CN101407542A - Preparations and uses of streptomycin-carrier protein coupled product and streptomycin antibody - Google Patents

Abstract

The invention discloses a coupling product of streptomycin and carrier protein, and a preparation method of a streptomycin antibody and an application thereof, which relate to a coupling method of the carrier protein such as keyhole limpet hemocyanin, human serum albumin, cow serum albumin, ovalbumin and the like with streptomycin, and immune BALB/C mouse spleen cells are fused with SP2/0 mouse myeloma cells by streptomycin immunogens coupled with the carrier protein, and the streptomycin coupled with the carrier protein is used as a coating antigen for screening positive hybridoma and hybridoma that can steady passage and excrete anti-specificity streptomycin monoclonal antibody can be obtained by the cell clone, and ascites monoclonal antibody is prepared. The prepared monoclonal antibody is used for building a direct competition ELISA method with high specificity, sensitivity and accuracy to the streptomycin and immunity colloidal gold test strips. The coupling of the streptomycin and the carrier protein and the preparation method of the streptomycin monoclonal antibody can provide services for detecting streptomycin residue in foods quickly.

Description

The Preparation method and use of Streptomycin sulphate and carrier protein couplet product and Streptomycin sulphate antibody

Technical field

The invention belongs to the immunochemistry biological technical field, especially relate to the Preparation method and use of a kind of Streptomycin sulphate and carrier protein couplet product and Streptomycin sulphate antibody.

Background technology

Streptomycin sulphate is an aminoglycosides antibiotics, is succeeded in developing in 1945 by U.S. Waksman institute, is widely used in the humans and animals treatment of diseases: the last Streptomycin sulphate of people is used for the treatment of diseases that tuberculosis, the plague and Gram-negative bacteria cause; Streptomycin sulphate has better curative effect to the multiple disease of chicken, sheep, ox, pig as infectious coryza of chicken, pig acute bronchitis, pig dysentery characterized by white mucous stool, cow endometritis, colibacillosis etc. on the animal, therefore often uses as fodder additives.But Streptomycin sulphate has many side effects: 1) anaphylaxis; 2) to the 8th pair of cranial nerve infringement; 3) to the inhibition of marrow; 4) has the potential carcinogenesis.Therefore, the application of Streptomycin sulphate on the people recently acted on similar microbiotic by other gradually and substitutes.But China is subjected to ordering about of economic interests and relevant detection supervision system unsound because the imperfection of animal doctor's medication system and some are cultured factory, and the abuse phenomenon of Streptomycin sulphate in livestock industry, Apiculture and fishery are produced is very general.The hygienic requirements of milk in the industry standard of the pollution-free food of implementing by the issue of Ministry of Agriculture calendar year 2001, the highest permission residue criterion of Streptomycin sulphate is 200ng/ml; The abuse of Streptomycin sulphate in livestock industry causes the delay of animal drug disposition or accumulates, and enters the human body and the ecosystem in residual mode.It mainly is chronic, at a specified future date and cumulative to the harm of human body and environment, as carcinogenic, the life long hair educated exert an influence, destroy the normal immunity system of people, make the intravital pathogenic micro-organism of people produce resistance etc.The existence of antibiotics resistance bacterial strain human beings'health with serious threat, and the easier transfer that impels resistant gene of the microbiotic of clinical inferior treatment level, such as animal being carried out the treatment of low-level Streptomycin sulphate, its excrement intestinal flora develops at last other medicines is also produced resistance by the Streptomycin sulphate sensitivity being become gradually anti-streptomycin.In secular life, the normal microflora of human and animal's enteron aisle has become the storage vault of drug resistant gene exactly, and constantly drug resistant gene is transferred to pathogenic bacterium, and intersection is propagated in humans and animals, especially it is even more serious to be discharged in the environment harm of resistant organism, can cause the rapid transfer of drug resistant gene.Along with people's living standard and to the raising of abuse of antibiotics recognizing dangers, people pay much attention to residues of antibiotics in the food, like edible residual livestock product and the fishery products of antibiotic-free.

