CN103645310B - A kind of Macrodantin chemiluminescence detection kit - Google Patents

Abstract

The present invention relates to a kind of detection Macrodantin chemiluminescence detection kit, comprise concentrated washing lotion, negative controls, positive reference substance, chemical luminescence for liquid, furadantin standard substance, the furadantin specific antibody of horseradish peroxidase-labeled and wrapped by the luminescent screen of furadantin; The furadantin specific antibody of described horseradish peroxidase-labeled is, horseradish peroxidase-labeled taking furadantin-BSA as the anti-furadantin polyclonal antibody of immunogen immune new zealand white rabbit gained; The furadantin specific antibody of horseradish peroxidase-labeled is prepared with Over-voltage protection; The present invention detects that Macrodantin chemiluminescence detection kit is highly sensitive, sensing range width, easy and simple to handle fast and nontoxic pollution-free; Metabolite and the carrier conjugation of furadantin is adopted to prepare envelope antigen, more economical; The preparation of horseradish peroxidase marker, adopts single stage method, and method easy steps is more simple, is beneficial to suitability for industrialized production.

Description

A kind of Macrodantin chemiluminescence detection kit

Technical field

The present invention relates to a kind of test kit, specifically a kind of Macrodantin chemiluminescence detection kit.

Background technology

In detection feed, the technology of microbiotic mainly contains following several method at present:

(1) Physico-chemical tests method

After the nineties in 20th century, most Physico-chemical tests method measuring furadantin mainly relies on liquid chromatography technology to be separated, next is look/mass spectrometric hyphenated technique, the detection method such as vapor-phase chromatography, high performance thin-layer chromatography, because of its distinctive performance separately, slightly apply in antibiotics leftover detection. Drug residue in chromatography detection feed will through sample preparation (comprising the steps such as the extraction of sample, deproteinated, centrifugal, chromatography column purification, derivatize), the separation of medicine and the detection of medicine. Physico-chemical tests method utilizes special reaction or character that the group in antibiotic molecule has to measure its content, can carry out qualitative and quantitative analysis and drug identification, can be used as the confirmation method of fodder antibiotics detection. This method detection sensitivity is higher, but instrument and testing cost height, trace routine complicated, more consuming time between etc.

(2) immune analysis method

Immune analysis method is the analytical procedure based on the specificity of antigen and antibody, reversibility association reaction, and current drug residue immuno analytical method mainly divides three major types: the method for relatively independent analytical procedure, immuno analytical method and conventional physical and chemical analysis technology coupling, immunity receptor method. Dutch vanweeman, schurrs in 1973 and Sweden Engvall, Perlmann propose enzyme-linked immunosorbent assay respectively, and over more than 30 year, microbiotic in immunological method detection food has been carried out big quantity research by Chinese scholars. But owing to microbiotic is haptens, antigen constructing technology difficulty is big, immunologic opsonin is strong, testing cost height, the companies such as current IDEXX are to exempt from the research in analytical method comparatively ripe, and this technology domestic is still in researchdevelopment. Current mainly with enzyme linked immunosorbent assay, radioimmunoassay luminescence method, fluorescent immune method, chemoluminescence method etc.; The methods such as enzyme linked immunosorbent assay, radioimmunoassay luminescence method, fluorescent immune method compare with luminous phase chemistry luminescence method, and it is lower that its enzyme exempts from sensitivity.

Summary of the invention

The present invention seeks to the deficiency for solving the problems of the technologies described above, it is provided that a kind of Macrodantin chemiluminescence detection kit, it has highly sensitive, sensing range width, the advantage such as fast easy and simple to handle.

The present invention solves the problems of the technologies described above the technical scheme adopted to be: a kind of Macrodantin chemiluminescence detection kit, comprises concentrated washing lotion, negative controls, positive reference substance, chemical luminescence for liquid, furadantin standard substance, the furadantin specific antibody of horseradish peroxidase-labeled and has wrapped by the luminescent screen of furadantin;

Described concentrated washing lotion is: containing the phosphate buffered saline buffer of Tween-20 and sodium-chlor;

Described negative controls is: the furadantin after more than the 5 parts Sterile Filtrations deposited separately is detected as negative pig urine mixture;

Described positive reference substance is: the pig urine mixture of the furadantin test positive after more than the 5 parts Sterile Filtrations deposited separately;

Described chemical luminescence for liquid, comprises A liquid and B liquid; A liquid making method is: the Tris-HCl damping fluid first configuring 0.2mol/LpH7.8, adds luminol,3-aminophthalic acid cyclic hydrazide and sodium tetraphenylborate in this damping fluid, mixes and can obtain A liquid; B liquid making method is: the Tris-HCl damping fluid first configuring 0.2mol/LpH7.8, adds 0.1%H in this damping fluid by weight₂O₂Mix and can obtain B liquid;

