO that the present invention provides one kind comprising preserving number：The hybridization of C2015129 The ELISA kit of monoclonal antibody secreted by tumor cell strain SC20150701A.

The kit is to detect the ELISA kit of Furaxone metabolite in aquatic products.

Inventor is using said monoclonal antibody and coating antigen as core reagent and other conventional reagents, to enzyme linked immunological Method is verified, and realizes the purpose of the present invention, so as to complete the present invention.

Monoclonal antibody prepared by the present invention can detect the ELISA kit of Furaxone metabolite in aquatic products in preparation Middle application.

Hybridoma SC20150701A prepared by the present invention can prepare detect aquatic products in Furaxone metabolite ELISA kit in apply.

Kit prepared by the present invention can be applied in detecting aquatic products in Furaxone metabolite retention analysis.

Main advantages of the present invention are：

(1) the monoclonal antibody affinity that prepared by the present invention is high, high specificity.

(2) enzyme-linked immunoassay method (ELISA) that the present invention establishes is easy, easy to operate, and high sensitivity, accuracy and essence Density is good.

More detailed technical solution is as described embodiments.

Description of the drawings

Fig. 1：The ultraviolet scanning atlas of CNPAOZ-KLH immunogenes prepared by the present invention.

Fig. 2：The ultraviolet scanning atlas of CPAOZ-BSA coating antigens prepared by the present invention.

Fig. 3：Using the indirect competitive ELISA reaction normal curve graph that AOZ is established as standard items, reference sign：X-axis is AOZ concentration of standard solution logarithms, Y-axis are the OD value divided by " zero " hole OD value (B/B of AOZ standard solutions₀)。

Specific implementation mode

Below by embodiment, the invention will be further described.

The preparation of embodiment 1 immunogene and coating antigen

1g Furaxone metabolites (AOZ), -2 nitro-benzaIdehyde of 1.65g 5- hydroxyls are taken, is allowed to dissolve with absolute ethyl alcohol, It is stirred to react 5h at room temperature, 4 DEG C of refrigerators are stood overnight, and filtering is dissolved with n,N-Dimethylformamide (DMF), is added 100mg's NaOH, 4- bromobutyrates 600mg is slowly added dropwise；It waits for after reaction, water being added, is extracted with ethyl acetate, merge organic Phase, filtering, filtrate are added a small amount of DMF and are allowed to after dissolving after being evaporated, 1N NaOH 40mL are added, are stirred to react at room temperature 2h filters reaction solution HCL tune pH=3 to get haptens CNPAOZ, structural formula is as follows：

0.2g AOZ are taken, is stirred at room temperature with the methanol of 2mL or so and is allowed to dissolve；0.15g 3- carboxyl benzaldehydes are taken, It is allowed to dissolve with 2mL methanol, is slowly dropped under stiring in above-mentioned AOZ solution, is stirred to react 5h at room temperature, filter, drying filter For slag up to CPAOZ, structural formula is as follows：

Take haptens CNPAOZ 10mg, dicyclohexylcarbodiimide (DCC) 8mg, N- hydroxysuccinimide (NHS) 5mg, DMF 1mL are allowed to dissolve, and reaction are stirred at room temperature for 24 hours, centrifuging and taking supernatant is slowly dropped to KLH solution (5mg blood indigo plant eggs later Be dissolved in 5mL distilled water in vain) in 4 DEG C reaction for 24 hours.Phosphate buffer (abbreviation PBS, preparation method：Accurately weigh NaCl 8.00g KH₂PO₄0.20g, Na₂HPO₄·12H₂O 2.90g, KCl 0.20g, a small amount of deionized water dissolving are settled to 1000mL) 5d dialyse up to immunogene CNPAOZ-KLH.

It takes haptens CPAOZ 8mg, DCC 8mg, NHS 5mg, DMF 1mL to be allowed to dissolve, reaction is stirred at room temperature for 24 hours, it Centrifuging and taking supernatant is slowly dropped in bovine serum albumin(BSA) (BSA) solution of 20mg in 4 DEG C of reactions for 24 hours afterwards.Use phosphate-buffered Liquid dialyses 5d up to coating antigen CPAOZ-BSA.

