CN102936584A - Semicarbazide derivative monoclonal antibody and applications thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody and applications thereof, especially to a semicarbazide (nitrofural metabolite) derivative monoclonal antibody and applications thereof, and belongs to the technical field of immunochemistry. The semicarbazide derivative monoclonal antibody 9B10' is produced by mouse hybridoma cell line 9B10, wherein a subtype is IgG1 type. According to the present invention, the monoclonal antibody 9B10' produced by the mouse hybridoma cell line 9B10 can be abundantly produced, and can be used to prepare an enzyme linked immunoassay kit and colloidal gold test paper strips for semicarbazide residue detection so as to achieve a purpose of rapid and sensitive detection of semicarbazide residues in honey and animal tissues.

Description

The monoclonal antibody of semicarbazide derivative and application thereof

Technical field

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

Background technology

Nitrofural (nitrofurazone) belongs to the itrofurans medicine, is a kind of broad spectrum antibiotic, because of cheap, effective, be used as medicine and fodder additives and control disease for poultry, fowl, aquaculture.Pharmacophoric group-nitro in nitrofural molecule major cause that to be it produce as pharmacological action and toxicity, simultaneously also so that nitrofural have teratogenesis, carinogenicity and bring out the side effect such as mammalian cell chromosome damage.European Union classifies nitrofural as the forbidding medicine in nineteen ninety-five.In No. 193 bulletin of the Ministry of Agriculture's in April, 2002 issue, nitrofural is put in " veterinary drug and other compound inventory of food animal forbidding ", in all food animals, forbid, again metabolites of nitrofuran in the fishery products was included in the residual Supervisory Surveillance Program in 2003.But because nitrofural has good treatment and preventive effect, still in aquaculture, poultry, fowl, be widely used at present, thereby cause increasingly sternness of monoconal antibody mediated furacilinum residue in the food.Therefore, healthy for Protection of consumer, strengthen Chinese animal derived food quarantine dynamics, the outlet that extends trade, residual detection is very important to Urea,amino-in the animal derived food (SEM).

Nitrofural prototype medicine is metabolized to Urea,amino-in animal body very soon, the meta-bolites of combination then can retain long period of time in tissue, be regarded as the marker of monoconal antibody mediated furacilinum residue, so usually analyze monoconal antibody mediated furacilinum residue by analyzing its meta-bolites SEM.The detection method that is used at present monoconal antibody mediated furacilinum residue marker SEM has the methods such as liquid-liquid extraction-high performance liquid chromatography, Solid-Phase Extraction-high performance liquid chromatography (SPE-HPLC), liquid chromatography mass combined instrument.But the required instrument of these instrumental methods is expensive, and length consuming time to having relatively high expectations of operator, is not suitable for promoting in grass-roots unit, is not suitable for high-throughout sample rapid screening.Immunochemical analyses method particularly enzyme-linked immunosorbent analytical technique (ELISA) has the advantages such as quick, highly sensitive, simple to operate, that specificity is good, is fit to high-throughout sample screening.

This research is carried out the derivatize experiment with the small-molecule drug Urea,amino-with terephthalaldehydic acid, introduce active group-carboxyl, and by the complete antigen of mixed anhydride method with this derivative and carrier protein couplet acquisition synthetic, the complete antigen that this method is synthetic, effect by crosslinked arm terephthalaldehydic acid, the small-molecule drug Urea,amino-fully can be come out, make body more easily identify antigenic determinant, improve the immunogenicity of antigen.With the complete antigen immunity Balb/c mouse of preparation, obtain to identify the monoclonal antibody of semicarbazide derivative by merging screening, thereby lay the foundation for the immunological method of Furacilin metabolite residue detection.

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

Hybridoma cell strain 9B10 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.5072, and Classification And Nomenclature is the hybridoma cell strain of anti-semicarbazide derivative monoclonal antibody.

By the monoclonal antibody called after 9B10 ' of above hybridoma cell strain secretion, its preparation process is as follows:

A. prepare immunogen and coating antigen: adopt the terephthalaldehydic acid method with carrier proteins (BSA) and the coupling of Urea,amino-small molecules, synthetic immunogen and coating antigen;

B. animal immune injection: with the Balb/c mouse as immune animal, abdominal injection immunogen and booster immunization;

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-semicarbazide derivative monoclonal antibody of stably excreting hybridoma cell strain 9B10;

E. prepare and monoclonal antibody purification: to mouse peritoneal injection hybridoma 9B10, gather ascites, ascites is carried out the liquid chromatography (LC) purifying, obtain semicarbazide derivative monoclonal antibody 9B10 '.

Wherein, the described carrier proteins of step a is bovine serum albumin and oralbumin, and corresponding immunogen is SEM-BSA, and coating antigen is SEM-OVA; In the described terephthalaldehydic acid method, the time of linking agent activated micromolecular derivative is 1-2 hour, reacts 10-15 hour with Urea,amino-small molecules derivative and carrier protein couplet.Each time immunization dosage among the described step b is 50-100 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.