The residual detection of Streptomycin sulphate at present mainly contains instrumental analysis and two kinds of methods of immunoassay, wherein instrumental method is mainly used high performance liquid chromatography (HPLC), gas-chromatography (GC), since its exist instrument costliness, operation loaded down with trivial details, waste time and energy, the expense height, can not be on a large scale problem such as scene detection, also require the technician to have certain operation and analytical skill, thereby can not finely apply.That Enzyme Linked Immunoadsorbent Assay (ELISA) has is easy, quick, can detect on a large scale, the advantage of sensitive and with low cost and on-the-spot detection, be particluarly suitable for producing and the process of circulation in antibiotic remainss such as Streptomycin sulphate in livestock product and the fishery products are detected.Colloidal gold immuno-chromatography test paper strip is widely used, the existing multiple test strip that detects people, animal, pathogenic, cancer antigen, hormone, medicine and pesticide residue, this method has following advantage: simple, convenient, quick, almost everybody can use; Do not need any equipment; The result accurately and reliably, high specificity, highly sensitive; The low-cost high-efficiency benefit; Preservation is convenient etc.Therefore immuno-chromatographic test paper strip has become one of developing direction of multiple material immunology detection at present.This patent is used the hybridoma cell strain that hybridoma technology is set up energy stably excreting anti-streptomycin monoclonal antibody, and preparation has high-affinity, high specific, highly sensitive Streptomycin sulphate monoclonal antibody, with this monoclonal antibody is Streptomycin sulphate residue detection test kit and the colloidal gold immuno-chromatography test paper strip that the core exploitation has independent intellectual property right, cheapness, is the residual rapid detection service of Streptomycin sulphate in China's animal-derived food.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, the Preparation method and use of a kind of Streptomycin sulphate and carrier protein couplet product and Streptomycin sulphate antibody is provided.

The preparation method of Streptomycin sulphate and carrier protein couplet product comprises the steps:

A) 50-1600mg Streptomycin sulphate and 30-300mg carboxymethyl azanol are dissolved in the Milli-Q ultrapure water or distilled water that 2-5ml contains the 5-20mg sodium acetate, stirring reaction 15-30 hour, synthetic Streptomycin sulphate-oxime compound;

B) be that methyl alcohol/chloroform/17% ammoniacal liquor of 50: 25: 25 is moving phase with volume ratio, carry out silica gel thin-layer chromatography disengaging latch mycin-oxime compound, Streptomycin sulphate-oxime compound is retained in and adds near the line-transect, scrape the silica gel that adds the line-transect place, extract Streptomycin sulphate--oxime compound with Milli-Q ultrapure water or distilled water, extract removes silica gel with filter paper filtering, with freeze drier dry filter liquid, obtains the Streptomycin sulphate-oxime compound of purifying;

C) 10-100mg Streptomycin sulphate-oxime compound and 1-8g carbodiimide being dissolved in 3-30ml contains in 20% alcoholic acid Milli-Q ultrapure water or the distilled water, adding 1-10ml contains the Milli-Q ultrapure water or the distilled water of 10-100mg carrier proteins, transfer pH to 5-6.5, at stirring reaction 10-36 hour, synthetic Streptomycin sulphate-carrier protein couplet product;

D) Streptomycin sulphate-carrier protein couplet product is put into dialysis tubing, dialyses 1-3 days at 4 ℃ with the phosphate buffered saline buffer of pH7.2;

E) Streptomycin sulphate-carrier protein couplet product after the dialysis carries out UV scanning, and Analysis and Identification coupled product and concentration determination are preserved standby in-20 ℃ of refrigerators after the packing.

Described carrier proteins is keyhole limpet hemocyanin, human serum albumin, bovine serum albumin, bovine gamma globulin(BGG) or ovalbumin.

Streptomycin sulphate-carrier protein couplet product is used for the residual immunological method check and analysis of preparation, livestock product Streptomycin sulphate of Streptomycin sulphate antibody.

Anti-streptomycin MONOCLONAL ANTIBODIES SPECIFIC FOR method comprises the steps:

1) Streptomycin sulphate-carrier protein couplet product is as the immunogen immune BALB/C mice;

2) splenocyte and the SP2/0 rat bone marrow tumour cell of getting immunized mice merges under 50% polyoxyethylene glycol fusogen, and HAT screening culture medium screening hybridoma detects the cell of secretion anti-streptomycin with indirect ELISA method;

3) adopt limiting dilution assay to carry out cell clone,, obtain to stablize the hybridoma cell strain of the justacrine anti-streptomycin monoclonal antibody specific that goes down to posterity through the indirect ELISA screening positive clone; Hybridoma is injected into the BALB/C mice abdominal cavity and prepares odd contradictive hydroperitoneum;

4) cross reacting rate of employing direct competitive ELISA method mensuration anti-streptomycin monoclonal antibody and Streptomycin sulphate, Vibriomycin, kantlex, gentamicin, penicillin G, peace penicillin G, tsiklomitsin, paraxin, sucrose, glucose, selecting only has specific immune response with Streptomycin sulphate, Vibriomycin, and detection sensitivity reaches the monoclonal antibody of 0.5-3ng/mL, can be used for the residual detection of Streptomycin sulphate in the livestock product.

Described immunogen is Streptomycin sulphate-carrier protein couplet product.Immunogenic carrier proteins is keyhole limpet hemocyanin, human serum albumin, bovine serum albumin, bovine gamma globulin(BGG) or ovalbumin.

Described immunization method is: with around Streptomycin sulphate-carrier protein couplet product immunity age body weight 18-20gBALB/C female mice: 1mg/ml antigen 0.5-0.7ml mixes with equal-volume Fu Shi Freund's complete adjuvant, after fully emulsified, through back of the body subcutaneous abdomen multi-point injection 0.2-0.3m/ only, interval 3-4 week, get with one exempt from equivalent antigen and isopyknic freund 's incomplete adjuvant fully emulsified after, abdominal injection 0.2-0.3ml/ only for the second time, carry out 4-6 time booster immunization altogether, the end exempts to carry out abdominal injection with the doubling dose antigen that does not add adjuvant, and extracting spleen cell merges after 3 days.

Described odd contradictive hydroperitoneum preparation method is: get BALB/C mice about age in 6-10 week, and abdominal injection 0.3-0.5ml pristane, pneumoretroperitoneum injected 5-10 * 10 in 7-10 days ⁵Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3-5min of 3000-5000rpm collects supernatant liquor, is monoclonal antibody ascites.

The Streptomycin sulphate monoclonal antibody is used for detecting the residual immunological method of livestock product Streptomycin sulphate.It is described that to be used for detecting the residual immunological method of livestock product Streptomycin sulphate be competitive ELISA or immunity test strip bar.

Advantage of the present invention is: 1) the invention provides a kind of Streptomycin sulphate immunogen coupling method that can preparation detects the residual monoclonal antibody of Streptomycin sulphate in the food, it is simple to operation; 2) provide a kind of can the acquisition to detect residual special, the sensitive MONOCLONAL ANTIBODIES SPECIFIC FOR method of Streptomycin sulphate, its method is simply effective; 3) utilize the prepared monoclonal antibody of the present invention, can be used for the residual detection of livestock product Streptomycin sulphate effectively.

Description of drawings

The UV scanning result of Fig. 1 coupled product.Str is an aqueous streptomycin; BSA is a bovine serum albumin solution; Str-BSA is Streptomycin sulphate and BSA coupled product.

Embodiment

The invention provides that a kind of Streptomycin sulphate artificial antigen is synthetic, Antibody Preparation and the immunological method of residue detection in food thereof.With the high specificity of the immunogen preparing Streptomycin sulphate of preparation, highly sensitive, good stability, can mass-produced monoclonal antibody, and set up with these monoclonal antibodies and to have detected residual fast diagnosis method-competitive ELISA and the immuno-chromatographic test paper strip of Streptomycin sulphate in the food with high degree of specificity, susceptibility and exactness, can be used for the residual detection of livestock product Streptomycin sulphate effectively, be the food safety service of China.

1. the preparation method of Streptomycin sulphate and carrier protein couplet product comprises the steps:

A) 50-1600mg Streptomycin sulphate and 30-300mg carboxymethyl azanol are dissolved in the Milli-Q ultrapure water or distilled water that 2-5ml contains the 5-20mg sodium acetate, stirring reaction 15-30 hour, synthetic Streptomycin sulphate-oxime compound;

B) be that methyl alcohol/chloroform/17% ammoniacal liquor of 50: 25: 25 is moving phase with volume ratio, carry out silica gel thin-layer chromatography disengaging latch mycin-oxime compound, Streptomycin sulphate-oxime compound is retained in and adds near the line-transect, scrape the silica gel that adds the line-transect place, extract Streptomycin sulphate--oxime compound with Milli-Q ultrapure water or distilled water, extract removes silica gel with filter paper filtering, with freeze drier dry filter liquid, obtains the Streptomycin sulphate-oxime compound of purifying;

C) 10-100mg Streptomycin sulphate-oxime compound and 1-8g carbodiimide being dissolved in 3-30ml contains in 20% alcoholic acid Milli-Q ultrapure water or the distilled water, add 1-10ml and contain Milli-Q ultrapure water or the distilled water that the 10-100mg carrier proteins is a bovine serum albumin (BSA), transfer pH to 5-6.5, at stirring reaction 10-36 hour, synthetic Streptomycin sulphate-BSA coupled product;

D) Streptomycin sulphate-BSA coupled product is put into dialysis tubing, dialyses 1-3 days at 4 ℃ with the phosphate buffered saline buffer of pH7.2;

E) Streptomycin sulphate-BSA coupled product, Streptomycin sulphate, the BSA solution after the dialysis carries out UV scanning, Analysis and Identification coupled product and concentration determination, analyze the UV scanning figure obviously different with the scanning pattern of BSA and Streptomycin sulphate (as Fig. 1) of finding coupled product, illustrate that Streptomycin sulphate is successful with the BSA coupling, preserve in-20 ℃ of refrigerators after the coupled product packing, be used for the Streptomycin sulphate artificial antigen in the residual immunological method of Antibody Preparation and detection food Streptomycin sulphate.

2. Streptomycin sulphate MONOCLONAL ANTIBODIES SPECIFIC FOR method

1) immune animal

With around Streptomycin sulphate and the immunity of BSA conjugate age body weight 18-20g BALB/C female mice: 1mg/ml extracts Streptomycin sulphate to be mixed with equal-volume Fu Shi Freund's complete adjuvant with BSA conjugate 0.5-0.7ml, after fully emulsified, through back of the body subcutaneous abdomen multi-point injection 0.2-0.3ml/ only, interval 3-4 week, get with one exempt from equivalent antigen and isopyknic freund 's incomplete adjuvant fully emulsified after, abdominal injection 0.2-0.3ml/ only for the second time, carry out booster immunization altogether 5 times, the end exempts to carry out abdominal injection with the doubling dose antigen that does not add adjuvant, and extracting spleen cell merges after 3 days.

2) cytogamy

Get above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) in 5-10: 1 ratio, mixing in the RPMI-1640 of serum-free (Gibco) substratum, the centrifugal 5min of 1500rpm, remove substratum, with 50%PEG (polyoxyethylene glycol, Sigma) as fusogen, under 37 ℃ of following water-baths, add 0.5-0.7ml, make it merge 2min, RPMI-1640 substratum with serum-free stops the centrifugal 5min of fusion back 1500rpm, and precipitation suspends with HAT screening culture medium (Sigma), and branch installs to 96 holes and contains in the cell plate of feeder cell, 37 ℃, cultivate in the cell cultures vessel of 5%CO2.

3) screening in hybridoma, positive hole and clone thereof

Cell is cultivated after 5 days in cultivating vessel, change liquid once with the HAT screening culture medium, changed liquid with the HT substratum on the 10th day, by the time at the bottom of the fused cell coverage hole during 5%-50%, carry out indirect ELISA method with Streptomycin sulphate coupling ovalbumin (Str-OVA) as envelope antigen and screen positive hole, obtain more than 101 the positive hole that Streptomycin sulphate is responded altogether, positive rate is 10%.Select 6 cell holes that are strong positive reaction, carry out the limiting dilution assay clone, obtain the hybridoma cell strain that the 10A12 strain can be secreted the specific antibody of anti-streptomycin.Through subculture in vitro separately more than 6 months with repeatedly behind the cryopreservation resuscitation, cell strain all can well be grown, and stably excreting antibody.After enlarged culturing, be used for ascites preparation and liquid nitrogen and preserve.