Described furadantin standard substance are the diluent that concentration is respectively 0ng/ml, 0.1ng/ml, 0.3ng/ml, 0.9ng/ml, 2.7ng/ml, 8.1ng/ml furadantin;

The furadantin specific antibody of described horseradish peroxidase-labeled is, horseradish peroxidase-labeled taking furadantin-BSA as the anti-furadantin polyclonal antibody of immunogen immune new zealand white rabbit gained; Prepare the furadantin specific antibody of horseradish peroxidase-labeled with Over-voltage protection, reaction system is: first with the NaIO of the horseradish peroxidase solution of 1ml concentration 5mg/ml and 0.15ml concentration 0.1mol/L₄Solution mix even after, it is placed in 4 DEG C of lucifuges 0.5 hour, then the ethylene glycol adding 0.25ml shakes even room temperature lucifuge 1 hour, then 4 DEG C of dialysed overnight it are placed in, afterwards with 0.2ml concentration 0.2mol/L, pH value is the carbonate bag of 9.6 is 9.2-9.5 by solution by the hydroformylation horseradish peroxidase solution adjust pH that obtains after dialysis, anti-for 5mg furadantin polyclonal antibody is dissolved in 1.0ml concentration 0.01mol/L, add immediately after the coating buffer of pH9.6 in the horseradish peroxidase solution after above-mentioned hydroformylation and react 1 hour in 37 DEG C, then add the NaBH of 0.1ml concentration 3.5mg/ml₄Solution, mixed even places 2 hours in 4 DEG C, and >=1:1000 colourless or be with brown clarified liq of after carrying out purifying with gel permeation chromatography, selecting to tire, is the furadantin specific antibody of horseradish peroxidase-labeled;

The preparation method of described furadantin-BSA immunogen is:

Prepared by step one, immunogen:

Step one, claim 10mg para-amino benzoic acid to dissolve in 1.1mL0.2mol/LHCl, it is placed in 0-4 DEG C and is stirred to and dissolves completely, obtain para-amino benzoic acid solution; Then the NaNO of 6mg is taken₂It is dissolved in the distilled water of 0.35mL, it is placed in 0-4 DEG C and is stirred to and dissolves completely, obtain NaNO₂Solution; By NaNO₂Dropwise joins in para-amino benzoic acid solution, and lucifuge reacts 1 hour, obtains mixed solution A;

Step 2, the furadantin taking 0.3-1.2mg are dissolved in 10mL containing in the borate buffer solution of NaCl, are stirred to and dissolve completely, obtain mixing solutions B at being placed in 0-4 DEG C; Dropwise joining in mixing solutions B by above-mentioned steps gained mixed solution A, lucifuge reacts 2 hours, obtains orange solution C;

In the described borate buffer solution containing NaCl, the concentration of borax is 0.05mol/L, and pH value is 8.5, and is the NaCl of 0.15mol/L containing concentration;

Step 3, in orange solution C, add H₃BO₃Crystal regulates its pH value to 7.4, then adds 94mg bovine serum albumin, 80mg carbodiimide and 4mgN-N-Hydroxysuccinimide, is then placed in stirring at room temperature 2 hours, obtains orange solution D;

Step 4, above-mentioned steps gained orange solution D is transferred in dialysis tubing, it is the PBS of 7.4 with 0.01mol/L, pH, dialyse five days at 0-4 DEG C, within every 12 hours, change a PBS; By the solution freeze-drying after dialysis, obtain faint yellow solid powder and it is immunogen furadantin-BSA, be placed on-20 DEG C of preservations, for subsequent use.

Described wrapping by the preparation method of the luminescent screen of furadantin is:

Step one, furadantin and ovalbumin carried out coupling obtain envelope antigen:

(1) NaNO of 9mg is taken₂It is dissolved in the distilled water of 0.3mL, obtains NaNO₂Solution; Taking 10mg furadantin is dissolved in the hydrochloric acid of 1.6mL0.2mol/L, and 0-4 DEG C is stirred to and dissolves completely, obtains furadantin solution; Then by gained NaNO₂Dropwise adds in gained furadantin solution, and lucifuge reacts 0.5 hour;

(2), after reaction terminates, adding 25mg amine sulfate until releasing without nitrogen, namely obtaining diazotizing furadantin solution;

(3) claiming 70mg ovalbumin to be dissolved in the 0.1mol/L of 4mL, pH value is in the PBS phosphate buffer solution of 7.5, obtains ovalbumin solution; Then the diazotizing furadantin dropwise described in above-mentioned steps is joined in ovalbumin solution, obtain mixing solutions E; Then with the sodium hydroxide of 1mol/L by the pH of mixing solutions E to being adjusted to 7.5, at being then placed in 0-4 DEG C, lucifuge reacts 18 hours;