The conventional method for preparing the semicarbazide derivative monoclonal antibody mainly is the amino coupled by carboxyl and the carrier proteins of medicine, the present invention carries out derivatize with the Urea,amino-small molecules by terephthalaldehydic acid and with the amino coupled of carboxyl and the carrier proteins of derived products, thereby expose Urea,amino-structure, in the later stage screening process, can obtain the antibody of pin semicarbazide derivative.

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

The invention has the beneficial effects as follows: by the monoclonal antibody 9B10 ' of mouse hybridoma cell strain 9B10 generation, this antibody and other nitrofurans cross reacting rate are low, 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 Furacilin metabolite, reach quick, special and detect delicately the residual purpose of Furacilin metabolite in honey and the animal tissues, lay the foundation for detecting residual ELISA test kit, the Radioactive colloidal gold product development of Furacilin metabolite.

Description of drawings

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

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

Embodiment

Embodiment one

Reagent and material are prepared

Nitrofural (Town in Shanghai spectrum scientific instrument company limited, C15571000), SEM (Town in Shanghai spectrum scientific instrument company limited, C16933500), terephthalaldehydic acid (Sigma 124 91-5), DMF(sigma, D4551), 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.

Experimental procedure of the present invention is as follows:

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

Artificial antigen of the present invention adopts terephthalaldehydic acid with the Urea,amino-derivatize, and utilizes carboxyl and the amino on the carrier proteins on the semicarbazide derivative to carry out coupling, thus synthetic nitrofural antigen.The semicarbazide derivative structural formula is as follows:

One, complete antigen is synthetic

1,15 ℃ of concussion reaction 24 h in 1.5 mL pyridine solutions with 2 mg Urea,amino-standard substance and 2.7 mg terephthalaldehydic acids.

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 bath 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 24 h.

4, dialyse among 0.01 M pH, 7.4 PBS.

Two, the complete antigen coupling ratio is measured:

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

1, carrier proteins BSA or OVA are dissolved in 0.1 M, in the sodium hydrogen carbonate solution of pH 8.5, are mixed with 0 mg/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 1N HCL.

5,335 nm places measure the OD value of each concentration standard product, see Table 1, according to OD value drawing standard curve and calculate 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
								OD 335Value	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
								OD 335 Value	0	0.015	0.032	0.045	0.06	0.076	0.092

By upper table institute's column data drawing standard curve such as Fig. 1 and Fig. 2, 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, SEM-BSA, the OD value of SEM-OVA sample is respectively 0.069,0.064.

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

n=B×（100-A/k）%

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

K: standard protein is in 335 OD of nm place value slope of standard curve under the different concns

The coupling ratio of sample SEM-BSA is 35 * (100-0.069/0.0008) %=5

The coupled ratio of sample SEM-OVA is 20 * (100-0.064/0.0008) %=4

By the method, the coupling ratio that calculates immunogen SEM-BSA is 5, and the coupling ratio of coating antigen SEM-OVA is 4.

                                      

B. animal immune:

Adopt the Balb/c mouse as immune animal, immunogen is SEM-BSA, each immunizing dose 〉=50 a μ g/ mouse, and immunity is more than twice.

The screening 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.

C. prepare hybridoma:

Get the splenocyte of above-mentioned immune Balb/c mouse, the PEG 4000 with 50% makes fusogen, and immune spleen cell and SP2/0 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 9B10 of stably excreting monoclonal antibody;

One, set up the step of indirect elisa method in this step as follows:

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

(1) coated: the carbonate buffer solution with pH9.6 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 OD ₄₅₀Value.

Criterion: be about 1.0 according to the OD value, determine that the best coated extent of dilution of coating antigen is 1:160000, the antibody optimum dilution degree is 1:12800.The best effort concentration of antigen, antibody sees Table 2.

Determining of table 2 antigen-antibody optimum dilution degree

Two, set up the step of indirect competitive ELISA method in this step 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 1 h for 37 ℃;

(4) washing is the same;

(5) application of sample: will add the semicarbazide derivative standard substance 50 μ L/ holes of proper concn in the enzyme plate hole, adjacent two hole first; Add again the good sheep anti mouse-HRP 50 μ L/ holes of dilution; Add at last the antibody 50 μ L/ holes of the good anti-semicarbazide derivative of dilution, enzyme plate slightly makes to shake mixing, hatches 1 h 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 10 min;

(8) termination reaction: the H that adds 2 M ₂SO ₄The stop buffer termination reaction, 50 μ L/ holes, and on microplate reader, read OD ₄₅₀Value.

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).

D. prepare and monoclonal antibody purification titration:

One, Preparation and identification monoclonal antibody:

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.In the cryopreservation tube frozen be logarithmic phase can the stably excreting monoclonal antibody hybridoma cell strain 9B10.

Two, ascites preparation and purifying:

With sterilization paraffin immunity Balb/c mouse, mouse peritoneal injection hybridoma 9B10 after 7 days, injected dose is 1.5 * 10 ⁶Individual/as only, to gather ascites, and carried 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 mMTris-HCL, 1 M NaCL, and pH 8.2,1 ml/ pipe is collected antibody.