4) preparation of monoclonal antibody ascites and purifying

Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5-10 * 10 in 7-10 days ⁵Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3-5min of 3000-5000rpm collects supernatant liquor, is monoclonal antibody ascites.Get 1 times of volume ascites and add 2 times of volume 0.06M pH 4.8 acetate buffer solutions dilutions, add sad (30ul/ml ascites), the following edged of room temperature stirs, and clarifies 1 hour for 4 ℃, the centrifugal 20min of 12000rpm, collect supernatant, use 50% saturated ammonium sulphate immunoglobulin (Ig) again, placed 2 hours for 4 ℃, the centrifugal 20min of 3000-5000rpm, precipitation is dissolved with the PBS solution of 2 times of volumes, promptly obtains the ascites antibody of purifying after 4 ℃ of mobile dialysis 24 hours ,-70 ℃ of preservations.

5) subgroup identification of monoclonal antibody and ascites titration

With the odd contradictive hydroperitoneum of purifying and the anti-BALB/C mice IgG of standard, the IgG of Sigma company ₁, IgG _2a, IgG _2b, IgG ₃, IgM antibody, do two-way agar diffusion test, the result is that the antibody type of 10A12 and subclass are IgG ₁, the light chain of monoclonal antibody is the κ chain.Detect odd contradictive hydroperitoneum with conventional indirect ELISA method and tire, the result tires all 10 for above-mentioned ascites ^-6More than.

3. detect the foundation of the residual direct competitive ELISA method of Streptomycin sulphate

1) preparation of horseradish peroxidase (HRP)-Streptomycin sulphate binding substances:

A) 50-1600mg Streptomycin sulphate and 30-300mg carboxymethyl azanol are dissolved in the Milli-Q ultrapure water 2-5ml that contains the 5-20mg sodium acetate, stirring at room reaction 15-30 hour, synthetic Streptomycin sulphate-oxime compound;

B) be that methyl alcohol/chloroform/17% ammoniacal liquor of 50: 25: 25 is moving phase with volume ratio, carry out silica gel thin-layer chromatography disengaging latch mycin-oxime compound, Streptomycin sulphate-oxime compound is retained in and adds near the line-transect, scrape the silica gel that adds the line-transect place, extract Streptomycin sulphate-oxime compound with the Milli-Q ultrapure water, extract removes silica gel with filter paper filtering, promptly obtains the Streptomycin sulphate-oxime compound of purifying with freeze drier dry filter liquid;

C) 10-100mg Streptomycin sulphate-oxime compound and 1-8g carbodiimide (EDC) are dissolved in 3-30ml and contain in the 20% alcoholic acid Milli-Q ultrapure water, the solution that adds the 1-10mlMilli-Q ultrapure water preparation that contains 10-100mg horseradish peroxidase (HRP), transfer pH to 5-6.5, at room temperature stirring reaction 10-36 hour;

D) horseradish peroxidase (HRP)-Streptomycin sulphate coupled product is put into dialysis tubing, dialyses 1-3 days at 4 ℃ with the phosphate buffered saline buffer (PBS) of pH7.2;

E) coupled product, Streptomycin sulphate, the HRP solution after the dialysis carries out UV scanning, identify coupled product, it is obviously different with the scanning pattern of HRP and Streptomycin sulphate to analyze the UV scanning figure of finding coupled product, illustrate that Streptomycin sulphate is successful with HRP (enzyme mark Streptomycin sulphate haptens) coupling, preserve in-20 ℃ of refrigerators after the coupled product packing, be used to detect the direct competitive ELISA method of Streptomycin sulphate.

2) direct competitive ELISA method condition optimizing:

With square formation test method(s) antagonist and the haptenic dilution of carrying out a series of concentration of enzyme mark, determine direct competitive ELISA method antibody and enzyme mark the suitableeest haptenic working concentration.OD ₄₅₀Value is about 1.0, and the concentration combination that the antibody antigen consumption is less is antibody-antigenic best effort concentration.At first, monoclonal antibody is diluted to a series of concentration with PBS (0.01M, pH 7.4), adds 96 hole enzyme plates (100 μ L/well) respectively successively, 37 ℃ of following incubation 2h, PBST washing 3 times; With the PBS sealing that contains 2.0% skimmed milk, every hole 300 μ L, 37 ℃ of incubation 0.5h, wash 3 times with PBST the back; Add the enzyme mark haptens that is diluted to different concns in advance with PBS, mixing on micro-oscillator, 37 ℃ of following incubation 1h, PBST washing 3 times; Add substrate solution (100 μ L/well), 37 ℃ of incubation 15min add stop buffer (50 μ L/well), measure the OD in each hole with microplate reader ₄₅₀Value.