(4) transferring in dialysis tubing by reacted for above-mentioned steps lucifuge reaction solution, with 0.01mol/L, pH value is the PBS of 7.4, dialyses five days at 0-4 DEG C, within every 12 hours, changes a PBS; Then by centrifugal for dialyzate 3000 revs/min 15 minutes, freeze-drying supernatant liquor, obtains faint yellow solid powder and is envelope antigen furadantin-OVA, be placed on-20 DEG C of preservations, for subsequent use;

Step 2, bag quilt:

(1) with 0.05mol/L, pH value be 9.6 carbonate bag by solution, envelope antigen is made into the solution of 1.5 �� g/mL, and the �� L that adds 100 in the reacting hole of each polystyrene board, spend the night at 4 DEG C afterwards, abandon solution in hole next day, wash 3 times with lavation buffer solution, each 5 minutes;

(2) close the above-mentioned polystyrene board having wrapped quilt by lock solution, 250 �� L/ holes, 37 DEG C hatch 1 hour after washing, obtain wrapping by the luminescent screen of furadantin.

A detection method for Macrodantin chemiluminescence detection kit, its concrete detecting step is:

Step one, add sample: by the furadantin standard solution of each concentration and sample solution, joining with the add-on in 50 �� L/ holes wraps by the reacting hole of the luminescent screen of furadantin, then every hole adds 100 �� L employing coating buffers with the furadantin specific antibody of the horseradish peroxidase-labeled of 1:7000 dilution proportion, then hatches 0.5 hour for 37 DEG C;

Step 2, washing: inclining to and wrap by liquid in the hole of the luminescent screen of furadantin, every hole adds washing soln 250 �� L, washs 3 times, pats dry;

Step 3, add luminous substrate liquid: after the A liquid of luminous substrate and B liquid are mixed mixture, add 100 �� L luminous substrate liquid mixtures wrapping every hole in the luminescent screen by furadantin,

Step 4, detection: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument;

Step 5, result judge: get standard substance log concentration and do X-coordinate, and standard substance detection luminous value logarithm does ordinate zou, does typical curve, and the concentration of each sample can be calculated from typical curve.

Useful effect is:

1, Macrodantin chemiluminescence detection kit of the present invention, highly sensitive, sensing range width, easy and simple to handle fast, nontoxic pollution-free, optical signal time length length and instrument used simple, the chemiluminescence detection kit of economic detection furadantin, provide its preparation method simultaneously, and detect the method for furadantin microbiotic in sample. The chemical luminescence ELISA detection kit of antibiotics leftover detection have highly sensitive, easy fast, accurately, security is good and the feature of usage period length, comparing with traditional colorimetric ELISA method, analytical procedure is easy fast, sensitivity can improve an order of magnitude. Play a significant role in being expected in feed Nitrofurantoin residue and detecting.

2, Macrodantin chemiluminescence detection kit of the present invention adopts the metabolite of furadantin and carrier conjugation to prepare envelope antigen, and more economical, step is more simple, is beneficial to suitability for industrialized production; The preparation of horseradish peroxidase marker, adopts single stage method, and method is simple, is more suitable for suitability for industrialized production, and obtains good effect through verification experimental verification.

Accompanying drawing explanation

Fig. 1 is the process flow sheet that the present invention prepares Macrodantin chemiluminescence detection kit;

Fig. 2 is the result standard graphic representation of embodiments of the invention detection;

Embodiment

A kind of Macrodantin chemiluminescence detection kit, comprises concentrated washing lotion, negative controls, positive reference substance, chemical luminescence for liquid, furadantin standard substance, the furadantin specific antibody of horseradish peroxidase-labeled and has wrapped by the luminescent screen of furadantin; Concrete preparation process is as shown in fig. 1;

Described concentrated washing lotion is: containing the phosphate buffered saline buffer of Tween-20 and sodium-chlor;

Described negative controls is: the furadantin after more than the 5 parts Sterile Filtrations deposited separately is detected as negative pig urine mixture;

Described positive reference substance is: the pig urine mixture of the furadantin test positive after more than the 5 parts Sterile Filtrations deposited separately;

Described chemical luminescence for liquid, comprises A liquid and B liquid; A liquid making method is: the Tris-HCl damping fluid first configuring 0.2mol/LpH7.8, adds luminol,3-aminophthalic acid cyclic hydrazide and sodium tetraphenylborate in this damping fluid, mixes and can obtain A liquid; B liquid making method is: the Tris-HCl damping fluid first configuring 0.2mol/LpH7.8, adds 0.1%H in this damping fluid by weight₂O₂Mix and can obtain B liquid;

Described furadantin standard substance are the diluent that concentration is respectively 0ng/ml, 0.1ng/ml, 0.3ng/ml, 0.9ng/ml, 2.7ng/ml, 8.1ng/ml furadantin;

The preparation method of the furadantin specific antibody of described horseradish peroxidase-labeled is:

Prepared by step one, immunogen:

(1) claim 10mg para-amino benzoic acid to dissolve in 1.1mL0.2mol/LHCl, it is placed in 0-4 DEG C and is stirred to and dissolves completely, obtain para-amino benzoic acid solution; Then the NaNO of 6mg is taken₂It is dissolved in the distilled water of 0.35mL, it is placed in 0-4 DEG C and is stirred to and dissolves completely, obtain NaNO₂Solution; By NaNO₂Dropwise joins in para-amino benzoic acid solution, and lucifuge reacts 1 hour, obtains mixed solution A;

(2) furadantin taking 0.3-1.2mg is dissolved in 10mL containing in the borate buffer solution of NaCl, is stirred to and dissolves completely, obtain mixing solutions B at being placed in 0-4 DEG C; Dropwise joining in mixing solutions B by above-mentioned steps gained mixed solution A, lucifuge reacts 2 hours, obtains orange solution C;

In the described borate buffer solution containing NaCl, the concentration of borax is 0.05mol/L, and pH value is 8.5, and is the NaCl of 0.15mol/L containing concentration;

(3) in orange solution C, H is added₃BO₃Crystal regulates its pH value to 7.4, then adds 94mg bovine serum albumin, 80mg carbodiimide and 4mgN-N-Hydroxysuccinimide, is then placed in stirring at room temperature 2 hours, obtains orange solution D;

(4) above-mentioned steps gained orange solution D is transferred in dialysis tubing, it is the PBS of 7.4 with 0.01mol/L, pH, dialyse five days at 0-4 DEG C, within every 12 hours, change a dialyzate; By the solution freeze-drying after dialysis, obtain faint yellow solid powder and it is immunogen furadantin-BSA, be placed on-20 DEG C of preservations, for subsequent use.

Step 2, employing new zealand white rabbit are as immune animal, taking furadantin-BSA as immunogen, first immunisation dosage is 100 �� g/ rabbit, during first immunisation, immunogen is dissolved in and makes emulsifying agent into isopyknic physiological saline and Freund's complete adjuvant, nape portion multi-point injection, booster immunization dosage reduces by half in isopyknic physiological saline and Freund's incomplete adjuvant mixing and emulsifying, first immunisation and second immunisation interval 14 days, later every immunity in 2 weeks once immunity five times altogether, do not add adjuvant for the last time; Last immunity Culling heart blood after 7 days, centrifugal obtains anti-furadantin polyclonal antibody.

The preparation of step 3, horseradish peroxidase marker

Preparing enzyme labelling furadantin antibody with Over-voltage protection, reaction system is: first with the NaIO of the horseradish peroxidase solution of 1ml concentration 5mg/ml and 0.15ml concentration 0.1mol/L₄Solution mix even after, it is placed in 4 DEG C of lucifuges 0.5 hour, then the ethylene glycol adding 0.25ml shakes even room temperature lucifuge 1 hour, then 4 DEG C of dialysed overnight it are placed in, afterwards with 0.2ml concentration 0.2mol/L, pH value is the carbonate bag of 9.6 is 9.2-9.5 by solution by the hydroformylation horseradish peroxidase solution adjust pH that obtains after dialysis, anti-for 5mg furadantin polyclonal antibody is dissolved in 1.0ml concentration 0.01mol/L, add immediately after the coating buffer of pH9.6 in the horseradish peroxidase solution after above-mentioned hydroformylation and react 1 hour in 37 DEG C, then add the NaBH of 0.1ml concentration 3.5mg/ml₄Solution, mixed even places 2 hours in 4 DEG C, and >=1:1000 colourless or be with brown clarified liq of after carrying out purifying with gel permeation chromatography, selecting to tire, is the furadantin specific antibody of horseradish peroxidase-labeled;

Described wrapping by the preparation method of the luminescent screen of furadantin is:

Step one, furadantin and ovalbumin carried out coupling obtain envelope antigen:

(1) NaNO of 9mg is taken₂It is dissolved in the distilled water of 0.3mL, obtains NaNO₂Solution; Taking 10mg furadantin is dissolved in the hydrochloric acid of 1.6mL0.2mol/L, and 0-4 DEG C is stirred to and dissolves completely, obtains furadantin solution; Then by gained NaNO₂Dropwise adds in gained furadantin solution, and lucifuge reacts 0.5 hour;

(2), after reaction terminates, adding 25mg amine sulfate until releasing without nitrogen, namely obtaining diazotizing furadantin solution;

(3) claiming 70mg ovalbumin to be dissolved in the 0.1mol/L of 4mL, pH value is in the PBS phosphate buffer solution of 7.5, obtains ovalbumin solution; Then the diazotizing furadantin dropwise described in above-mentioned steps is joined in ovalbumin solution, obtain mixing solutions E; Then with the sodium hydroxide of 1mol/L by the pH of mixing solutions E to being adjusted to 7.5, at being then placed in 0-4 DEG C, lucifuge reacts 18 hours;

(4) transferring in dialysis tubing by reacted for above-mentioned steps lucifuge reaction solution, with 0.01mol/L, pH value is the PBS of 7.4, dialyses five days at 0-4 DEG C, within every 12 hours, changes a PBS; Then by centrifugal for dialyzate 3000 revs/min 15 minutes, freeze-drying supernatant liquor, obtains faint yellow solid powder and is envelope antigen furadantin-OVA, be placed on-20 DEG C of preservations, for subsequent use;

Step 2, bag quilt:

(1) with 0.05mol/L, pH value be 9.6 carbonate bag by solution, envelope antigen is made into the solution of 1.5 �� g/mL, and the �� L that adds 100 in the reacting hole of each polystyrene board, spend the night at 4 DEG C afterwards, abandon solution in hole next day, wash 3 times with lavation buffer solution, each 5 minutes;

(2) close the above-mentioned polystyrene board having wrapped quilt by lock solution, 250 �� L/ holes, 37 DEG C hatch 1 hour after washing, obtain wrapping by the luminescent screen of furadantin.

A detection method for Macrodantin chemiluminescence detection kit, its concrete detecting step is:

Step one, add sample: joined with the add-on in 50 �� L/ holes by the furadantin standard solution of each concentration and wrap by the reacting hole of the luminescent screen of furadantin, then every hole adds 100 �� L employing coating buffers with the furadantin specific antibody of the horseradish peroxidase-labeled of 1:7000 dilution proportion, then hatches 0.5 hour for 37 DEG C;

Step 2, washing: inclining to and wrap by liquid in the hole of the luminescent screen of furadantin, every hole adds washing soln 250 �� L, washs 3 times, pats dry;

Step 3, add luminous substrate liquid: after the A liquid of luminous substrate and B liquid are mixed mixture, add 100 �� L luminous substrate liquid mixtures wrapping every hole in the luminescent screen by furadantin,

Step 4, detection: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument;

Step 5, result judge: get standard substance log concentration and do X-coordinate, and standard substance detection luminous value logarithm does ordinate zou, does typical curve, and the concentration of each sample can be calculated from typical curve.

Embodiment

A kind of Macrodantin chemiluminescence detection kit, comprises concentrated washing lotion, negative controls, positive reference substance, chemical luminescence for liquid, furadantin standard substance, the furadantin specific antibody of horseradish peroxidase-labeled and has wrapped by the luminescent screen of furadantin;

Described concentrated washing lotion is: containing the phosphate buffered saline buffer of Tween-20 and sodium-chlor;

Described negative controls is: the furadantin after more than the 5 parts Sterile Filtrations deposited separately is detected as negative pig urine mixture;

Described positive reference substance is: the pig urine mixture of the furadantin test positive after more than the 5 parts Sterile Filtrations deposited separately;

Described chemical luminescence for liquid, comprises A liquid and B liquid; A liquid making method is: the Tris-HCl damping fluid first configuring 0.2mol/LpH7.8, adds luminol,3-aminophthalic acid cyclic hydrazide and sodium tetraphenylborate in this damping fluid, mixes and can obtain A liquid; B liquid making method is: the Tris-HCl damping fluid first configuring 0.2mol/LpH7.8, adds 0.1%H in this damping fluid by weight₂O₂Mix and can obtain B liquid;

Described furadantin standard substance are the diluent that concentration is respectively 0ng/ml, 0.1ng/ml, 0.3ng/ml, 0.9ng/ml, 2.7ng/ml, 8.1ng/ml furadantin;

The preparation method of the furadantin specific antibody of described horseradish peroxidase-labeled is:

Prepared by step one, immunogen:

(1) claim 10mg para-amino benzoic acid to dissolve in 1.1mL0.2mol/LHCl, it is placed in 0-4 DEG C and is stirred to and dissolves completely, obtain para-amino benzoic acid solution; Then the NaNO of 6mg is taken₂It is dissolved in the distilled water of 0.35mL, it is placed in 0-4 DEG C and is stirred to and dissolves completely, obtain NaNO₂Solution; By NaNO₂Dropwise joins in para-amino benzoic acid solution, and lucifuge reacts 1 hour, obtains mixed solution A;

(2) furadantin taking 0.3-1.2mg is dissolved in 10mL containing in the borate buffer solution of NaCl, is stirred to and dissolves completely, obtain mixing solutions B at being placed in 0-4 DEG C; Dropwise joining in mixing solutions B by above-mentioned steps gained mixed solution A, lucifuge reacts 2 hours, obtains orange solution C;

In the described borate buffer solution containing NaCl, the concentration of borax is 0.05mol/L, and pH value is 8.5, and is the NaCl of 0.15mol/L containing concentration;

(3) in orange solution C, H is added₃BO₃Crystal regulates its pH value to 7.4, then adds 94mg bovine serum albumin, 80mg carbodiimide and 4mgN-N-Hydroxysuccinimide, is then placed in stirring at room temperature 2 hours, obtains orange solution D;

(4) above-mentioned steps gained orange solution D is transferred in dialysis tubing, it is the PBS of 7.4 with 0.01mol/L, pH, dialyse five days at 0-4 DEG C, within every 12 hours, change a dialyzate; By the solution freeze-drying after dialysis, obtain faint yellow solid powder and it is immunogen furadantin-BSA, be placed on-20 DEG C of preservations, for subsequent use.