Three, the detection behind the antibody purification

1, the 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;

2, the monoclonal antibody mensuration of tiring: with the coated elisa plate of the coating antigen SC-OVA of 0.1 μ g/ml, 9B10 ' the monoclonal antibody of purifying is carried out 1:20000,1:40000,1:80000,1:160000,1:320000,1:640000,1:1280000 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 9B10 ' the results are shown in Table 3 at last.

Table 3 antibody titer is measured

Positive judgement standards: P/ N 〉=2.1

9B10 ' antibody test result: when antibody purification concentration was 1 mg/ml, tiring to reach 1.28 * 10 ⁶Above;

E. the 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 with the size of affinity costant Ka, and 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 40000,80000,160000,320000, each is coated with 4 after 640000,1280000 times of dilutions, 100 μ L/ holes, 37 ℃, incubation 2 h, washing, sealing;

2, antibody is carried out 1600,3200,6400,12800,25600,51200,102400 times of dilutions are added on respectively in 2,100 μ 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 640000 times of dilutions according to the big or small definite best antigen concentration of OD value, and optimum antibody concentration is 51200 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 640000 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 Scatchard formula

Calculate its slope value,

In the formula, 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 9B10 ' is 2.1 * 10 ¹⁰

Embodiment two

Medicine cross reaction test

Undertaken by the monoclonal antibody screening indirect competitive ELISA method of setting up among the embodiment one, Urea,amino-the test that is at war with of the metabolite derivative of monoclonal antibody and Urea,amino-haptens analog such as furadantin, Furaltadone, Nifurazolidone, these metabolite derivative standard substance are diluted to different concns carry out indirect competitive ELISA, draw and suppress curve, calculate the IC of competition thing ₅₀Value and cross reacting rate, the result shows the cross reacting rate of this antibody and other three kinds of furans metabolite derivatives all less than 0.1%, concrete outcome sees Table 4.

Table 4 semicarbazide derivative and other nitrofurans derivative cross reacting rate

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

Embodiment three

The present embodiment is that monoclonal antibody 9B10 ' can be used for honey and the residual detection of animal tissues's Furacilin metabolite setting up the applicating example that detects the residual ELISA method of Furacilin metabolite among the present invention.Take shrimp (market purchase) as example, illustrate that to detect the residual key step of Furacilin metabolite in the shrimp as follows:

1, sample pre-treatments

(1) the shrimp sample takes by weighing the equal pledge of 1.0 ± 0.05 g with homogenizer homogeneous 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 K2HPO4,0.4 mL, 1 M NaOH, the ethyl acetate of 2 g NaCl and 6 mL, thermal agitation 5min;

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

The ethyl acetate upper strata of (6) taking out 3mL 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; The centrifugal 5min of (20 ~ 25 ℃) 4000 rpm at room temperature;

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

2, the detection principle of test kit is the indirect competitive ELISA method among the present invention, coating antigen SEM-OVA is coated on the microwell plate, add respectively 4.05 of 50 μ L, 1.35,0.45,0.15,0.05 and semicarbazide derivative or the sample of 0 ng/mL, the monoclonal antibody 9B10 ' that adds 50 μ L semicarbazide derivatives, in the sample residual derivatize Urea,amino-with coated antigen simultaneously with anti-Urea,amino-antibody competition be combined, add 50 μ L ELIAS secondary antibody, the TMB colour developing is read OD in microplate reader after the colour developing ₄₅₀, in the sample Urea,amino-content and sample absorbance be negative correlation, compare with typical curve, can judge that when the reference value that provides is provided the OD value Urea,amino-that contains in the sample surpasses detectability.

Embodiment four

The present embodiment is the applicating example of monoclonal antibody 9B10 ' in the residual colloidal gold strip of preparation Furacilin metabolite among the present invention, mainly is that to be applied to detect in honey, the animal tissues Furacilin metabolite residual.

Reaction principle adopts competition law that Furacilin metabolite is carried out half-quantitative detection, the derivatize Urea,amino-molecule that exists in the sample is moving past the antibody 9B10 ' combination of Cheng Zhongxian and gold grain mark along test strip, the coating antigen and the Urea,amino-that are fixed on the NC film are competed the joining gold labeling antibody simultaneously, the colour developing of T line strong and weak with sample in residual Urea,amino-content be inversely proportional to, if residual without Urea,amino-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 embodiment.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.?Bioconjμgate?techniqμes.?Academic?Press,?2008:216-219.

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

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

Claims (4)

1. a hybridoma cell strain 9B10 who produces the monoclonal antibody of semicarbazide derivative is that mouse hybridoma cell is CGMCC No.5072.

2. the monoclonal antibody 9B10 ' that produces of described hybridoma cell strain 9B10 according to claim 1.

3. described monoclonal antibody 9B10 ' 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 9B10 ' in the detection Urea,amino-is residual.

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