3) antibody affinity and detection sensitivity

Under optimized conditions, on wrapping in advance by the enzyme plate of good monoclonal antibody, the Streptomycin sulphate standard specimen that adds series concentration, repeat in every concentration 3 holes, if do not add Streptomycin sulphate contrast and solvent blank contrast, 50 μ L/well add 0.89 μ g/ml enzyme mark haptens, 50 μ L again, 37 ℃ of following incubation 1h behind the concussion 1min, PBST washing 4 times; Add tmb substrate solution (100 μ L/well), 37 ℃ of incubation 15min add 2M sulfuric acid stop buffer (50 μ L/well), measure the OD in each hole with microplate reader ₄₅₀Value.Semilog drawing standard curve with inhibiting rate and Streptomycin sulphate concentration.Inhibiting rate I calculation formula is as follows:

$I\%=\frac{(\mathrm{OD}\max -\mathrm{OD}\min )-(\mathrm{ODx}-\mathrm{OD}\min )}{(\mathrm{OD}\max -\mathrm{OD}\min )}\×100$

In the formula: OD _MaxLight absorption value during-not dosing; OD _xLight absorption value when-pesticide concentration is x; OD _MinThe light absorption value in-blank hole.

Reach 50% pesticide concentration (IC with inhibiting rate ₅₀) represent the affinity of antibody to Streptomycin sulphate, with IC ₁₀Detection sensitivity as the ELISA method.Under the elisa assay condition of optimizing, Streptomycin sulphate is set up typical curve, to investigate its detection sensitivity in optimized reaction system.Get IC according to competitive ELISA reaction normal curve calculation ₅₀Be 10.32ng/ml, IC ₁₀Be 0.5ng/ml, linearity range 1.51-85.95ng/m.

4) specificity of Streptomycin sulphate monoclonal antibody

Under optimized conditions, Streptomycin sulphate standard substance, Vibriomycin, kantlex, penicillin G, gentamicin, tsiklomitsin, paraxin, sucrose, glucose are done serial dilution, carry out direct competitive ELISA with monoclonal antibody respectively, the production standard curve, and on curve, find out the concentration of inhibiting rate 50%, and the concentration of above-mentioned several material 50% inhibiting rate, calculate all kinds of antibiotic cross reacting rates then.Cross reacting rate CR method of calculation are CR%=Streptomycin sulphate IC ₅₀/ other microbiotic IC ₅₀* 100.The result shows that the cross reacting rate of 10A12 monoclonal antibody and Vibriomycin is 100%, with the cross reacting rate of kantlex, penicillin G, gentamicin, tsiklomitsin, paraxin, sucrose, glucose all less than 0.1%.The specificity that the monoclonal antibody for preparing is described is very high, only Streptomycin sulphate and dihydrostreptomycin is had strong specific reaction.

4. the preparation of immuno-chromatographic test paper strip

The golden mark of the preparation of Radioactive colloidal gold and monoclonal antibody:

The colloid gold particle preparation adds 1% trisodium citrate 1ml in the 100ml deionized water, boil the back and add 1% hydrochloro-auric acid 1ml rapidly, continues to boil 15min, after the cooling, preserves standby down for 4 ℃.The big or small average out to 30nm of the colloid gold particle that generally, prepares.

The golden mark of monoclonal antibody:

Get the colloidal gold solution 100ml that has prepared, transfer pH to 8.0 with the 0.1mol/L solution of potassium carbonate.Add monoclonal antibody 2mg while stirring, stir 15min, dropwise add 2.5ml 25mg/ml Macrogol 2000 0 (PEG20000) again, stir 20min.The centrifugal 20min of 20000r/min abandons supernatant liquor, adds 10ml pH 7.4PBS damping fluid (containing 0.4mg/ml PEG) and cleans 2 times.Precipitation is dissolved with the PBS damping fluid (pH 7.4) that 5ml contains 2%BSA, and after filtering with 0.22 μ m sterilizing filter, 4 ℃ of preservations are standby.

