CN101942414B - Hybridoma cell line and chloramphenicol-resistant monoclonal antibody produced by same - Google Patents

Abstract

The invention relates to a chloramphenicol-resistant monoclonal antibody, which is mainly used for detecting chloramphenicol residue in livestock products and milk products. The chloramphenicol-resistant monoclonal antibody is secreted from a hybridoma cell line 7C10; and the preparation method comprises the following steps of: coupling EDC-activated chloramphenicol and carrier protein to obtain immunogen and coating antigen; performing intraperitoneal injection of mouse immunogen and enhancing the immunity; screening out a mouse who is subjected to immunity enhancement and has high serum titer; fusing spleen cells and SP2/0 myeloma cells; performing sub-clone to obtain the monoclonal antibody hybridoma cell line 7C10 capable of stably secreting the chloramphenicol; performing intraperitoneal injection on the mouse with the hybridoma cell line 7C10; collecting ascitic fluid; and performing liquid-phase chromatographic purification to obtain the chloramphenicol-resistant monoclonal antibody 7C10'. The chloramphenicol-resistant monoclonal antibody has the advantages of high specificity, high flexibility and affinity, large-scale production and effect of quickly and sensitively detecting the chloramphenicol residue in foods such as milk, meats, aquatic products and the like.

Description

The chloramphenicol resistance monoclonal antibody of a kind of hybridoma cell strain and generation thereof

Technical field

The present invention relates to chloramphenicol resistance monoclonal antibody specific, the hybridoma cell strain that produces the chloramphenicol resistance monoclonal antibody, the preparation method and the application in chlorine detection mycin drug residue thereof of chloramphenicol resistance monoclonal antibody specific, belong to the immunochemical technique field.

Background technology

Paraxin (Chloramphenicol) is a kind of broad-spectrum antibacterial agent.But get in the bacterial cell through fat-soluble disperse, mainly act on the ribosomal 50s subunit of bacterium 70s, suppress transpeptidase; The growth of peptide chain is obstructed; Suppressed the formation of peptide chain, thereby stoped proteinic synthesizing, strong to the bacteriostatic action of Gram-negative bacteria than gram-positive microorganism.Paraxin once was widely used in the infection of the various sensitive organisms of treatment, and the back has serious adverse reaction because of it to hemopoietic system, so strict control has been made in its clinical application at present.The main adverse reaction of paraxin is to suppress the marrow hemopoiesis function: 1. reversible pancytopenia, wherein granulocyte at first descends.2. irreversible aplastic anemia, its principle maybe be relevant with the identical 70S ribosome with bacterium in the plastosome in paraxin suppresses myeloid element.Paraxin also can produce gastrointestinal reaction and superinfection.In addition, can cause anaphylaxis such as fash and vasodilation under the few cases.

In order to reduce the harm of this medicine to the mankind, Food and Argriculture OrganizationFAO just forbade the use of paraxin in 1994.The chloramphenicol residue standard is " zero tolerance " in many national regulation animal derived foods such as European Union, the U.S., promptly must not detect.China also put into effect relevant regulations in 1999, classified paraxin as forbidden drugs, in food, must not detect.But because paraxin is cheap; Evident in efficacy; Remain incessant after repeated prohibition in clinical use of culturing with use; In order to strengthen, need the monoclonal antibody of preparation high specificity, mass producible chloramphenicol resistance, to adopt residual in animal food of paraxin enzyme-linked immunosorbent assay test kit and colloidal gold chromatography rapid detection paraxin to the residue detection and the supervision of paraxin medicine in animal food.Yet itself be difficult to induce body to produce antibody as the haptenic paraxin of small molecules, must its complete antigen with carrier protein couplet acquisition synthetic be just had stronger antigenicity.The screening of positive colony all directly affects the preparation effect of antibody after the coupling effect of small molecules antigen and carrier proteins and the cytogamy, is the key of Antibody Preparation.

Summary of the invention

The objective of the invention is shortcoming to above prior art existence; Provide a kind of and paraxin and analogue cross reaction thereof weak, avidity and highly sensitive and have specific, mass producible chloramphenicol resistance monoclonal antibody and this a chloramphenicol resistance MONOCLONAL ANTIBODIES SPECIFIC FOR method.