Step 2, employing new zealand white rabbit are as immune animal, taking furadantin-BSA as immunogen, first immunisation dosage is 100 �� g/ rabbit, during first immunisation, immunogen is dissolved in and makes emulsifying agent into isopyknic physiological saline and Freund's complete adjuvant, nape portion multi-point injection, booster immunization dosage reduces by half in isopyknic physiological saline and Freund's incomplete adjuvant mixing and emulsifying, first immunisation and second immunisation interval 14 days, later every immunity in 2 weeks once immunity five times altogether, do not add adjuvant for the last time; Last immunity Culling heart blood after 7 days, centrifugal obtains anti-furadantin polyclonal antibody.

The preparation of step 3, horseradish peroxidase marker

Preparing enzyme labelling furadantin antibody with Over-voltage protection, reaction system is: first with the NaIO of the horseradish peroxidase solution of 1ml concentration 5mg/ml and 0.15ml concentration 0.1mol/L₄Solution mix even after, it is placed in 4 DEG C of lucifuges 0.5 hour, then the ethylene glycol adding 0.25ml shakes even room temperature lucifuge 1 hour, then 4 DEG C of dialysed overnight it are placed in, afterwards with 0.2ml concentration 0.2mol/L, pH value is the carbonate bag of 9.6 is 9.2-9.5 by solution by the hydroformylation horseradish peroxidase solution adjust pH that obtains after dialysis, anti-for 5mg furadantin polyclonal antibody is dissolved in 1.0ml concentration 0.01mol/L, add immediately after the coating buffer of pH9.6 in the horseradish peroxidase solution after above-mentioned hydroformylation and react 1 hour in 37 DEG C, then add the NaBH of 0.1ml concentration 3.5mg/ml₄Solution, mixed even places 2 hours in 4 DEG C, and >=1:1000 colourless or be with brown clarified liq of after carrying out purifying with gel permeation chromatography, selecting to tire, is the furadantin specific antibody of horseradish peroxidase-labeled;

Described wrapping by the preparation method of the luminescent screen of furadantin is:

Step one, furadantin and ovalbumin carried out coupling obtain envelope antigen:

(1) NaNO of 9mg is taken₂It is dissolved in the distilled water of 0.3mL, obtains NaNO₂Solution; Taking 10mg furadantin is dissolved in the hydrochloric acid of 1.6mL0.2mol/L, and 0-4 DEG C is stirred to and dissolves completely, obtains furadantin solution; Then by gained NaNO₂Dropwise adds in gained furadantin solution, and lucifuge reacts 0.5 hour;

(2), after reaction terminates, adding 25mg amine sulfate until releasing without nitrogen, namely obtaining diazotizing furadantin solution;

(3) claiming 70mg ovalbumin to be dissolved in the 0.1mol/L of 4mL, pH value is in the PBS phosphate buffer solution of 7.5, obtains ovalbumin solution; Then the diazotizing furadantin dropwise described in above-mentioned steps is joined in ovalbumin solution, obtain mixing solutions E; Then with the sodium hydroxide of 1mol/L by the pH of mixing solutions E to being adjusted to 7.5, at being then placed in 0-4 DEG C, lucifuge reacts 18 hours;

(4) transferring in dialysis tubing by reacted for above-mentioned steps lucifuge reaction solution, with 0.01mol/L, pH value is the PBS of 7.4, dialyses five days at 0-4 DEG C, within every 12 hours, changes a PBS; Then by centrifugal for dialyzate 3000 revs/min 15 minutes, freeze-drying supernatant liquor, obtains faint yellow solid powder and is envelope antigen furadantin-OVA, be placed on-20 DEG C of preservations, for subsequent use;

Step 2, bag quilt:

(1) with 0.05mol/L, pH value be 9.6 carbonate bag by solution, envelope antigen is made into the solution of 1.5 �� g/mL, and the �� L that adds 100 in the reacting hole of each polystyrene board, spend the night at 4 DEG C afterwards, abandon solution in hole next day, wash 3 times with lavation buffer solution, each 5 minutes;

(2) close the above-mentioned polystyrene board having wrapped quilt by lock solution, 250 �� L/ holes, 37 DEG C hatch 1 hour after washing, obtain wrapping by the luminescent screen of furadantin.

The concrete detecting step of a kind of Macrodantin chemiluminescence detection kit:

Step one, sample pre-treatments

Feed pre-treating process: take 1.0 �� 0.05g feed sample; Adding 5ml deionized water, fully vibration is to dissolving; Take out 50ul supernatant and it is used as analysis.