The assembling of immuno-chromatographic test paper strip:

With a film machine Streptomycin sulphate of proper concn-BSA antigen and sheep anti-mouse igg are sprayed on the NC film, respectively as detection line (T) and control line (C), at 37 ℃ of oven drying 8h.In kind, the golden mark Streptomycin sulphate monoclonal antibody for preparing is coated on the Radioactive colloidal gold pad.Sample pad, Radioactive colloidal gold pad, nitrocellulose filter and absorbent pad are sticked on the base plate successively, thereby form a successive micro-filtration system.The plate that posts is cut into the wide bar of 4mm, make the detection reagent plate in the template of packing into, lucifuge, airtight, normal temperature are preserved standby.

Draw sample solution to be checked with dropper, drip 3 (about 100ul) and in the well of mentioned reagent plate, pick up counting behind the application of sample; The result should read in 3～5 minutes, and the other times interpretation is invalid; The color of Qu T, C line compares to determine the result according to the observation, and when the colour developing of T line was dark or more equal than C line, the result was judged to be feminine gender.When the colour developing of T line color was more shallow than C line, detected result was positive.

The Vetstrep standard substance are configured to the analytical sample solution of different concns: 0,1,3,5,7,10,15,20,30,40,50,60,70,80,90,100ng/ml, measure with test strip, each sample is done 3 repetitions, observations behind the 5min, detected result shows, the content of Streptomycin sulphate left drug meets or exceeds 1ng/ml in sample, because most of monoclonal antibodies are by the residual competition combination of the Streptomycin sulphate in the sample, the Streptomycin sulphate monoclonal antibody that the envelope antigen of T line is attached to just seldom or do not have, the colour developing of T line is obviously shallow or do not develop the color than C line, and the result is positive; When the residual content of Streptomycin sulphate in the sample was lower than 1ng/ml, T line colour generation was consistent or more shallow than C line with the C line, and the result is negative; No matter be the positive or negative findings, Radioactive colloidal gold-Streptomycin sulphate monoclonal antibody binding substances can combine with the sheep anti-mouse igg at C line place, forms a mauve band.If the C line does not develop the color, no matter be that the T line has or not, result at this moment is all invalid.

The stability test of detection reagent plate:

Detect same sample with same batch of different detection reagent plates, and measure same sample with different batches detection reagent plate, the developing time of its nature controlling line, detection line and shade and net result interpretation are basic identical.The agent plate of same batch was placed 37 ℃, room temperature, 4 ℃ of airtight preservations 3 months, per 2 weeks each detect each 20 parts in positive and negative milk sample, the result shows that big variation does not take place detected result along with time and variation of temperature.Be equivalent to the estimation of 1 week according to 37 ℃ of next skies, this agent plate can at room temperature be preserved 12 months.

Claims (10)

1. the preparation method of Streptomycin sulphate and carrier protein couplet product is characterized in that comprising the steps:

A) 50-1600mg Streptomycin sulphate and 30-300mg carboxymethyl azanol are dissolved in the Milli-Q ultrapure water or distilled water that 2-5ml contains the 5-20mg sodium acetate, stirring reaction 15-30 hour, synthetic Streptomycin sulphate-oxime compound;

B) be that methyl alcohol/chloroform/17% ammoniacal liquor of 50: 25: 25 is moving phase with volume ratio, carry out silica gel thin-layer chromatography disengaging latch mycin-oxime compound, Streptomycin sulphate-oxime compound is retained in and adds near the line-transect, scrape the silica gel that adds the line-transect place, extract Streptomycin sulphate--oxime compound with Milli-Q ultrapure water or distilled water, extract removes silica gel with filter paper filtering, with freeze drier dry filter liquid, obtains the Streptomycin sulphate-oxime compound of purifying;

C) 10-100mg Streptomycin sulphate-oxime compound and 1-8g carbodiimide being dissolved in 3-30ml contains in 20% alcoholic acid Milli-Q ultrapure water or the distilled water, adding 1-10ml contains the Milli-Q ultrapure water or the distilled water of 10-100mg carrier proteins, transfer pH to 5-6.5, at stirring reaction 10-36 hour, synthetic Streptomycin sulphate-carrier protein couplet product;

D) Streptomycin sulphate-carrier protein couplet product is put into dialysis tubing, dialyses 1-3 days at 4 ℃ with the phosphate buffered saline buffer of pH7.2;

E) Streptomycin sulphate-carrier protein couplet product after the dialysis carries out UV scanning, and Analysis and Identification coupled product and concentration determination are preserved standby in-20 ℃ of refrigerators after the packing.