Another object of the present invention provides the hybridoma cell strain of this specific anti paraxin monoclonal antibody of secretion.

The objective of the invention is to realize through following technical scheme:

A kind of hybridoma cell strain 7C10 that produces the chloramphenicol resistance monoclonal antibody, its preserving number is CGMCCNo.3574.

A kind of by hybridoma cell strain 7C10 excretory chloramphenicol resistance monoclonal antibody 7C10 '.

The preparation method of above-mentioned chloramphenicol resistance monoclonal antibody 7C10 ' may further comprise the steps:

A. prepare immunogen and coating antigen:

Paraxin is with carbodiimide hydrochloride (EDC) activation, then with carrier proteins and the coupling of activatory paraxin get immunogen and coating antigen (Greg T.Hermanson.Bioconjugate techniques.AcademicPress, 2008:216-219.);

B. animal immune injection:

With the Balb/c mouse as immune animal, abdominal injection immunogen and booster immunization;

C. screen immune serum:

Balb/c mice serum antibody titer behind the detection booster immunization filters out the high mouse of serum titer and carries out booster immunization;

D. prepare hybridoma:

The splenocyte and the SP2/O myeloma cell of mouse of booster immunization of learning from else's experience carries out cytogamy, through at least 2 subclones, obtains the monoclonal antibody hybridoma cell strain 7C10 of stably excreting chloramphenicol resistance;

E. prepare and monoclonal antibody purification 7C10 ':

With hybridoma 7C10 mouse is carried out abdominal injection, gather ascites and obtain chloramphenicol resistance monoclonal antibody 7C10 ' through the LC purifying.

Wherein, among the step a, be carrier proteins with bovine serum albumin or human serum albumin, get immunogen CAP-BSA or CAP-HAS with the paraxin linked reaction; With the oralbumin is carrier proteins, gets coating antigen CAP-OVA with the paraxin linked reaction.

The reaction times of said carbodiimide hydrochloride (EDC) activation paraxin, paraxin and carrier protein couplet reaction times were at least 10 hours less than 20min.

Each immunization dosage among the said step b is at least 50 a μ g/ mouse.

A further object of the present invention provides the qualitative or detection by quantitative that this chloramphenicol resistance monoclonal antibody 7C10 ' is applied to residual chloromycetin.

The invention has the beneficial effects as follows: produce high specificity, sensitivity and the high chloramphenicol resistance monoclonal antibody of avidity through mouse hybridoma cell strain 7C10; Low with other chloromycetin series antibiotics cross reacting rate; Can mass production; Can be used for preparing the enzyme-linked immunosorbent assay test kit and the colloidal gold strip of chlorine detection mycin, reach fast and detect milk, the effect of the paraxin drug residue in the food such as meat and fishery products delicately.

Description of drawings

Below in conjunction with accompanying drawing the present invention is further described.

Fig. 1 is a BSA protein standard substance mass spectrum; Fig. 2 is CAP-BSA immunogen mass spectrometric detection figure;

Fig. 3 is an OVA protein standard substance mass spectrum; Fig. 4: CAP-OVA coating antigen mass spectrometric detection figure; Fig. 5 is a HSA protein standard substance mass spectrum.

Fig. 6: CAP-HSA immunogen mass spectrometric detection figure; Fig. 7 is that the hypotype of monoclonal antibody 7C10 ' is identified figure;

Fig. 8 is the canonical plotting of monoclonal antibody 7C10 ' and paraxin reaction.

Produce the hybridoma cell strain 7C10 of chloramphenicol resistance monoclonal antibody; Its preserving number is CGMCCNo.3574; Sent China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 31st, 2009; Institute of Microorganism, Academia Sinica), its preserving number is CGMCC No.3574.