The preparation of 2 monoclonal antibody of embodiment

2.1 mouse immune

The CNPAOZ-KLH conjugates prepared using embodiment 1 are immunized Balb/C female mices and (are purchased from Hubei Province as immunogene Disease prevention and control center).With the albumen containing 50 μ g immunogenes with isometric Freund's complete adjuvant emulsification when first immunisation Emulsion injection subcutaneously carries out fundamental immunity in the nape part of mouse, contains 50 with what incomplete Freund's adjuvant emulsified every 15 days later The protein emulsion booster immunization of μ g immunogenes.From third time is immune, the immune rear 7th day tail blood for adopting mouse, detaches blood every time Clearly, with common indirect elisa method (with reference to Dong state is big and Liu Xianjin, the methods of 2001 reports) detection serum antibody titer.

2.2 cell fusions and screening

3 days mouse peritoneals to immuno-competent inject 100 μ g immunogene CNPAOZ-KLH reinforced immunologicals before fusion.According to The count results of myeloma cell, take myeloma cell to be mixed with immune spleen cell；5min is centrifuged with 1500r/min, will be centrifuged After being emptied on pipe to the greatest extent, tips upside down on blotting paper, drain water droplet；50% polyethylene glycol (PEG) for there are 1mL pre-temperatures to 37 DEG C will be inhaled LmL measuring pipettes are inserted into tube bottom, and slowly plus on PEG to cell mixing, side edged is gently mixed, and 90sec is stood after adding, and use RPMI -1640 basic culture solutions (being purchased from Hyclone companies) of pre-temperature to 37 DEG C are slowly added on fused cell along tube wall, Bian Jia Centrifuge tube is gently shaken on side, and lid is tightened after adding, and slowly overturns several times, mixing；5min is centrifuged with 1500r/min, is discarded supernatant, Gently fused cell is stirred with the HAT complete mediums (being purchased from Hyclone companies) containing feeder cells and is resuspended.It will merge thin Born of the same parents' suspension is inoculated in 96 porocyte culture plates, is placed in CO₂It is cultivated in incubator.According to the growing state of cell, cell is taken to train Supernatant is supported, the CPAOZ-BSA conjugates prepared using inventor filter out secretion AOZ antibody as coating antigen, using ELISA method Positive cell hole.Limiting dilution assay cloning utilized to the positive cell hole that screens, it is final establish obtain one plant it is stable Secrete and can identify that the hybridoma of the monoclonal antibody of Furaxone metabolite (AOZ), applicant are thin by the hybridoma Born of the same parents are named as hybridoma cell strain SC20150701A, and the Chinese Wuhan Wuhan Universitys Chinese Typical Representative is delivered on July 20th, 2015 Culture collection (CCTCC) preservation, preserving number are CCTCC NO：C2015129.

2.3 ascites prepare and identification

It takes within 7 days before inoculation Balb/c number of mice that 0.5ml incomplete Freund's adjuvants are only injected intraperitoneally to be pre-processed.Use base Basal culture medium suspension preserving number is CCTCC NO：The hybridoma of C2015129 is inoculated in mouse peritoneal.Wait for that mouse web portion is bright It is aobvious to expand collection ascites, it is obtained using conventional sad ammonium sulfate method (with reference to Yang Liguo etc., the method for 2011 reports) purifying Monoclonal antibody.Monoclonal antibody subtype identifying test paper item is used to determine the monoclonal antibody of gained for IgG1 κ hypotypes.

The foundation of 3 ELISA detection method of embodiment

The determination of 3.1 optimum reaction conditions

(the results are shown in Table 1) is titrated by square formation, is increased with the dilution of antibody in peridium concentration, OD values decline unknown Aobvious, the affinity of the coating antigen and antibody that illustrate the present invention is not strong, this is advantageous to the competition of drug, this is also selection of the present invention A reasons of the CPAOZ-BSA as coating antigen.Therefore the coating antigen CPAOZ-BSA of final choice 1ppm concentration is dense to be most preferably coated with Degree.

The determination of 1 best ELISA reaction conditions of table

The foundation of 3.2 standard curves

AOZ standard items are configured to 6 concentration gradients such as 0,0.05,0.1,0.2,0.4,0.8 μ g/L, each concentration 2 Parallel hole, does indirect competitive ELISA, draws standard curve (see Fig. 3), the regression equation of racing ELISA detecting method and The index of correlation is：Y=-0.5330x+0.1820, r²=0.9996, IC₅₀=0.26ppb, the range of linearity are 0.05-0.8ppb.

3.3 antibody specificity

Respectively by haptens CPAOZ, AOZ, derivative reagent 2- nitrobenzaldehydes doubling dilution at gradient concentration, by being established Indirect competitive ELISA method, measure its IC₅₀Value, with NPAOZ standard items (being purchased from sigma companies) IC₅₀Value comparison is handed over Fork reaction, the results are shown in Table 2, identifications of the hybridoma SC20150701A that as can be seen from Table 2 prepared by the present invention to NPAOZ Preferably, but other medicines cannot then be identified.

Monoclonal antibody specificity prepared by 2 present invention of table measures

Competitor	IC50(μg/L)	Cross reacting rate (%)
			NPAOZ	0.42	100
CPAOZ	＞ 1000	＜ 0.1
			AOZ	＞ 1000	＜ 0.1
2- nitrobenzaldehydes	＞ 1000	＜ 0.1

4 ELISA kit of embodiment assembles

4.1 kit material requesteds

96 1 piece of hole elisa Plates；6 bottles of AOZ titers, concentration are formulated as 0,0.05,0.1,0.2,0.4,0.8 μ g/L respectively； 1 bottle of AOZ antibody liquids；1 bottle of horseradish peroxidase-labeled sheep anti-mouse igg (being purchased from Wuhan Fei Yi Co., Ltds)；Cleaning solution is (accurate Weigh NaCl 8.00g, KH₂PO₄0.20g, Na₂HPO₄·12H₂O 2.90g, KCl 0.20g, a small amount of deionized water dissolving are added Tween 200.50mL, are settled to 1000mL；) 1 bottle of (10 × concentration)；((PBS accurately weighs NaCl to phosphate buffer 8.00g KH₂PO₄0.20g, Na₂HPO₄·12H₂O 2.90g, KCl 0.20g, a small amount of deionized water dissolving are settled to 1000mL；) 1 bottle of (10 × concentration)；Substrate developing solution A (accurately weighs TMB 160mg, tetrabutyl hydroboration ammonia 21mg, is added 10ml dimethylacetamide amine solvents mixing) 1 bottle of (TMB)；Substrate developing solution B (accurately weighs citric acid 13.70g, trisodium citrate 10.14g and carbamide peroxide 282.00mg, a small amount of ultrapure water dissolution, and it is settled to 1000mL；) 1 bottle of carbamide peroxide；It terminates 1 bottle of liquid (accurate to measure concentrated sulfuric acid 100mL, to be slowly dropped in 900mL deionized waters).

4.2 the processing of aquatic products sample to be detected

Weigh fresh water shrimp (such as Procambius clarkii, clear shrimp etc., kind is unlimited) or the flesh of fish (such as grass carp, silver carp, Wuchang Fish etc., kind is unlimited) 0.1M HCl 2mL, 0.01M derivatization reagents is successively added in 1 ± 0.01g in 50mL plastic centrifuge tubes (being purchased from sigma companies) 0.1mL, it is permanent in 37 DEG C of insulating boxs with the abundant vortex mixed 1min of vortex instrument so that tissue dispersion is uniform Temperature is incubated 16h or so, and 1M NaOH 0.2mL, ethyl acetate 8mL is successively added, with the abundant vortex mixed 1min of vortex instrument, 4000r/min centrifuges 5min.It takes supernatant 4mL in 5mL plastic centrifuge tubes, is dried up in 60 DEG C or so water-bath nitrogen, respectively first Be added n-hexane 0.5mL, PBS buffer solution 0.5mL afterwards, 5min centrifuged in 4000r/min after vortex, remove clear liquid for analyze inspection It surveys.

4.3 kit measurement programs

(1) hermetic bag is cut off, ELISA Plate time is taken out and warms to room temperature.

(2) add AOZ titers or sample to be tested：It is added in kit and provides the AOZ standard items of each concentration or by kit In the 50 μ L to micropore of detection liquid that method is handled in specification.

(3) add antibody：It is (the AOZ antibody liquid phosphate buffers provided in kit is dilute that AOZ antibody working solution is added Release 400 times of uses) it is sufficiently mixed in 50 μ L to each micropore, horseradish peroxidase-labeled sheep anti-mouse igg working solution is added It is sufficiently mixed, is placed in wet box in 100 μ L to each micropore, 30min is incubated in 37 DEG C of incubators.

(4) it washs：The liquid in hole is poured out, wash 5 times and is patted dry, Substrate cocktail, which is added, in every hole (accurately draws bottom 100 μ L substrate developing solution A, mixing is added in object developing solution B 10mL；) 100 μ L, it after being sufficiently mixed, is placed in wet box, at 37 DEG C 15~20min is incubated in incubator；

(5) it terminates：50 μ L of terminate liquid are added in per hole, optical density (OD) value is measured at 450nm.

4.4 result judgement

By AOZ titers or the average value (B) of sample liquid light absorption value divided by the light absorption value (B of 0 gauge orifice₀) multiplied by with 100%, you can calculate inhibiting rate.Within the scope of 0.05ppb-0.8ppb, using the inhibiting rate of AOZ titers as ordinate, mark The logarithm of quasi- liquid concentration is that abscissa draws standard curve, obtains regression equation.The inhibiting rate of sample is substituted into regression equation, meter Measured concentration is calculated, the coefficient of dilution (when using this kit measurement, it is desirable that the extension rate of sample is 4 times), as sample are multiplied by The actual concentrations of AOZ in product.Calculation formula is as follows：

Sensitivity, the preci-sion and accuracy experiment of 5 kit of the present invention of embodiment

The sensitivity test of 5.1 kits of the present invention

The residue of veterinary drug enzyme linked immunological kit issued according to the Ministry of Agriculture of China is put on record with reference to judgment criteria (agriculture doctor's hair [2005] No. 17,2005) drug (Furaxone metabolite (AOZ)) remains minimum detection limit in the investigation detection tissue provided in Method, i.e., the measurement average value of 20 parts blank samples adds 3 times of standard deviation methods to be minimum detection limit in tissue.Measure 20 portions of shrimp The minimum detection limit of Furaxone metabolite (AOZ) in (cultivar origin is shown in embodiment 4) and the flesh of fish (cultivar origin is shown in embodiment 4) For 0.06~0.08 μ g/kg.

The precision of 5.2 kits of the present invention

5 multiple holes are arranged in the standard items of each concentration, are repeated 5 times, using the coefficient of variation of standard items inhibiting rate as index, comment As a result the repeatability and precision of valence detection method show that the coefficient of variation of each concentration is equal<7% (table 3).

The precision of 3 ELISA method of the present invention of table is tested

AOZ(μg/L)	0.05	0.1	0.2	0.4	0.8
						The coefficient of variation (%)	3.67	4.64	4.33	5.72	5.99

The accuracy test of 5.3 kits of the present invention

Blank tissue samples are taken, are distinguished according to maximum residue limit of the drug (Furaxone metabolite) in various tissues It is added experiment, makes the μ g/kg of a concentration of 0.1,0.2 and 0.4 for adding drug (Furaxone metabolite) in tissue, each sample 5 Duplicate Samples are arranged in product concentration.Then sample treatment is carried out, drug (furazolidone metabolite is measured with common ELISA method Object) concentration, 3 batches are repeated, calculates the rate of recovery, the rate of recovery is calculated by following formula, and calculates within-run and between-run analysis coefficient.

TIANZHU XINGNAO Capsul is tested in 4 aquatic products tissue of table

AOZ is respectively added to freshwater fish (cultivar origin is shown in embodiment 4) and fresh water shrimp by the present invention, and (cultivar origin is shown in Embodiment 4) in, TIANZHU XINGNAO Capsul and batch interior and interassay coefficient of variation measurement result are shown in Table 4.The drug rate of recovery is 81.16% Between~128.73%, in batch and differences between batches are respectively less than 25%.

Claims (7)

1. a kind of monoclonal antibody that can identify Furaxone metabolite, which is characterized in that it is CCTCC NO that it, which is by preserving number,： Secreted by the anti-AOZ hybridomas SC20150701A of C2015129.

2. a kind of hybridoma SC20150701A for the monoclonal antibody that can identify Furaxone metabolite, is deposited in China Type Tissue Collection, deposit number are CCTCC NO：C2015129.

3. including the kit of monoclonal antibody described in claim 1.

4. kit according to claim 3, the kit is Furaxone metabolite in detection aquatic products ELISA kit.

5. the ELISA kit of monoclonal antibody described in claim 1 Furaxone metabolite in preparing detection aquatic products In application.

6. the Furaxone metabolites in preparing detection aquatic products of the hybridoma SC20150701A described in claim 2 Application in ELISA kit.

7. application of the kit in detecting aquatic products in Furaxone metabolite retention analysis described in claim 3 or 4.