2. according to the preparation method of described a kind of Streptomycin sulphate of claim 1 and carrier protein couplet product, it is characterized in that described carrier proteins is keyhole limpet hemocyanin, human serum albumin, bovine serum albumin, bovine gamma globulin(BGG) or ovalbumin.

3. the Streptomycin sulphate of method preparation and the purposes of carrier protein couplet product according to claim 1 is characterized in that described Streptomycin sulphate-carrier protein couplet product is used for the preparation of Streptomycin sulphate antibody, the residual immunological method check and analysis of livestock product Streptomycin sulphate.

4. an anti-streptomycin MONOCLONAL ANTIBODIES SPECIFIC FOR method is characterized in that comprising the steps:

1) Streptomycin sulphate-carrier protein couplet product is as the immunogen immune BALB/C mice;

2) splenocyte and the SP2/0 rat bone marrow tumour cell of getting immunized mice merges under 50% polyoxyethylene glycol fusogen, and HAT screening culture medium screening hybridoma detects the cell of secretion anti-streptomycin with indirect ELISA method;

3) adopt limiting dilution assay to carry out cell clone,, obtain to stablize the hybridoma cell strain of the justacrine anti-streptomycin monoclonal antibody specific that goes down to posterity through the indirect ELISA screening positive clone; Hybridoma is injected into the BALB/C mice abdominal cavity and prepares odd contradictive hydroperitoneum;

4) cross reacting rate of employing direct competitive ELISA method mensuration anti-streptomycin monoclonal antibody and Streptomycin sulphate, Vibriomycin, kantlex, gentamicin, penicillin G, peace penicillin G, tsiklomitsin, paraxin, sucrose, glucose, selecting only has specific immune response with Streptomycin sulphate, Vibriomycin, and detection sensitivity reaches the monoclonal antibody of 0.5-3ng/mL, can be used for the residual detection of Streptomycin sulphate in the livestock product.

5. a kind of anti-streptomycin MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 4 is characterized in that described immunogen is according to claims 1 method link coupled Streptomycin sulphate-carrier protein couplet product.

6. a kind of anti-streptomycin MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 4 is characterized in that described immunogenic carrier proteins is keyhole limpet hemocyanin, human serum albumin, bovine serum albumin, bovine gamma globulin(BGG) or ovalbumin.

7. a kind of anti-streptomycin MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 4, it is characterized in that described immunization method is: with around Streptomycin sulphate-carrier protein couplet product immunity age body weight 18-20gBALB/C female mice: 1mg/ml antigen 0.5-0.7ml mixes with equal-volume Fu Shi Freund's complete adjuvant, after fully emulsified, through back of the body subcutaneous abdomen multi-point injection 0.2-0.3m/ only, interval 3-4 week, get with one exempt from equivalent antigen and isopyknic freund 's incomplete adjuvant fully emulsified after, abdominal injection 0.2-0.3ml/ only for the second time, carry out 4-6 time booster immunization altogether, the end exempts to carry out abdominal injection with the doubling dose antigen that does not add adjuvant, and extracting spleen cell merges after 3 days.

8. a kind of anti-streptomycin MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 4, it is characterized in that described odd contradictive hydroperitoneum preparation method is: get BALB/C mice about age in 6-10 week, abdominal injection 0.3-0.5ml pristane, pneumoretroperitoneum injected 5-10 * 10 in 7-10 days ⁵Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3-5min of 3000-5000rpm collects supernatant liquor, is monoclonal antibody ascites.

9. the purposes as the Streptomycin sulphate monoclonal antibody of claim 4 method preparation is characterized in that the Streptomycin sulphate monoclonal antibody is used for detecting the residual immunological method of livestock product Streptomycin sulphate.

10. the purposes of a kind of Streptomycin sulphate monoclonal antibody according to claim 9 is characterized in that described to be used for detecting the residual immunological method of livestock product Streptomycin sulphate be competitive ELISA or immunity test strip bar.

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