Embodiment

Embodiment 1

Reagent and material are prepared

Chloramphenicol sodium succinate (sigma, C3787); Bovine serum albumin (BSA) (Amresco, 0332); Human serum albumin (HSA) (Sigma, A9511); Oralbumin (OVA) (Sigma, A5503); Carbodiimide hydrochloride (EDC) (Sigma, E6383); N-hydroxy-succinamide (NHS) (Sigma, 130672); N, and dinethylformamide (DMF) (Sigma, D4551); Freund's complete adjuvant (Sigma, F5881); Freund's incomplete adjuvant (Sigma, F5506); TMB (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); DMSO 99.8MIN. (DMSO) (Applichem, 0231); Macrogol 4000 (PEG4000) (Sigma, P7306); DMEM high glucose medium (Gibco, 11995); Foetal calf serum (Gibco, C2027050).

Laboratory animal and cell: Balb/c mouse (6-8 week age, female), available from Yangzhou University's animal center; SP2/O (murine myeloma cell) is infected with the immune Research center by biophysics institute of the Chinese Academy of Sciences to be provided.

Experimental procedure of the present invention is following:

A. prepare immunogen CAP-BSA, CAP-HSA and coating antigen CAP-OVA:

(1) chloramphenicol sodium succinate of getting 30mg is dissolved in the MES damping fluid of 300 μ l pH4.7;

(2) take by weighing 50mg carbodiimide hydrochloride (EDC) be dissolved in the 200 μ l zero(ppm) water A liquid;

(3) the A drop is added in the chloramphenicol sodium succinate solution of step (1), 25 ℃ of lucifuge concussions, reaction is more than 20 minutes.

(4) with BSA, HAS, each 20mg of OVA are dissolved in respectively in the MES buffered soln of 400 μ l pH4.7, and the reaction product of step (3) adds respectively in each solution, and 25 ℃ of lucifuge reactions are more than 10 hours;

(5) above-mentioned reaction product is dialysed respectively, leaves heart 30min with 13000, collect supernatant, obtain immunogen CAP-BSA, CAP-HSA and coating antigen CAP-OVA, be stored in-20 ℃ subsequent use.

(6) to BSA, HSA, the immunogen CAP-BSA after OVA protein standard substance and the dialysis, CAP-HSA and coating antigen CAP-OVA carry out mass spectrometric detection respectively, and the result sees Fig. 1～Fig. 6, can judge the coupling success from the diagram result.

B. animal immune:

Adopt the Balb/c mouse as immune animal, immunogen is CAP-HSA or CAP-BSA, each immunizing dose >=50 a μ g/ mouse, and more than twice, each dosage is identical.

C. screen immune serum:

Serum antibody titer is surveyed with indirect elisa method and indirect competitive ELISA method in the immune the last time back of above immunized mice 7-10 days.Choose the high mouse of serum titer and carry out booster immunization, immunity is got mouse spleen after three days and is carried out next step experiment.

D. prepare hybridoma:

Get the splenocyte of above-mentioned immune Balb/c mouse, the polyoxyethylene glycol with 50% (PEG) 4000 is made fusogen, and immune mouse spleen cell and SP2/O myeloma cell are carried out cytogamy in 2: 1～10: 1 ratio.Adopt indirect elisa method to detect the cells and supernatant that merges the back 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 7C10 of stably excreting monoclonal antibody.

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

Best antigen coated dilution selection, adopt the square formation method to confirm coating antigen concentration:

(1) encapsulate: with the carbonate buffer solution of pH9.6 coating antigen is diluted to a series of concentration and adds in the enzyme plates, 100 μ L/ holes, 4 ℃ encapsulate and spend the night;

(2) washing: washings is the PBS that contains the pH7.4 of 1 ‰ tween 20s, with washing plate machine washing plate 3 times, and on thieving paper, enzyme plate is clapped dried;

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

(4) washing is the same;

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

(6) washing is the same;

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

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

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

Criterion:, confirm that it is 1: 400000 that coating antigen the best encapsulates extent of dilution according to positive serum OD value and P/N >=2.1.

