CN103645310A - Macrodantin chemiluminescence detection kit - Google Patents

Abstract

The invention relates to a chemiluminescence detection kit used for detecting macrodantin. The chemiluminescence detection kit comprises a wash concentrate, a negative reference substance, a positive reference substance, a chemiluminescence liquid, macrodantin standard substance, horse radish peroxidase labelled macrodantin specific antibody, and a luminescence test plate coated with macrodantin. The horse radish peroxidase labelled macrodantin specific antibody is horse radish peroxidase labelled anti-macrodantin polyclonal antibody obtained via immunization of New Zealand white rabbits, wherein macrodantin-BSA is taken as an immunogen; and the horse radish peroxidase labelled macrodantin specific antibody is prepared via adoption of sodium periodate method. The chemiluminescence detection kit used for detecting macrodantin is high in sensitivity, and wide in detection range; is simple and convenient for operation; and possesses no toxicity. No pollution is caused by the chemiluminescence detection kit. According to the chemiluminescence detection kit, the envelope antigen is prepared via coupling of a macrodantin metabolite and a carrier, so that cost is low. The horse radish peroxidase labelled macrodantin specific antibody is prepared via one-step method, so that the preparation method and preparation steps are simpler, and it is beneficial for industrialized production.

Description

A kind of furantoin chemiluminescence detection kit

Technical field

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

Background technology

The antibiotic technology in feed that detects at present mainly contains following several method:

(1) physics and chemistry detection method

After the nineties in 20th century, most physics and chemistry detection methods of measuring furantoin mainly rely on liquid chromatography technology to carry out separation, 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, aspect antibiotics leftover detection, slightly apply.Chromatography detect medicament residue in feed to pass through sample preparation (comprise sample extraction, take off the steps such as albumen, centrifugal, chromatographic column purification, derivatization), the separation of medicine and the detection of medicine.Physics and chemistry detection method utilizes special reaction or the 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 that fodder antibiotics detects.This method detection sensitivity is higher, but instrument and testing cost are high, trace routine is complicated, compared with time-consuming etc.

(2) immune analysis method

Immune analysis method is that to take specificity, the reversibility association reaction of antigen and antibody be basic analytical approach, and medicament residue immuno analytical method is mainly divided three major types at present: method, the immunity receptor method of relatively independent analytical approach, immuno analytical method and the coupling of conventional physical and chemical analysis technology.Dutch van weeman, schurrs in 1973 and Sweden Engvall, Perlmann propose respectively enzyme linked immunosorbent assay, and over more than 30 year, Chinese scholars detects microbiotic in food to immunological method and carried out large quantity research.But because microbiotic is haptens, antigen constructing technology difficulty is large, immunologic opsonin is strong, testing cost is high, at present the company such as IDEXX is comparatively ripe in the research of exempting from aspect analytic approach, and domestic this technology is still in research and development.Current is mainly with enzyme linked immunosorbent assay, radioimmunoassay luminescence method, fluorescent immune method, chemoluminescence method etc.; Method and the comparisons of luminous phase chemistry luminescence method such as enzyme linked immunosorbent assay, radioimmunoassay luminescence method, fluorescent immune method, it is lower that its enzyme is exempted from sensitivity.

Summary of the invention

The present invention seeks to the deficiency for solving the problems of the technologies described above, a kind of furantoin chemiluminescence detection kit be provided, its have highly sensitive, sensing range is wide, the advantage such as fast easy and simple to handle.

The present invention solves the problems of the technologies described above adopted technical scheme to be: a kind of furantoin chemiluminescence detection kit, comprises the luminous plaque of the furantoin specific antibody of concentrated washing lotion, negative control product, positive reference substance, chemical luminescence for liquid, furantoin standard items, horseradish peroxidase-labeled and coated furantoin;

Described concentrated washing lotion is: the phosphate buffer that contains Tween-20 and sodium chloride;

Described negative control product are: the furantoin after 5 parts of aseptic filtrations of depositing separately above detects negative pig urine potpourri;

Described positive reference substance is: the pig urine potpourri of the furantoin test positive after 5 parts of aseptic filtrations of depositing separately above;

Described chemical luminescence for liquid, comprises A liquid and B liquid; A liquid making method is: first configure the Tris-HCl damping fluid of 0.2 mol/L pH7.8, add luminol and tetraphenylboron sodium in this damping fluid, mix and can obtain A liquid; B liquid making method is: first configure the Tris-HCl damping fluid of 0.2 mol/L pH7.8, add by weight 0.1% H in this damping fluid ₂o ₂mix and can obtain B liquid;

Described furantoin standard items are the dilution that concentration is respectively 0ng/ml, 0.1ng/ml, 0.3 ng/ml, 0.9 ng/ml, 2.7 ng/ml, 8.1ng/ml furantoin;

The furantoin specific antibody of described horseradish peroxidase-labeled is, horseradish peroxidase-labeled take furantoin-BSA as the anti-furantoin polyclonal antibody of immunogen immune new zealand white rabbit gained, furantoin specific antibody by sodium periodate legal system for horseradish peroxidase-labeled, reaction system is: first use the horseradish peroxidase solution of 1ml concentration 5mg/ml and the NaIO of 0.15ml concentration 0.1 mol/L ₄after solution mixes, be placed in 4 ℃ of lucifuges 0.5 hour, then add the ethylene glycol of 0.25ml to shake up room temperature lucifuge 1 hour, then be placed in 4 ℃ of dialysed overnight, use afterwards 0.2ml concentration 0.2 mol/L, pH value is that the coated solution of 9.6 carbonate is 9.2-9.5 by the hydroformylation horseradish peroxidase solution adjust pH obtaining after dialysis, the anti-furantoin polyclonal antibody of 5mg is dissolved in to 1.0ml concentration 0.01 mol/L, after the coating buffer of pH9.6, add immediately in the horseradish peroxidase solution after above-mentioned hydroformylation in 37 ℃ of reactions 1 hour, then the NaBH that adds 0.1ml concentration 3.5mg/ml ₄solution, mixes in 4 ℃ of placements 2 hours, with gel permeation chromatography, carries out after purifying, selects the colourless of tire>=1:1000 or is with brown supernatant liquid, is the furantoin specific antibody of horseradish peroxidase-labeled,

The immunogenic preparation method of described furantoin-BSA is:

Step 1, immunogene preparation:

Step 1, title 10mg p-aminobenzoic acid dissolve in 1.1mL 0.2 mol/L HCl, are placed in 0-4 ℃ and are stirred to dissolving completely, obtain p-aminobenzoic acid solution; Then take the NaNO of 6mg ₂be dissolved in the distilled water of 0.35mL, be placed in 0-4 ℃ and be stirred to dissolving completely, obtain NaNO ₂solution; By NaNO ₂solution dropwise joins in p-aminobenzoic acid solution, and lucifuge reaction 1 hour, obtains mixed solution A;

Step 2, the furantoin that takes 0.3-1.2mg are dissolved in 10mL containing in the borate buffer solution of NaCl, are placed at 0-4 ℃ and are stirred to completely and dissolve, and obtain mixed solution B; Above-mentioned steps gained mixed solution A solution is dropwise joined in mixed solution B, and lucifuge reaction 2 hours, obtains orange colour solution C;

Described containing in the borate buffer solution of NaCl, the concentration of borax is 0.05mol/L, and pH value is 8.5, and contains the NaCl that concentration is 0.15mol/L;

Step 3, in orange colour solution C, add H ₃bO ₃crystal regulates its pH value to 7.4, then adds 94mg bovine serum albumin(BSA), 80mg carbodiimide and 4mg N-hydroxy-succinamide, is then placed in stirring at room 2 hours, obtains orange colour solution D;

Step 4, above-mentioned steps gained orange colour solution D is transferred in bag filter, with 0.01mol/L, the PBS damping fluid that pH is 7.4 is dialysed five days at 0-4 ℃, within every 12 hours, changes a PBS damping fluid; Solution freeze-drying by after dialysis, obtains faint yellow solid powder and is immunogene furantoin-BSA, is placed on-20 ℃ of preservations, standby.

The preparation method of the luminous plaque of described coated furantoin is:

Step 1, furantoin and ovalbumin carried out to coupling obtain immunogene:

(1) take the NaNO of 9mg ₂be dissolved in the distilled water of 0.3mL, obtain NaNO ₂solution; Take in the hydrochloric acid that 10mg furantoin is dissolved in 1.6mL 0.2mol/L, 0-4 ℃ is stirred to completely and dissolves, and obtains furantoin solution; Then by gained NaNO ₂solution dropwise adds in gained furantoin solution, lucifuge reaction 0.5 hour;

(2) after reaction finishes, add 25mg sulfuric acid amine until emit without nitrogen, obtain diazotizing furantoin solution;

(3) claim 70mg ovalbumin to be dissolved in the 0.1mol/L of 4mL, in the PBS phosphate buffer solution that pH value is 7.5, obtain ovalbumin solution; Then the diazotizing furantoin solution described in above-mentioned steps is dropwise joined in ovalbumin solution, obtain mixed solution E; Then with the NaOH of 1mol/L by the pH of mixed solution E to being adjusted to 7.5, be then placed at 0-4 ℃, lucifuge is reacted 18 hours;

(4) the reacted reactant liquor of above-mentioned steps lucifuge is transferred in bag filter, with 0.01mol/L, the PBS damping fluid that pH value is 7.4 is dialysed five days at 0-4 ℃, within every 12 hours, changes a PBS damping fluid; Then by centrifugal 15 minutes of 3000 revs/min of dislysates, freeze-drying supernatant, obtained faint yellow solid powder and is envelope antigen furantoin-OVA, is placed on-20 ℃ of preservations, standby;

Step 2, coated:

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

(2), by the above-mentioned coated polystyrene board of lock solution sealing, 250 μ L/ holes, hatch washing after 1 hour for 37 ℃, obtain being coated with the luminous plaque of furantoin.

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

Step 1, application of sample: by furantoin standard solution and the sample solution of each concentration, addition with 50 μ L/ holes joins in the reacting hole of the luminous plaque that is coated with furantoin, then every hole adds 100 μ L to adopt coating buffer with the furantoin specific antibody of the horseradish peroxidase-labeled of 1:7000 dilution proportion, then hatches 0.5 hour for 37 ℃;

Step 2, washing: liquid in the hole of the luminous plaque that coated furantoin of inclining, every hole adds wash solution 250 μ L, washs 3 times, pats dry;

Step 3, add luminous substrate liquid: will the A liquid of luminous substrate and B liquid after mixing potpourri, in the luminous plaque of coated furantoin, every hole adds 100 μ L luminous substrate liquid mixtures,

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

Step 5, result judgement: get standard items concentration logarithm and do horizontal ordinate, standard items detect luminous value logarithm and do ordinate, do typical curve, and the concentration of each sample can be calculated from typical curve.

Beneficial effect is:

1, furantoin chemiluminescence detection kit of the present invention, highly sensitive, sensing range is wide, easy and simple to handle fast, nontoxic pollution-free, the light signal duration is long and instrument is simple, the chemiluminescence detection kit of economic detection furantoin, its preparation method is provided simultaneously, and has detected the antibiotic method of furantoin in sample.The chemical luminescence ELISA detection kit of antibiotics leftover detection have highly sensitive, easy fast, accurately, security is good and long-lived feature, with traditional colorimetric ELISA method comparison, analytical approach is easy fast, sensitivity can improve an order of magnitude.Being expected to Nitrofurantoin residue in feed plays a significant role in detecting.

2, furantoin chemiluminescence detection kit of the present invention adopts metabolin and the carrier coupling of furantoin to prepare envelope antigen, and more economical, step is more simple, is beneficial to suitability for industrialized production; The preparation of horseradish peroxidase-labeled thing, adopts single stage method, and method is simple, is more suitable for suitability for industrialized production, and has obtained good effect through verification experimental verification.

Accompanying drawing explanation

Fig. 1 is the process chart that the present invention prepares furantoin chemiluminescence detection kit;

Fig. 2 is the result standard curve map that embodiments of the invention detect.

Embodiment

A furantoin chemiluminescence detection kit, comprises the luminous plaque of the furantoin specific antibody of concentrated washing lotion, negative control product, positive reference substance, chemical luminescence for liquid, furantoin standard items, horseradish peroxidase-labeled and coated furantoin; Concrete preparation process as shown in fig. 1;

Described concentrated washing lotion is: the phosphate buffer that contains Tween-20 and sodium chloride;

Described negative control product are: the furantoin after 5 parts of aseptic filtrations of depositing separately above detects negative pig urine potpourri;

Described positive reference substance is: the pig urine potpourri of the furantoin test positive after 5 parts of aseptic filtrations of depositing separately above;

Described chemical luminescence for liquid, comprises A liquid and B liquid; A liquid making method is: first configure the Tris-HCl damping fluid of 0.2 mol/L pH7.8, add luminol and tetraphenylboron sodium in this damping fluid, mix and can obtain A liquid; B liquid making method is: first configure the Tris-HCl damping fluid of 0.2 mol/L pH7.8, add by weight 0.1% H in this damping fluid ₂o ₂mix and can obtain B liquid;

Described furantoin standard items are the dilution that concentration is respectively 0ng/ml, 0.1ng/ml, 0.3 ng/ml, 0.9 ng/ml, 2.7 ng/ml, 8.1ng/ml furantoin;

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

Step 1, immunogene preparation:

(1) claim 10mg p-aminobenzoic acid to dissolve in 1.1mL 0.2 mol/L HCl, be placed in 0-4 ℃ and be stirred to dissolving completely, obtain p-aminobenzoic acid solution; Then take the NaNO of 6mg ₂be dissolved in the distilled water of 0.35mL, be placed in 0-4 ℃ and be stirred to dissolving completely, obtain NaNO ₂solution; By NaNO ₂solution dropwise joins in p-aminobenzoic acid solution, and lucifuge reaction 1 hour, obtains mixed solution A;

(2) furantoin that takes 0.3-1.2mg is dissolved in 10mL containing in the borate buffer solution of NaCl, is placed at 0-4 ℃ and is stirred to completely and dissolves, and obtains mixed solution B; Above-mentioned steps gained mixed solution A solution is dropwise joined in mixed solution B, and lucifuge reaction 2 hours, obtains orange colour solution C;

Described containing in the borate buffer solution of NaCl, the concentration of borax is 0.05mol/L, and pH value is 8.5, and contains the NaCl that concentration is 0.15mol/L;

(3) in orange colour solution C, add H ₃bO ₃crystal regulates its pH value to 7.4, then adds 94mg bovine serum albumin(BSA), 80mg carbodiimide and 4mg N-hydroxy-succinamide, is then placed in stirring at room 2 hours, obtains orange colour solution D;

(4) above-mentioned steps gained orange colour solution D is transferred in bag filter, with 0.01mol/L, the PBS damping fluid that pH is 7.4 is dialysed five days at 0-4 ℃, within every 12 hours, changes a dislysate; Solution freeze-drying by after dialysis, obtains faint yellow solid powder and is immunogene furantoin-BSA, is placed on-20 ℃ of preservations, standby.

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

The preparation of step 3, horseradish peroxidase-labeled thing

By sodium periodate legal system, for enzyme labeling furantoin antibody, reaction system is: first use the horseradish peroxidase solution of 1ml concentration 5mg/ml and the NaIO of 0.15ml concentration 0.1 mol/L ₄after solution mixes, be placed in 4 ℃ of lucifuges 0.5 hour, then add the ethylene glycol of 0.25ml to shake up room temperature lucifuge 1 hour, then be placed in 4 ℃ of dialysed overnight, use afterwards 0.2ml concentration 0.2 mol/L, pH value is that the coated solution of 9.6 carbonate is 9.2-9.5 by the hydroformylation horseradish peroxidase solution adjust pH obtaining after dialysis, the anti-furantoin polyclonal antibody of 5mg is dissolved in to 1.0ml concentration 0.01 mol/L, after the coating buffer of pH9.6, add immediately in the horseradish peroxidase solution after above-mentioned hydroformylation in 37 ℃ of reactions 1 hour, then the NaBH that adds 0.1ml concentration 3.5mg/ml ₄solution, mixes in 4 ℃ of placements 2 hours, with gel permeation chromatography, carries out after purifying, selects the colourless of tire>=1:1000 or is with brown supernatant liquid, is the furantoin specific antibody of horseradish peroxidase-labeled,

The preparation method of the luminous plaque of described coated furantoin is:

Step 1, furantoin and ovalbumin carried out to coupling obtain immunogene:

(1) take the NaNO of 9mg ₂be dissolved in the distilled water of 0.3mL, obtain NaNO ₂solution; Take in the hydrochloric acid that 10mg furantoin is dissolved in 1.6mL 0.2mol/L, 0-4 ℃ is stirred to completely and dissolves, and obtains furantoin solution; Then by gained NaNO ₂solution dropwise adds in gained furantoin solution, lucifuge reaction 0.5 hour;

(2) after reaction finishes, add 25mg sulfuric acid amine until emit without nitrogen, obtain diazotizing furantoin solution;

(3) claim 70mg ovalbumin to be dissolved in the 0.1mol/L of 4mL, in the PBS phosphate buffer solution that pH value is 7.5, obtain ovalbumin solution; Then the diazotizing furantoin solution described in above-mentioned steps is dropwise joined in ovalbumin solution, obtain mixed solution E; Then with the NaOH of 1mol/L by the pH of mixed solution E to being adjusted to 7.5, be then placed at 0-4 ℃, lucifuge is reacted 18 hours;

(4) the reacted reactant liquor of above-mentioned steps lucifuge is transferred in bag filter, with 0.01mol/L, the PBS damping fluid that pH value is 7.4 is dialysed five days at 0-4 ℃, within every 12 hours, changes a PBS damping fluid; Then by centrifugal 15 minutes of 3000 revs/min of dislysates, freeze-drying supernatant, obtained faint yellow solid powder and is envelope antigen furantoin-OVA, is placed on-20 ℃ of preservations, standby;

Step 2, coated:

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

(2), by the above-mentioned coated polystyrene board of lock solution sealing, 250 μ L/ holes, hatch washing after 1 hour for 37 ℃, obtain being coated with the luminous plaque of furantoin.

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

Step 1, application of sample: the addition by the furantoin standard solution of each concentration with 50 μ L/ holes joins in the reacting hole of the luminous plaque that is coated with furantoin, then every hole adds 100 μ L to adopt coating buffer with the furantoin specific antibody of the horseradish peroxidase-labeled of 1:7000 dilution proportion, then hatches 0.5 hour for 37 ℃;

Step 2, washing: liquid in the hole of the luminous plaque that coated furantoin of inclining, every hole adds wash solution 250 μ L, washs 3 times, pats dry;

Step 3, add luminous substrate liquid: will the A liquid of luminous substrate and B liquid after mixing potpourri, in the luminous plaque of coated furantoin, every hole adds 100 μ L luminous substrate liquid mixtures,

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

Step 5, result judgement: get standard items concentration logarithm and do horizontal ordinate, standard items detect luminous value logarithm and do ordinate, do typical curve, and the concentration of each sample can be calculated from typical curve.

Embodiment

A furantoin chemiluminescence detection kit, comprises the luminous plaque of the furantoin specific antibody of concentrated washing lotion, negative control product, positive reference substance, chemical luminescence for liquid, furantoin standard items, horseradish peroxidase-labeled and coated furantoin;

Described concentrated washing lotion is: the phosphate buffer that contains Tween-20 and sodium chloride;

Described negative control product are: the furantoin after 5 parts of aseptic filtrations of depositing separately above detects negative pig urine potpourri;

Described positive reference substance is: the pig urine potpourri of the furantoin test positive after 5 parts of aseptic filtrations of depositing separately above;

Described chemical luminescence for liquid, comprises A liquid and B liquid; A liquid making method is: first configure the Tris-HCl damping fluid of 0.2 mol/L pH7.8, add luminol and tetraphenylboron sodium in this damping fluid, mix and can obtain A liquid; B liquid making method is: first configure the Tris-HCl damping fluid of 0.2 mol/L pH7.8, add by weight 0.1% H in this damping fluid ₂o ₂mix and can obtain B liquid;

Described furantoin standard items are the dilution that concentration is respectively 0ng/ml, 0.1ng/ml, 0.3 ng/ml, 0.9 ng/ml, 2.7 ng/ml, 8.1ng/ml furantoin;

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

Step 1, immunogene preparation:

(1) claim 10mg p-aminobenzoic acid to dissolve in 1.1mL 0.2 mol/L HCl, be placed in 0-4 ℃ and be stirred to dissolving completely, obtain p-aminobenzoic acid solution; Then take the NaNO of 6mg ₂be dissolved in the distilled water of 0.35mL, be placed in 0-4 ℃ and be stirred to dissolving completely, obtain NaNO ₂solution; By NaNO ₂solution dropwise joins in p-aminobenzoic acid solution, and lucifuge reaction 1 hour, obtains mixed solution A;

(2) furantoin that takes 0.3-1.2mg is dissolved in 10mL containing in the borate buffer solution of NaCl, is placed at 0-4 ℃ and is stirred to completely and dissolves, and obtains mixed solution B; Above-mentioned steps gained mixed solution A solution is dropwise joined in mixed solution B, and lucifuge reaction 2 hours, obtains orange colour solution C;

Described containing in the borate buffer solution of NaCl, the concentration of borax is 0.05mol/L, and pH value is 8.5, and contains the NaCl that concentration is 0.15mol/L;

(3) in orange colour solution C, add H ₃bO ₃crystal regulates its pH value to 7.4, then adds 94mg bovine serum albumin(BSA), 80mg carbodiimide and 4mg N-hydroxy-succinamide, is then placed in stirring at room 2 hours, obtains orange colour solution D;

(4) above-mentioned steps gained orange colour solution D is transferred in bag filter, with 0.01mol/L, the PBS damping fluid that pH is 7.4 is dialysed five days at 0-4 ℃, within every 12 hours, changes a dislysate; Solution freeze-drying by after dialysis, obtains faint yellow solid powder and is immunogene furantoin-BSA, is placed on-20 ℃ of preservations, standby.

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

The preparation of step 3, horseradish peroxidase-labeled thing

By sodium periodate legal system, for enzyme labeling furantoin antibody, reaction system is: first use the horseradish peroxidase solution of 1ml concentration 5mg/ml and the NaIO of 0.15ml concentration 0.1 mol/L ₄after solution mixes, be placed in 4 ℃ of lucifuges 0.5 hour, then add the ethylene glycol of 0.25ml to shake up room temperature lucifuge 1 hour, then be placed in 4 ℃ of dialysed overnight, use afterwards 0.2ml concentration 0.2 mol/L, pH value is that the coated solution of 9.6 carbonate is 9.2-9.5 by the hydroformylation horseradish peroxidase solution adjust pH obtaining after dialysis, the anti-furantoin polyclonal antibody of 5mg is dissolved in to 1.0ml concentration 0.01 mol/L, after the coating buffer of pH9.6, add immediately in the horseradish peroxidase solution after above-mentioned hydroformylation in 37 ℃ of reactions 1 hour, then the NaBH that adds 0.1ml concentration 3.5mg/ml ₄solution, mixes in 4 ℃ of placements 2 hours, with gel permeation chromatography, carries out after purifying, selects the colourless of tire>=1:1000 or is with brown supernatant liquid, is the furantoin specific antibody of horseradish peroxidase-labeled,

The preparation method of the luminous plaque of described coated furantoin is:

Step 1, furantoin and ovalbumin carried out to coupling obtain immunogene:

(1) take the NaNO of 9mg ₂be dissolved in the distilled water of 0.3mL, obtain NaNO ₂solution; Take in the hydrochloric acid that 10mg furantoin is dissolved in 1.6mL 0.2mol/L, 0-4 ℃ is stirred to completely and dissolves, and obtains furantoin solution; Then by gained NaNO ₂solution dropwise adds in gained furantoin solution, lucifuge reaction 0.5 hour;

(2) after reaction finishes, add 25mg sulfuric acid amine until emit without nitrogen, obtain diazotizing furantoin solution;

(3) claim 70mg ovalbumin to be dissolved in the 0.1mol/L of 4mL, in the PBS phosphate buffer solution that pH value is 7.5, obtain ovalbumin solution; Then the diazotizing furantoin solution described in above-mentioned steps is dropwise joined in ovalbumin solution, obtain mixed solution E; Then with the NaOH of 1mol/L by the pH of mixed solution E to being adjusted to 7.5, be then placed at 0-4 ℃, lucifuge is reacted 18 hours;

(4) the reacted reactant liquor of above-mentioned steps lucifuge is transferred in bag filter, with 0.01mol/L, the PBS damping fluid that pH value is 7.4 is dialysed five days at 0-4 ℃, within every 12 hours, changes a PBS damping fluid; Then by centrifugal 15 minutes of 3000 revs/min of dislysates, freeze-drying supernatant, obtained faint yellow solid powder and is envelope antigen furantoin-OVA, is placed on-20 ℃ of preservations, standby;

Step 2, coated:

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

(2), by the above-mentioned coated polystyrene board of lock solution sealing, 250 μ L/ holes, hatch washing after 1 hour for 37 ℃, obtain being coated with the luminous plaque of furantoin.

A kind of concrete detecting step of furantoin chemiluminescence detection kit:

Step 1, sample pre-treatments

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

Step 2, detecting step

(1) application of sample: to the Cistofuran metabolite series standard concentration solution and the sample solution that add 50uL/ hole in luminous plaque, then add Cistofuran metabolite antibody working fluid 50uL/ hole, add fresh dilution horseradish peroxidase-furantoin antibody (1:7000) 100 μ L/ holes, 37 ℃ 0.5 hour;

(2) washing: the middle liquid that portals that inclines, the cleansing solution to adding 300uL/ hole in luminous plaque, pats dry after standing 5min, in triplicate;

(3) add luminescent solution: every hole adds luminescent solution lOOuL;

(4) result judgement: get standard items concentration logarithm and do horizontal ordinate, standard items detect luminous value logarithm and do ordinate, do typical curve, result is as Fig. 2, figure Plays curve y=-1.0354x+4.7213, R ²=0.9983; The concentration of each sample can be calculated from typical curve, and result is as table 1.

Table 1:

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

Claims (2)

1. a furantoin chemiluminescence detection kit, is characterized in that: the luminous plaque that comprises the furantoin specific antibody of concentrated washing lotion, negative control product, positive reference substance, chemical luminescence for liquid, furantoin standard items, horseradish peroxidase-labeled and coated furantoin;

Described concentrated washing lotion is: the phosphate buffer that contains Tween-20 and sodium chloride;

Described negative control product are: the furantoin after 5 parts of aseptic filtrations of depositing separately above detects negative pig urine potpourri;

Described positive reference substance is: the pig urine potpourri of the furantoin test positive after 5 parts of aseptic filtrations of depositing separately above;

Described chemical luminescence for liquid, comprises A liquid and B liquid; A liquid making method is: first configure the Tris-HCl damping fluid of 0.2 mol/L pH7.8, add luminol and tetraphenylboron sodium in this damping fluid, mix and can obtain A liquid; B liquid making method is: first configure the Tris-HCl damping fluid of 0.2 mol/L pH7.8, add by weight 0.1% H in this damping fluid ₂o ₂mix and can obtain B liquid;

Described furantoin standard items are the dilution that concentration is respectively 0ng/ml, 0.1ng/ml, 0.3 ng/ml, 0.9 ng/ml, 2.7 ng/ml, 8.1ng/ml furantoin;

The furantoin specific antibody of described horseradish peroxidase-labeled is, horseradish peroxidase-labeled take furantoin-BSA as the anti-furantoin polyclonal antibody of immunogen immune new zealand white rabbit gained, furantoin specific antibody by sodium periodate legal system for horseradish peroxidase-labeled, reaction system is: first use the horseradish peroxidase solution of 1ml concentration 5mg/ml and the NaIO of 0.15ml concentration 0.1 mol/L ₄after solution mixes, be placed in 4 ℃ of lucifuges 0.5 hour, then add the ethylene glycol of 0.25ml to shake up room temperature lucifuge 1 hour, then be placed in 4 ℃ of dialysed overnight, use afterwards 0.2ml concentration 0.2 mol/L, pH value is that the coated solution of 9.6 carbonate is 9.2-9.5 by the hydroformylation horseradish peroxidase solution adjust pH obtaining after dialysis, the anti-furantoin polyclonal antibody of 5mg is dissolved in to 1.0ml concentration 0.01 mol/L, after the coating buffer of pH9.6, add immediately in the horseradish peroxidase solution after above-mentioned hydroformylation in 37 ℃ of reactions 1 hour, then the NaBH that adds 0.1ml concentration 3.5mg/ml ₄solution, mixes in 4 ℃ of placements 2 hours, with gel permeation chromatography, carries out after purifying, selects the colourless of tire>=1:1000 or is with brown supernatant liquid, is the furantoin specific antibody of horseradish peroxidase-labeled,

The immunogenic preparation method of described furantoin-BSA is:

Step 1, immunogene preparation:

Step 1, title 10mg p-aminobenzoic acid dissolve in 1.1mL 0.2 mol/L HCl, are placed in 0-4 ℃ and are stirred to dissolving completely, obtain p-aminobenzoic acid solution; Then take the NaNO of 6mg ₂be dissolved in the distilled water of 0.35mL, be placed in 0-4 ℃ and be stirred to dissolving completely, obtain NaNO ₂solution; By NaNO ₂solution dropwise joins in p-aminobenzoic acid solution, and lucifuge reaction 1 hour, obtains mixed solution A;

Step 2, the furantoin that takes 0.3-1.2mg are dissolved in 10mL containing in the borate buffer solution of NaCl, are placed at 0-4 ℃ and are stirred to completely and dissolve, and obtain mixed solution B; Above-mentioned steps gained mixed solution A solution is dropwise joined in mixed solution B, and lucifuge reaction 2 hours, obtains orange colour solution C;

Described containing in the borate buffer solution of NaCl, the concentration of borax is 0.05mol/L, and pH value is 8.5, and contains the NaCl that concentration is 0.15mol/L;

Step 3, in orange colour solution C, add H ₃bO ₃crystal regulates its pH value to 7.4, then adds 94mg bovine serum albumin(BSA), 80mg carbodiimide and 4mg N-hydroxy-succinamide, is then placed in stirring at room 2 hours, obtains orange colour solution D;

Step 4, above-mentioned steps gained orange colour solution D is transferred in bag filter, with 0.01mol/L, the PBS damping fluid that pH is 7.4 is dialysed five days at 0-4 ℃, within every 12 hours, changes a PBS damping fluid; Solution freeze-drying by after dialysis, obtains faint yellow solid powder and is immunogene furantoin-BSA, is placed on-20 ℃ of preservations, standby;

The preparation method of the luminous plaque of described coated furantoin is:

Step 1, furantoin and ovalbumin carried out to coupling obtain immunogene:

(1) take the NaNO of 9mg ₂be dissolved in the distilled water of 0.3mL, obtain NaNO ₂solution; Take in the hydrochloric acid that 10mg furantoin is dissolved in 1.6mL 0.2mol/L, 0-4 ℃ is stirred to completely and dissolves, and obtains furantoin solution; Then by gained NaNO ₂solution dropwise adds in gained furantoin solution, lucifuge reaction 0.5 hour;

(2) after reaction finishes, add 25mg sulfuric acid amine until emit without nitrogen, obtain diazotizing furantoin solution;

(3) claim 70mg ovalbumin to be dissolved in the 0.1mol/L of 4mL, in the PBS phosphate buffer solution that pH value is 7.5, obtain ovalbumin solution; