CN102936582A - Furazolidone metabolite derivative monoclonal antibody and applications thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody and applications thereof, especially to a furazolidone metabolite derivative monoclonal antibody and applications thereof, and belongs to the technical field of immunochemistry. The furazolidone metabolite derivative monoclonal antibody 1C3' is produced by mouse hybridoma cell line 1C3, wherein a subtype is IgG1 type. According to the present invention, the monoclonal antibody 1C3' produced by the mouse hybridoma cell line 1C3 can be abundantly produced, and can be used to prepare an enzyme linked immunoassay kit and colloidal gold test paper strips for furazolidone metabolite (AOZ) detection so as to achieve a purpose of rapid and sensitive detection of furazolidone drug metabolites in muscle, liver and aquatic products and establish a foundation for furazolidone metabolite residue detection kit and colloidal gold product development.

Description

The monoclonal antibody of Furaxone metabolite derivative and application thereof

Technical field

The present invention relates to a kind of monoclonal antibody and application thereof, the especially monoclonal antibody of Furaxone metabolite derivative and application thereof belongs to the immunochemical technique field.

Background technology

Nifurazolidone (Furazolidone, FZ), have another name called Trichofuron, it is a kind of itrofurans broad spectrum antibiotic of synthetic, multiple gram-positive microorganism and Gram-negative bacteria and some protozoon and fungi had suppress or killing effect, and efficacy stability, in livestock and poultry and aquaculture, be widely used.But toxicologic study is found, Nifurazolidone and metabolite 3-amino thereof-2-oxazolidone (3-amino-2-oxazolidinone, AOZ) very strong toxic side effect is all arranged, can carcinogenic, mutagenesis, can cause the various diseases such as hemolytic anemia, thrombopenic purpura, polyneuritis.Nineteen ninety-five, European Union forbade that at first Nifurazolidone uses in livestock and poultry and aquaculture, the countries such as the U.S., Japan, Australia classify Nifurazolidone as the forbidding medicine subsequently, classify Nifurazolidone as the forbidding medicine in " veterinary drug and other compound inventory of food animal forbidding " (agriculture and animal husbandry is sent out [2002] No. 1) that China Ministry of Agriculture promulgates in March, 2002.EU Committee has passed through the resolution of the 2003/181/EC council in 2003, the minimum of having set up for detection of the whole bag of tricks of the metabolite of total itrofurans medicine in poultry product and the fishery products requires performance limit value (Minimum Required Performance Limits, MRPL) be 1 μ g/kg, namely the content of European Union's metabolite of itrofurans medicine from the animal product of third country import must not surpass 1 μ g/kg.U.S. FDA had been announced 11 kinds of list of substance forbidding using in the import animal derived food in 2004, comprising Nifurazolidone.Therefore residual not only directly endanger user healthy of Nifurazolidone in food animal also seriously restricts China's animal food outlet simultaneously, caused huge financial loss.

At present, the method for the AOZ residue detection mainly contains physico-chemical analysis method and enzyme-linked immunosorbent assay (ELISA) etc.The physico-chemical analysis method mainly is high performance liquid chromatography (HPLC), liquid chromatograph mass spectrography (LC-MS) etc.The physico-chemical analysis method is highly sensitive, and the result is accurate, but sample needs the treatment and purification through series of complex, and loaded down with trivial details time-consuming, required plant and instrument is expensive, only has specific professional and technical personnel to operate, and can not carry out Site Detection, and poor in timeliness is difficult to promote.Immunochemical analyses method particularly enzyme-linked immunosorbent analytical technique (ELISA) has fast, highly sensitive, simple to operate, the advantages such as specificity is good, be fit to high-throughout sample screening, the applicant retrieves discovery, application number is 200610041480.1, name is called and adopts the ELISA method to detect the residual of Nifurazolidone in one piece of Chinese invention patent application of " furazolidone residual detecting method of mediated monoclonal antibody and application ", because Nifurazolidone in animal body very fast metabolism is AOZ, AOZ be combined with tissue protein and in vivo the residence time long, be regarded as the marker of furazolidone residual.Only can not judge the actual residual condition of Nifurazolidone by detecting parent drug.

Summary of the invention

The present invention wants the technical solution problem to be: for the shortcoming that above prior art exists, carry

Go out a kind of monoclonal antibody of Furaxone metabolite derivative, and this monoclonal antibody is applied to detect Furaxone metabolite.That the method has is easy and simple to handle, with low cost, the characteristics of high-throughput, high specificity.

Hybridoma cell strain 1C3 involved in the present invention has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 18th, 2011, deposit number is CGMCC No.5071, and Classification And Nomenclature is the hybridoma cell strain of anti-Furaxone metabolite derivative monoclonal antibody.

By the monoclonal antibody called after 1C3 ' that above-mentioned hybridoma cell strain 1C3 produces, its preparation method may further comprise the steps:

A. prepare immunogen and coating antigen: the aldehyde benzoic acid method is with carrier proteins and the coupling of furazolidone metabolite product small molecules, synthetic immunogen and coating antigen between employing;

B. animal immune injection: as immune animal, immunization route is the subcutaneous multi-point injection of nape section with the Balb/c mouse, and the superpower immunization route before merging is peritoneal immunity;

C. screen animal immune serum: with indirect enzyme-linked immunosorbent assay and competition indirect enzyme-linked immunosorbent assay screening immune mouse serum;

D. prepare hybridoma: get mouse boosting cell and SP2/0 myeloma cell and carry out cytogamy, through subclone obtain can the anti-Furaxone metabolite derivative of stably excreting monoclonal antibody hybridoma cell strain 1C3;

E. prepare and monoclonal antibody purification: to mouse peritoneal injection hybridoma 1C3, gather ascites, ascites is carried out the liquid chromatography (LC) purifying, obtain Furaxone metabolite derivative monoclonal antibody 1C3 '.

Wherein, the described carrier proteins of step a is bovine serum albumin and oralbumin, and corresponding immunogen is AOZ-BSA, and coating antigen is AOZ-OVA; In described the aldehyde benzoic acid method, the time of linking agent activated micromolecular derivative is 1-2 hour, reacts 10-15 hour with Furaxone metabolite small molecules derivative and carrier protein couplet.Each time immunization dosage among the described step b is 100 μ g/.The injection volume that among the described step e cell strain of monoclonal antibody is carried out in the immune mouse abdominal cavity is (1-5) * 10 ⁶Individual/only.

Identify that by hypotype the hypotype that draws monoclonal antibody of the present invention is the IgG1 type.

Usually the method for preparing the Furaxone metabolite monoclonal antibody mainly is the amino coupled by carboxyl and the carrier proteins of medicine, the present invention carries out derivatize with the Furaxone metabolite small molecules by an aldehyde benzoic acid and with the carboxyl of the derivatize product amino coupled by mixed anhydride method and carrier proteins, like this, the complete antigen that this method is synthetic, effect by aldehyde benzoic acid between crosslinked arm, the Furaxone metabolite small molecules fully can be come out, make the easier identification of body antigenic determinant, improve the immunogenicity of antigen, in the later stage screening process, can obtain the antibody for the Furaxone metabolite derivative.The Furaxone metabolite structural formula is as follows:

A further object of the present invention provides the application of this monoclonal antibody in preparation detection Furaxone metabolite, mainly is to be applied to prepare the residual detection kit of Furaxone metabolite and colloid gold test strip paper.

The present invention obtains AOZ and carrier protein couplet the complete antigen of synthetic.Complete antigen immunity Balb/c mouse with preparation, obtain to identify the monoclonal antibody of AOZ derivative by merging screening, its beneficial effect is: the monoclonal antibody 1C3 ' that produces by mouse hybridoma cell strain 1C3 is low with the derivative cross reacting rate of other nitrofurans metabolite, can produce in a large number, and can be used for preparing enzyme-linked immunosorbent assay test kit and the colloidal gold strip that detects Furaxone metabolite, reach quick, special, and detect delicately milk, the purpose that Furaxone metabolite in muscle and the fishery products is residual is for detecting the residual test kit of Furaxone metabolite, the Radioactive colloidal gold product development lays the foundation.

Description of drawings

Fig. 1 is immunogenic OD in the one embodiment of the invention ₃₃₅Typical curve.

Fig. 2 is the OD of coating antigen in the one embodiment of the invention ₃₃₅Typical curve.

Embodiment

Embodiment one

A. reagent and material are prepared

The Furaxone metabolite standard substance (AOZ, Town in Shanghai spectrum scientific instrument company limited, NF005), Nifurazolidone (AO, pharmaceutical biological product calibrating institute, 10046-200401); Between aldehyde benzoic acid (Alfa, B21277); DMF(sigma, D4551), sodium-acetate (sigma, S2889), diacetyl oxide (Chemical Reagent Co., Ltd., Sinopharm Group), bovine serum albumin (BSA) (Jackson, 001-000-173), oralbumin (OVA) (Sigma, A5503), Freund's complete adjuvant (Sigma, F5881), Freund's incomplete adjuvant (Sigma, F5506), tetramethyl benzidine (TMB) (Amresco, 0759), HAT(Sigma, H0262) and HT(Sigma, H0137), sodium-chlor (Amresco, 0241), Repone K (Amresco, 0395), potassium primary phosphate (Sigma, P9791), Sodium phosphate dibasic (Sigma, 71639), sheep anti-mouse igg-HRP(Jackson, 115-035-044), dimethyl sulfoxide (DMSO) (DMSO) (Applichem, 0231), Macrogol 4000 (PEG4000) (Sigma, P7306), DMEM high glucose medium (Gibco, 11995), foetal calf serum (Gibco, C20270).

Laboratory animal and cell: Balb/c mouse (6-8 week age, female), available from Yangzhou University's animal center, SP2/0(murine myeloma cell) infected with immune Research center professor Tang Jie by biophysics institute of the Chinese Academy of Sciences and to be so kind as to give.

B. prepare Furaxone metabolite derivative monoclonal antibody 1C3 '.

Concrete steps are as follows:

A. prepare immunogen AOZ-BSA and coating antigen AOZ-OVA:

Aldehyde benzoic acid was with furazolidone metabolite product small molecules derivatize between artificial antigen of the present invention was adopted, then utilize carboxyl and the amino on the carrier proteins on the furazolidone metabolite product small molecules derivative to carry out coupling, thus synthetic Furaxone metabolite antigen.

One, complete antigen is synthetic

1, with 15 ℃ of concussion reaction 24 h in 1.5 mL pyridine solutions of aldehyde benzoic acid between 2 mg Furaxone metabolite standard substance and 2.7 mg.

2, reaction solution is dissolved in behind the Rotary Evaporators evaporate to dryness in the 400 μ L DMF solution, adds Tributylamine 5 μ l, ice bath vibrates behind 10 min, adds isobutyl chlorocarbonate 3 μ l ice baths 1 h that vibrates.

3, under condition of ice bath, reaction product slowly is added dropwise to BSA solution or OVA solution, dropwises rear 4 ℃ of stirring reaction 24h.

4, dialyse among 0.01 M pH, 7.4 PBS.

Two, immunogen and coating antigen coupling ratio are measured:

Immunogen and coating antigen coupling ratio measuring method: trinitro-benzene-sulfonic acid method (TNBS method), detailed process is as follows:

1, carrier proteins BSA or OVA are dissolved in 0.1 M, in pH 8.5 sodium hydrogen carbonate solutions, are mixed with 0 μ g/mL, 20 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL, 100 μ g/mL, 120 μ g/mL are totally 7 concentration gradients (being used for the drawing standard curve); Simultaneously, testing sample is mixed with in the same way the concentration of 150 μ g/mL.

2, TNBS is dissolved in 0.1 M, in pH 8.5 sodium hydrogen carbonate solutions, is mixed with 0.01%(w/v) solution.

3,0.5 mL TNBS solution is added respectively in 6 standard specimens of 1 mL and 1 testing sample, mixing is hatched 2 h under 37 ℃.

4, in above-mentioned 7 samples, add 0.5 mL, 10% SDS and 0.25 mL, 1 N HCL.

5,335 nm places measure the OD value of each sample, and the drawing standard curve also calculates primary amine content in the testing sample.

The OD of table 1 BSA and OVA standard ₃₃₅

BSA protein standard strength of solution (μ g/mL)	0	20	40	60	80	100	120
								The OD value	0	0.014	0.031	0.046	0.06	0.075	0.09
OVA protein standard strength of solution (μ g/mL)	0	20	40	60	80	100	120
								The OD value	0	0.015	0.032	0.045	0.06	0.076	0.092

See Fig. 1 and Fig. 2 by upper table institute column data drawing standard curve, BSA, OVA standard protein are y=0.0008x at 335 OD of nm place value typical curves under the different concns

By ultraviolet determination, at 335 nm places, AOZ-BSA, the OD value of AOZ-OVA sample is respectively 0.053,0.072.

It is coupled than the mensuration formula according to TNBS method antigen,

n=B×（100-A/k）%

In the formula, B: carrier proteins free amino group number (BSA, OVA standard protein free amino group number are respectively 35,20), A: testing sample is in the OD value at 335 nm places, and K: standard protein is in 335 OD of nm place value slope of standard curve under the different concns.

The coupling ratio of sample AOZ-BSA is 35 * (100-0.053/0.0008) %=11

The coupled ratio of sample AOZ-OVA is 20 * (100-0.072/0.0008) %=2

By the method, the coupling ratio that calculates immunogen AOZ-BSA is 11, and the coupling ratio of coating antigen AOZ-OVA is 2.

B. animal immune:

Adopt the Balb/c mouse as immune animal, immunogen is AOZ-BSA, each immunizing dose 〉=50 a μ g/ mouse.

C. screen animal immune serum:

Above immunized mice used ELISA method and indirect competitive ELISA method to detect serum titer in rear 7-10 days in for the second time immunity ^[2]Choose the high mouse of serum titer and carry out booster immunization.

D. prepare hybridoma:

Get the splenocyte of above-mentioned immune Balb/c mouse, the PEG 4000 with 50% makes fusogen, and immune spleen cell and SP 2/0 myeloma cell are carried out cytogamy by a certain percentage.Adopt indirect elisa method to detect the cells and supernatant that merges rear survival, positive colony is carried out subclone, detect the cells and supernatant that mono-clonal growth hole is arranged, positive rate reaches 100%, obtains the hybridoma cell strain 1C3 of stably excreting monoclonal antibody;

The step of setting up indirect elisa method in this step is as follows:

Best antigen coated dilution selection, adopt the square formation volumetry to determine coating antigen concentration:

(1) coated: the carbonate buffer solution with pH 9.6 is diluted to coating antigen in a series of concentration adding enzyme plates 100 μ L/ holes, 4 ℃ of coated spending the night;

(2) washing: get rid of clean coating buffer, washings is the PBS that contains the pH 7.4 of 1 ‰ tweens, with washing machine-wash plate 3 times of plate, and pats dry at thieving paper;

(3) sealing: every hole adds the BSA confining liquid of 200 μ L, hatches 1 h for 37 ℃;

(4) wash same step (2);

(5) primary antibodie: add the antibody of series concentration, 1 h is hatched for 37 ℃ in 100 μ L/ holes;

(6) wash same step (2);

(7) ELIAS secondary antibody: add sheep anti mouse-HRP, 1 h is hatched for 37 ℃ in 100 μ L/ holes, washs same step (2);

(8) colour developing: every hole adds the TMB nitrite ion of 100 μ L, 20-25 ℃ of colour developing 10 min;

(9) termination reaction: the H that adds 2M ₂SO ₄The stop buffer termination reaction, 50 μ L/ holes, and on microplate reader, read the OD450 value.

Criterion: about 1.0, determine that the best coated extent of dilution of coating antigen is 1:40000 according to the OD value, the optimum antibody extent of dilution is 1:8000.The best effort concentration of antigen, antibody sees Table 2.

Determining of table 2 antigen-antibody optimum dilution degree

The step of setting up the indirect competitive ELISA method in this step is as follows:

(1) coated: the carbonate buffer solution with pH 9.6 is diluted to suitable concn with coating antigen, adds in the enzyme plate 100 μ L/ holes, coated spending the night;

(2) washing: get rid of clean coating buffer, washings is the PBS that contains the pH 7.4 of 1 ‰ tweens, with washing machine-wash plate 3 times of plate, and pats dry at thieving paper;

(3) sealing: every hole adds the BSA confining liquid of 200 μ L, hatches 1h for 37 ℃;

(4) washing is the same;

(5) application of sample: will add the Nifurazolidone pharmaceutical standards product 50 μ L/ holes of proper concn in the enzyme plate hole, adjacent two hole first; Adding the good sheep anti mouse-HRP 50 μ L/ holes of dilution; Add at last the good antibody 50 μ L/ holes of dilution, enzyme plate slightly makes to shake mixing, hatches 30 min for 25 ℃;

(6) washing is the same;

(7) colour developing: every hole adds the TMB nitrite ion of 100 μ L, 20-25 ℃ of colour developing 15 min;

(8) termination reaction: the H that adds 2 moL/L ₂SO ₄The stop buffer termination reaction, 50 μ L/ holes, and on microplate reader, read the OD450 value.

Criterion: at first, can find out from naked eyes, suppress the variation that Kong Yuwei suppresses the hole color, if it is more shallow or colourless to suppress the hole color, has illustrated that specific antibody produces, otherwise then produced without specific antibody.Secondly, can judge according to the OD value, suppress hole OD value less than not suppressing hole OD value, illustrate that specific antibody produces.Employing has the immune mouse spleen cell of inhibition serum to carry out cytogamy, and filters out the hybridoma cell strain of energy secreting specificity antibody.

The power of antibodies specific can be judged according to the size of competition inhibiting rate.

Competition inhibiting rate=1-B/B0(B adds the inhibition hole OD value of competing thing, and B0 is not for adding the positive control hole OD value of competing thing).

E. prepare and monoclonal antibody purification titration:

Preparation and identification monoclonal antibody at first: take out cryopreservation tube during cell recovery, melt in 37 ℃ of water-baths immediately, move into afterwards enlarged culturing in the culture dish, substratum is the DMEM substratum that contains 10% FBS.Wherein, in the cryopreservation tube frozen be logarithmic phase can the stably excreting monoclonal antibody hybridoma cell strain, called after 1C3.

With sterilization paraffin immunity Balb/c mouse, mouse peritoneal injection hybridoma 1C3 after 7 days, injected dose is 1.5 * 106/, gathers ascites, and carries out subsequently the monoclonal antibody purifying in 7-10 days.

Sad-the ammonium sulfate salting-out process monoclonal antibody purification, concrete steps are as follows:

(1) gets the ascites of 1 times of volume, add the NaAC-HAc damping fluid of 2 times of volumes;

(2) add 10% sad amount by every mL ascites, add good after, room temperature is shaken 30 min on shaking table, 4 ℃ leave standstill 2 h afterwards;

(3) with 4 ℃ of 12000 centrifugal 30 min of rpm, get supernatant, add 0.1 M PBS of 1/10 volume, transfer pH to 7.4 with NaOH subsequently;

(4) in supernatant, add isopyknic saturated ammonium sulphate solution, 4 ℃ leave standstill 1 h after, 4 ℃, centrifugal 10 min of 12000 rpm;

(5) abandon supernatant, suspend with an amount of PBS and precipitate;

(6) with monoclonal antibody suspension dialysis 3 times, be no less than 2 h at every turn;

(7) monoclonal antibody after the dialysis is done and is further purified, and liquid phase column chromatography elution buffer is 20 mM Tris-HCl, 1 M NaCl, and the every pipe of pH 8.2,1 mL is collected monoclonal antibody.

(8) monoclonal antibody behind the purifying carries out its purity of SDS-PAGE electrophoresis detection.Utilize ultraviolet spectrophotometer to measure its concentration, the ELISA method is measured monoclonal antibody and is tired packing, cryopreservation;

(9) the monoclonal antibody mensuration of tiring: with the coated elisa plate of the coating antigen AOZ-OVA of 0.1 μ g/mL, 1C3 ' the monoclonal antibody of purifying is carried out 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000 dilution, add in the enzyme plate hole, the sheep anti-mouse igg that adds the HRP mark after the reaction, with the TMB colour developing, the titration of 1C3 ' the results are shown in Table 3 at last.

Table 3 antibody titer is measured

Positive judgement standards: P/ N 〉=2.1

1C3 ' antibody test result: when antibody purification concentration was 1 mg/mL, tiring to reach 1.28 * 10 ⁵More than;

(10) mensuration of monoclonal antibody avidity

The ELISA method of introducing according to Gosling is carried out the mensuration of affinity of antibody, and the size of its avidity represents that with the size of affinity costant Ka formula is:

The unit of affinity costant is the inverse of concentration, that is: molconcentration (L/moL), and tightness degree of the higher expression antigen-antibody of its value combination is higher.Its testing process is as follows:

The first step, defined antigen, antibody the best use of concentration:

1, artificial antigen is carried out respectively 5000,10000,20000,40000, each is coated with 4 after 80000,160000 times of dilutions, 100 L/ holes, 37 ℃, incubation 2 h, washing, sealing;

2, antibody is carried out 1000,2000,4000,8000,16000,32000,64000 times of dilutions are added on respectively in 2,100 L/ holes, room temperature incubation 1 h;

3, the antibody in these two is moved into respectively in the second room temperature incubation 1 h;

4, these two washings are added ELIAS secondary antibody, continue to be ELISA, measure at last OD value A1;

5, press above-mentioned steps for rear two, measure at last OD value A2.

6, according to formula

Calculate f value, choose all f values all less than 10% antigen, antibody dilution is 80000 times of dilutions according to the big or small definite best antigen concentration of OD value, and optimum antibody concentration is 32000 times of dilutions.

Second step, mensuration avidity

1, a series of concentration antigens of preparation are 6;

2, add equivalent series concentration antigen in being added with the EP pipe of optimum concn antibody, totally 7 manage, last pipe adds antigenic dilution, spends the night under the room temperature;

3, antigen is carried out being coated with after 80000 times of dilutions simultaneously, spend the night under the room temperature, wash plate, sealing;

4, the reaction product of second step is got 100 μ L and add in the above plate hole that seals, carry out ELISA under the room temperature, measure at last OD value A;

5, according to the AOatchard formula

Calculate its slope value

Wherein, α is the concentration of free antigen, and V is the binding antibody site and the ratio of total antibody sites, and the size of gained avidity is affinity costant Ka, is the negative of slope value.The result who obtains is that the avidity of monoclonal antibody 1C3 ' is 5.4 * 10 ⁹

Embodiment two

Medicine cross reaction test

Undertaken by the monoclonal antibody screening indirect competitive ELISA method of setting up among the embodiment one, the test that is at war with of the metabolite derivative of the monoclonal antibody of Furaxone metabolite derivative and Nifurazolidone haptens analog such as furadantin, Furaltadone, Nifurazolidone, these standard substance are diluted to different concns carry out indirect competitive ELISA, draw and suppress curve, calculate IC50 value and the cross reacting rate of competition thing, the result shows that the cross reaction of this monoclonal antibody and four kinds of similar metabolic derivatives of Nifurazolidone is all very low, and concrete outcome sees Table 4.

Table 4 Furaxone metabolite and other nitrofurans metabolite cross reacting rate

Kind	%CR	Kind	%CR
				The Furaxone metabolite derivative	100%	The Cistofuran metabolite derivative	<0.1%
The AMOZ derivative	<0.1%	The Furaxone metabolite derivative	<0.1%

Embodiment three

Present embodiment is that monoclonal antibody 1C3 ' can be used for muscle setting up the applicating example that detects the residual ELISA method of Furaxone metabolite among the present invention, fishery products, and the residual detection of Furaxone metabolite in the milk.Take shrimp (market purchase) as example, illustrate that to detect the residual key step of Furaxone metabolite in the shrimp as follows:

1. sample pre-treatments

(1) shrimp sample with homogenizer homogeneous sample, takes by weighing the equal pledge of 1.0 ± 0.05 g (shrimp sample), adds 15 mL centrifuge tubes.

(2) distilled water of adding 4.0 mL, 0.5 mL, 1 M HCl and 100 μ l derivatization reagents, fully vibration.

(3) hatching 16 h(at 37 ℃ spends the night).

(4) add respectively 1.0 mL, 0.5 M K ₂HPO ₄, 0.4 mL, 1 M NaOH, the ethyl acetate of 2 g NaCl and 6 mL, thermal agitation 5 minutes.

(5) at room temperature the centrifugal 10min of (20 ~ 25 ℃) 4000 rpm.

The ethyl acetate upper strata of (6) taking out 3 mL dries up with nitrogen in 50 ℃ of water-baths in another 15 mL centrifuge tube.

(7) dissolve dry thing with 1.0 mL normal hexanes, add 1.0 mL redissolution liquid and fully shake mixing 5 min; Centrifugal 5 min of (20 ~ 25 ℃) 4000 rpm at room temperature.

(8) getting 50 μ L lower floor solution is used for analyzing.

2. detect

The detection principle of test kit is the indirect competitive ELISA method among the present invention, coating antigen AOZ-OVA is coated on the microwell plate, add respectively 4.05 of 50 μ L, 1.35,0.45,0.15,0.05 and the AOZ derivative of 0 ng/mL or the sample of derivatize, the monoclonal antibody 1C3 ' that adds the anti-Furaxone metabolite of 50 μ L, Furaxone metabolite residual in the sample is combined with the antibody competition of anti-Furaxone metabolite derivative with coated antigen simultaneously, add 50 μ L ELIAS secondary antibody, the TMB colour developing is read OD450 in microplate reader after the colour developing, and the content of Nifurazolidone medicine and sample absorbance are negative correlation in the sample, compare with typical curve, can judge that when the reference value that provides is provided the OD value Furaxone metabolite that contains in the sample surpasses detectability.

Embodiment four

Present embodiment is the applicating example of monoclonal antibody 1C3 ' in the residual colloidal gold strip of preparation Furaxone metabolite among the present invention, mainly is that to be applied to the Furaxone metabolite that detects in muscle, milk, the fishery products residual.

Reaction principle adopts competition law that Furaxone metabolite is carried out half-quantitative detection, the Furaxone metabolite derivative molecular that exists in the sample is moving past the antibody 1C3 ' combination of Cheng Zhongxian and gold grain mark along test strip, the coating antigen and the Furaxone metabolite derivative that are fixed on the NC film are competed the joining gold labeling antibody simultaneously, the content of residual Furaxone metabolite is inversely proportional in the colour developing power of T line and the sample, if residual without Furaxone metabolite in the sample, then golden labeling antibody all reacts with coating antigen, the T line colour developing of test strip.When C, T line represent feminine gender when all developing the color, when not developing the color, C line colour developing T line then is expressed as the positive, when the C line does not develop the color, T line colour developing or do not develop the color and represent that all test strip lost efficacy.

(1) concrete operation step is as follows:

1, sample pre-treatments: the treatment process of various samples is with the sample-pretreating method in the ELISA method in present method;

2, test strip is put on the clean smooth table top, draws testing sample solution with dropper, drip 1～2 on sample pad;

3, wait for the appearance of red-purple band, read test result in the time of standing and reacting 5-10 minute, later on judgement in 10 minutes is invalid.

In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.

  

Reference

1、Greg?T.?Hermanson.?Bioconjugate?techniques.?Academic?Press，2008:216-219.

2, Yang Liguo, " enzyme immunoassay technique ", press of Nanjing University, 1998.

3.?Gary?C.?Howard.?Making?and?Using?Antibodies:?A?Practical?Handbook.?CRC?Press，2006.127-130。

Claims (4)

1. a hybridoma cell strain 1C3 who produces the monoclonal antibody of Furaxone metabolite derivative is that mouse hybridoma cell is CGMCC No.5071.

2. the monoclonal antibody 1C3 ' that produces of described hybridoma cell strain 1C3 according to claim 1.

3. described monoclonal antibody 1C3 ' according to claim 2, it is characterized in that: described monoclonal antibody hypotype is the IgG1 type.

4. the according to claim 2 application of described monoclonal antibody 1C3 ' in detecting Furaxone metabolite.

Images