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

(1) encapsulate: the carbonate buffer solution with pH9.6 encapsulates 400000 times of dilutions of extent of dilution with coating antigen according to the best, add in the enzyme plate, and 100 μ L/ holes, 4 ℃ encapsulate and spend the night;

(2) washing: washings is the PBS that contains the pH7.4 of 1 ‰ tweens, with washing plate machine washing plate 3 times, and on thieving paper, enzyme plate is clapped dried;

(3) sealing: every hole adds the 0.5%BSA confining liquid of 200 μ L, hatches 1h for 37 ℃;

(4) washing is the same;

(5) application of sample: earlier in enzyme plate hole, adjacent two hole, add diluent, 50 μ L/ holes, the paraxin standard substance of adding proper concn in the adjacent hole, two holes therewith; 50 μ L/ holes last add dilution antibody well, 50 μ L/ holes toward institute in porose; Enzyme plate slightly shakes mixing, hatches 1h for 37 ℃;

(6) washing is the same;

(7) ELIAS secondary antibody: add the sheep anti mouse-HRP of dilution in 1: 10000,1h is hatched for 37 ℃ in 100 μ L/ holes, washs the same;

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

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

Criterion: at first, can find out, add paraxin and suppress the difference that Kong Yuwei adds paraxin inhibition hole color, if suppress hole lighter color or colourless, explain to the paraxin specific antibody to produce, otherwise then had the specific antibody generation from naked eyes.Secondly, also can judge, add paraxin and suppress hole OD value, explain to the paraxin specific antibody to produce less than adding paraxin inhibition hole OD value according to the OD value.Adopt detected result paraxin to have the immune mouse spleen cell of the serum that suppresses effect to carry out cytogamy, and filter out the hybridoma cell strain of ability secreting specificity antibody.

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

Competition inhibiting rate=1-B/B ₀(B adds the OD value that paraxin suppresses the hole, B ₀For not adding the OD value that paraxin suppresses the hole).

E. prepare and monoclonal antibody purification titration:

At first prepare and identify monoclonal antibody: take out frozen pipe during cell recovery, in 37 ℃ of water-baths, melt frozen thing immediately, will melt thing afterwards and move into the interior enlarged culturing of petridish, substratum is the DMEM substratum that contains 10%FBS.Wherein, in the frozen pipe frozen be in logarithmic phase can the stably excreting monoclonal antibody hybridoma cell strain 7C10.

With sterilization paraffin immunity Balb/c mouse, respectively every group of mouse peritoneal injected hybridoma 6611, ID>=10 after 7 days ⁶Individual/as only, to gather ascites, and obtained monoclonal antibody, called after 7C10 ' in 7-10 days.Employing is carried out the hypotype evaluation available from the mouse source monoclonal antibody hypotype identification kit (Mouse Monoclonal Antibody SubtypeIdentification Kit) of Pierce company to the resulting monoclonal antibody of the present invention; Its hypotype qualification result shows; Monoclonal antibody 7C10 ' is the IgG1 hypotype, and the result sees table 1 and Fig. 7.

Table 1. antibody subtype qualification result

Antibody subtype	7C10clone
		IgA	0.213
IgG3	0.205
		IgM	0.199
IgG1	1.397
		IgG2a	0.302
IgG2b	0.190
		The κ chain	0.895
The λ chain	0.208
		blank?control	0.201
negative	0.214

Monoclonal antibody purification (hereinafter to be referred as monoclonal antibody): with liquid chromatography above-mentioned ascites is handled, concrete steps are following:

(1) gets the ascites of 1 times of volume, add the NaAC-HAc damping fluid of 4 times of volumes;

(2) add the sad amount of 10-50% by every ml ascites, add good after, room temperature is shaken 30min on shaking table.4 ℃ leave standstill 3h afterwards;

(3) high speed centrifugation 30min gets supernatant, transfers pH to 7.4 with NaOH;

(4) the saturated ammonium sulphate solution of adding certain volume in supernatant, 4 ℃ leave standstill 1h.High speed centrifugation 10min afterwards;

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

(6) change the monoclonal antibody suspension over to the 12h that dialyses in the dialysis tubing;

(7) monoclonal antibody after the dialysis is done and is further purified, and used sample-loading buffer is 20mM Tris-HCL, and pH8.6, elution buffer are 20mMTris-HCL, 1MNaCL, and pH8.6, the 1ml/ pipe 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, tire cryopreservation;

(9) the monoclonal antibody mensuration of tiring: the coating antigen CAP-OVA with 0.05 μ g/mL encapsulates elisa plate, the 7C10 monoclonal antibody of purifying carried out 1: 8000, and 1: 16000,1: 32000; 1: 64000,1: 128000,1: 256000,1: 512000; 1: 1024000, dilution added in the enzyme plate hole encapsulate after the sealing with the 100uL/ hole, washes plate behind the 37 degree reaction 60min; Add the sheep anti mouse two anti-reactions of HRP mark, with the TMB colour developing, the result sees table 2 at last.

The titration result of table 2 7C10 '

Positive criterion: P/N>=2.1,7C10 ' antibody test result: when antibody purification concentration was 1mg/mL, tiring to reach 1.024 * 10 ⁶More than.

(10) specificity of detection purified monoclonal antibody: adopt the indirect competitive ELISA method of setting up in the steps d to carry out; Be coated with in the elisa plate of coupled antigen; Add 7C10 ' (the 0.02 μ g/mL) antibody behind the purifying, add the paraxin standard substance of different concns simultaneously, the chloramphenicol concentration of adding is respectively 5ng/mL, 2.5ng/mL, 1ng/mL, 0.5ng/mL, 0.25ng/mL, 0.1ng/mL, 0ng/mL; Replication 3 times, the result sees table 3.Natural logarithm with drug level is an X-coordinate, is ordinate zou drawing standard curve with the B/B0 value, and the result sees Fig. 8.The IC50 value of calculating 7C10 ' monoclonal antibody according to testing data is 0.91ng/ml.

Can find out when 7C10 ' monoclonal antibody is 0.1ng/mL in CAP concentration, still to have the good restraining effect from the 0D value and the IC50 value of Fig. 8 typical curve, the monoclonal antibody that screens among this explanation the present invention has very high specificity and susceptibility.

The inhibition effect of table 3 monoclonal antibody 7C10 '

(11) 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 representes that with the size of affinity costant Ka formula is: $\frac{V}{\mathrm{\α}}=K_a(1-V)$

The unit of affinity costant is the inverse of concentration, that is: molconcentration (L/moL), and the high more expression antigen-antibody bonded tightness degree of its value is high more.Its testing process is following:

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

1, artificial antigen is carried out 100000,200000,400000,500000 respectively, respectively encapsulate 4 after 600000,800000 times of dilutions, 100 μ L/ holes, 37 ℃, incubation 2h, washing, sealing;

2, antibody is carried out 20000,40000,80000,160000,320000,640000 times of dilutions are added on respectively in 2,100 μ L/ holes, room temperature incubation 1h;

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

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

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

6, according to formula $f=\frac{A_1\left(c\right)-A_2\left(c\right)}{A_1\left(c\right)}$

Calculate f value, choose all f values all less than 10% antigen, antibody dilution is 500000 times of dilutions according to the big or small definite best antigen concentration of 0D value, and optimum antibody concentration is 160000 times of dilutions.

Second step, mensuration avidity:

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

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

3, simultaneously antigen is carried out encapsulating after 208000 times of dilutions, spend the night under the room temperature, wash plate, sealing;

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

5, according to the Scatchard formula $\frac{V}{\mathrm{\α}}=K_a(1-V)$ 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 affinity costant of monoclonal antibody 7C10 ' is 1.6*10 ¹⁰

Embodiment 2 medicine cross reactions test

Monoclonal antibody screening indirect competitive ELISA method by setting up among the embodiment one is carried out the monoclonal antibody of paraxin and paraxin haptin analog such as chloramphenicol Base, chloramphenicol sodium succinate, thiamphenicol; Tsiklomitsin, qingfengmeisu qiong, Ampicillin Trihydrate; The florfenicol test that is at war with; These standard substance are diluted to different concns carry out indirect competitive ELISA, draw and suppress typical curve, calculate the IC of competition thing ₅₀Value and cross reacting rate; The cross reacting rate of this monoclonal antibody and chloramphenicol sodium succinate reaches 120%; With the crossing-over rate of other similar drug reaction below 5%; Therefore, among the present invention the monoclonal antibody of prepared chloramphenicol resistance can be used for the chlorine detection mycin simultaneously and chloramphenicol sodium succinate residual, concrete outcome is seen table 5.

The cross reaction of table 5 monoclonal antibody 7C10 ' medicine similar with it

Kind	％CR	Kind	％CR
				Paraxin
	100％	Thiamphenicol	1.52％
				Tsiklomitsin	Less than 0.01%	Qingfengmeisu qiong	Less than 0.01%
The Ampicillin Trihydrate	Less than 0.01%	Chloramphenicol sodium succinate	120％

Embodiment 3 monoclonal antibody 7C10 ' are in the application of the ELISA method that detects residual chloromycetin

Monoclonal antibody 7C10 ' can be applied to detect the ELISA method of residual chloromycetin, for example can be used for the detection of eel, shrimp, chicken residual chloromycetin.With eel (market is bought, river eel) is example, explains that the key step that detects residual chloromycetin in the eel is following:

1. sample pre-treatments: use 20% ethanol and eel sample to carry out homogenate than 3: 1 according to volume mass, then with homogenate at the centrifugal 10min of 4 degree 5000G, get middle layer liquid, can detect with behind 2 times of the PBS solution dilutions.2. the detection principle of test kit is the indirect competitive ELISA method among the present invention; Coating antigen OVA-CAP is encapsulated on microwell plate, add the paraxin standard substance or the sample of serial dilution, add the monoclonal antibody 7C10 ' of chloramphenicol resistance again; Paraxin residual in the sample combines with the antibody competition property of chloramphenicol resistance with the antigen that encapsulates simultaneously; Add ELIAS secondary antibody then, the TMB colour developing, OD is read in the colour developing back on ELIASA ₄₅₀, the content of paraxin and sample absorbance are negative correlation in the sample, compare with typical curve, can draw the content of corresponding residue paraxin.

Because the high specific of the monoclonal antibody of chloramphenicol resistance makes and compares with existing sample treatment, has simplified the pre-treatment step of sample, only needs 20% extraction using alcohol, 8 times of dilutions promptly can be used for detecting.Because the high-affinity of antibody makes the sensitivity that detects improve greatly, standard curve range is 0.1-5ng/ml.

The application of embodiment 4 monoclonal antibody 7C10 ' in the colloidal gold strip that detects residual chloromycetin

Present embodiment is the applicating example of monoclonal antibody 7C10 ' in the colloidal gold strip that detects residual chloromycetin among the present invention, mainly is to be applied to detect to be used for eel, shrimp, chicken residual chloromycetin.

Reaction principle is to adopt competition law that small-molecule drug paraxin is carried out half-quantitative detection; The paraxin molecule that exists in the sample is combining along moving past the antibody protein of Cheng Zhongxian with the gold grain mark on the test strip; The coating antigen and the paraxin that are fixed on the NC film are competed binding antibody simultaneously, and the content of residual chloramphenicol is inversely proportional in the colour developing power of T line and the sample, if do not have residual chloromycetin in the sample; Then golden labeling antibody all reacts with coating antigen, and the T line colour developing of test strip is the darkest.Be that C, T line all develop the color and depth unanimity, represent negative this moment, and when the colour developing of C line, the T line does not develop the color or develops the color and then is expressed as the positive when being weaker than the C line, does not develop the color as if the C line, representes that all test strip lost efficacy no matter the T line develops the color or do not develop the color.

(1) concrete operations step is following:

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

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

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

(2) confirming of test strip detectability:

Get test strip and lie against on the testing table, use the PBS solution that contains different concns paraxin respectively, drip on sample pad, react back 5-10min result of determination, confirm the detectability of test strip as analyte sample fluid.Test strip can be carried out different dilutions corresponding to the detection of all kinds of samples, and the detectability of test strip multiply by the detectability that all kinds of dilution of sample multiples are all kinds of samples.

Except that the foregoing description, 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.

Claims (3)

1. hybridoma cell strain 7C10 who produces the chloramphenicol resistance monoclonal antibody, its preserving number is CGMCCNo.3574.

2. hybridoma cell strain 7C10 excretory chloramphenicol resistance monoclonal antibody 7C10 ' by claim 1.

3. the chloramphenicol resistance monoclonal antibody 7C10 ' of claim 2 is applied to the qualitative or detection by quantitative of residual chloromycetin.

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