Step 2, detecting step

(1) sample is added: Cistofuran metabolite series standard strength solution and the sample solution adding 50uL/ hole in luminescent screen, then Cistofuran metabolite antibody working fluid 50uL/ hole is added, add diluted fresh horseradish peroxidase-furadantin antibody (1:7000) 100 �� L/ hole, 37 DEG C 0.5 hour;

(2) wash: incline the middle liquid that portals, and adds the washings in 300uL/ hole in luminescent screen, pat dry after leaving standstill 5min, repeat three times;

(3) luminescent solution is added: every hole adds luminescent solution lOOuL;

(4) result judges: gets standard substance log concentration and does X-coordinate, and standard substance detection luminous value logarithm does ordinate zou, does typical curve, and result is such as Fig. 2, figure standard curve y=-1.0354x+4.7213, R²=0.9983; The concentration of each sample can be calculated from typical curve, and result is such as table 1.

Table 1:

Furadantin standard substance (ng/ml)	Luminous value
		0.1	550000
0.3	183000
		0.9	60000
2.7	21000
		8.1	5500
24.3	1800

Claims (1)

1. a Macrodantin chemiluminescence detection kit, it is characterised in that: comprise concentrated washing lotion, negative controls, positive reference substance, chemical luminescence for liquid, furadantin standard substance, the furadantin specific antibody of horseradish peroxidase-labeled and wrapped by the luminescent screen of furadantin;

Described concentrated washing lotion is: containing the phosphate buffered saline buffer of Tween-20 and sodium-chlor;

Described negative controls is: the furadantin after more than the 5 parts Sterile Filtrations deposited separately is detected as negative pig urine mixture;

Described positive reference substance is: the pig urine mixture of the furadantin test positive after more than the 5 parts Sterile Filtrations deposited separately;

Described chemical luminescence for liquid, comprises A liquid and B liquid; A liquid making method is: the Tris-HCl damping fluid first configuring 0.2mol/LpH7.8, adds luminol,3-aminophthalic acid cyclic hydrazide and sodium tetraphenylborate in this damping fluid, mixes and can obtain A liquid; B liquid making method is: the Tris-HCl damping fluid first configuring 0.2mol/LpH7.8, adds 0.1%H in this damping fluid by weight₂O₂Mix and can obtain B liquid;

Described furadantin standard substance are the diluent that concentration is respectively 0ng/ml, 0.1ng/ml, 0.3ng/ml, 0.9ng/ml, 2.7ng/ml, 8.1ng/ml furadantin;

The furadantin specific antibody of described horseradish peroxidase-labeled is, horseradish peroxidase-labeled taking furadantin-BSA as the anti-furadantin polyclonal antibody of immunogen immune new zealand white rabbit gained; Prepare the furadantin specific antibody of horseradish peroxidase-labeled with Over-voltage protection, reaction system is: first with the NaIO of the horseradish peroxidase solution of 1ml concentration 5mg/ml and 0.15ml concentration 0.1mol/L₄Solution mix even after, it is placed in 4 DEG C of lucifuges 0.5 hour, then the ethylene glycol adding 0.25ml shakes even room temperature lucifuge 1 hour, then 4 DEG C of dialysed overnight it are placed in, afterwards with 0.2ml concentration 0.2mol/L, pH value is the carbonate bag of 9.6 is 9.2-9.5 by solution by the hydroformylation horseradish peroxidase solution adjust pH that obtains after dialysis, anti-for 5mg furadantin polyclonal antibody is dissolved in 1.0ml concentration 0.01mol/L, the carbonate bag of pH9.6 is reacted 1 hour in 37 DEG C in the horseradish peroxidase solution after above-mentioned hydroformylation by adding immediately after solution, then the NaBH of 0.1ml concentration 3.5mg/ml is added₄Solution, mixed even places 2 hours in 4 DEG C, and >=1:1000 colourless or be with brown clarified liq of after carrying out purifying with gel permeation chromatography, selecting to tire, is the furadantin specific antibody of horseradish peroxidase-labeled;

The preparation method of the furadantin specific antibody of described horseradish peroxidase-labeled is:

Prepared by step one, immunogen:

(1) claim 10mg para-amino benzoic acid to dissolve in 1.1mL0.2mol/LHCl, it is placed in 0-4 DEG C and is stirred to and dissolves completely, obtain para-amino benzoic acid solution; Then the NaNO2 taking 6mg is dissolved in the distilled water of 0.35mL, is placed in 0-4 DEG C and is stirred to and dissolves completely, obtains NaNO2 solution; Being joined by NaNO2 dropwise in para-amino benzoic acid solution, lucifuge reacts 1 hour, obtains mixed solution A;

(2) furadantin taking 0.3-1.2mg is dissolved in 10mL containing in the borate buffer solution of NaCl, is stirred to and dissolves completely, obtain mixing solutions B at being placed in 0-4 DEG C; Dropwise joining in mixing solutions B by above-mentioned steps gained mixed solution A, lucifuge reacts 2 hours, obtains orange solution C;

In the described borate buffer solution containing NaCl, the concentration of borax is 0.05mol/L, and pH value is 8.5, and is the NaCl of 0.15mol/L containing concentration;

(3) in orange solution C, add H3BO3 crystal and regulate its pH value to 7.4, then add 94mg bovine serum albumin, 80mg carbodiimide and 4mgN-N-Hydroxysuccinimide, be then placed in stirring at room temperature 2 hours, obtain orange solution D;

(4) above-mentioned steps gained orange solution D is transferred in dialysis tubing, it is the PBS of 7.4 with 0.01mol/L, pH, dialyse five days at 0-4 DEG C, within every 12 hours, change a dialyzate; By the solution freeze-drying after dialysis, obtain faint yellow solid powder and it is immunogen furadantin-BSA, be placed on-20 DEG C of preservations, for subsequent use;

Step 2, employing new zealand white rabbit are as immune animal, taking furadantin-BSA as immunogen, first immunisation dosage is 100 �� g/ rabbit, during first immunisation, immunogen is dissolved in isopyknic physiological saline and Freund's complete adjuvant makes emulsifying agent, nape portion multi-point injection, booster immunization dosage reduces by half in isopyknic physiological saline and Freund's incomplete adjuvant mixing and emulsifying, first immunisation and second immunisation interval 14 days, later every immunity in 2 weeks once immunity five times altogether, do not add adjuvant for the last time; Last immunity Culling heart blood after 7 days, centrifugal obtains anti-furadantin polyclonal antibody;

Described wrapping by the preparation method of the luminescent screen of furadantin is:

Step one, furadantin and ovalbumin carried out coupling obtain envelope antigen:

(1) NaNO of 9mg is taken₂It is dissolved in the distilled water of 0.3mL, obtains NaNO₂Solution; Taking 10mg furadantin is dissolved in the hydrochloric acid of 1.6mL0.2mol/L, and 0-4 DEG C is stirred to and dissolves completely, obtains furadantin solution; Then by gained NaNO₂Dropwise adds in gained furadantin solution, and lucifuge reacts 0.5 hour;

(2), after reaction terminates, adding 25mg amine sulfate until releasing without nitrogen, namely obtaining diazotizing furadantin solution;

(3) claiming 70mg ovalbumin to be dissolved in the 0.1mol/L of 4mL, pH value is in the PBS phosphate buffer solution of 7.5, obtains ovalbumin solution; Then the diazotizing furadantin dropwise described in above-mentioned steps is joined in ovalbumin solution, obtain mixing solutions E; Then with the sodium hydroxide of 1mol/L by the pH of mixing solutions E to being adjusted to 7.5, at being then placed in 0-4 DEG C, lucifuge reacts 18 hours;

(4) transferring in dialysis tubing by reacted for above-mentioned steps lucifuge reaction solution, with 0.01mol/L, pH value is the PBS of 7.4, dialyses five days at 0-4 DEG C, within every 12 hours, changes a PBS; Then by centrifugal for dialyzate 3000 revs/min 15 minutes, freeze-drying supernatant liquor, obtains faint yellow solid powder and is envelope antigen furadantin-OVA, be placed on-20 DEG C of preservations, for subsequent use;

Step 2, bag quilt:

(1) with 0.05mol/L, pH value be 9.6 carbonate bag by solution, envelope antigen is made into the solution of 1.5 �� g/mL, and the �� L that adds 100 in the reacting hole of each polystyrene board, spend the night at 4 DEG C afterwards, abandon solution in hole next day, wash 3 times with lavation buffer solution, each 5 minutes;

(2) close the above-mentioned polystyrene board having wrapped quilt by lock solution, 250 �� L/ holes, 37 DEG C hatch 1 hour after washing, obtain wrapping by the luminescent screen of furadantin;

The method of prepared Macrodantin chemiluminescence detection kit detection Nitrofurantoin residue, its concrete detecting step is:

Step one, add sample: by the furadantin standard solution of each concentration and sample solution, joining with the add-on in 50 �� L/ holes wraps by the reacting hole of the luminescent screen of furadantin, then every hole adds 100 �� L employing coating buffers with the furadantin specific antibody of the horseradish peroxidase-labeled of 1:7000 dilution proportion, then hatches 0.5 hour for 37 DEG C;

Step 2, washing: inclining to and wrap by liquid in the hole of the luminescent screen of furadantin, every hole adds washing soln 250 �� L, washs 3 times, pats dry;

Step 3, add luminous substrate liquid: after the A liquid of luminous substrate and B liquid are mixed mixture, add 100 �� L luminous substrate liquid mixtures wrapping every hole in the luminescent screen by furadantin,

Step 4, detection: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument;

Step 5, result judge: get standard substance log concentration and do X-coordinate, and standard substance detection luminous value logarithm does ordinate zou, does typical curve, and the concentration of each sample can be calculated from typical curve.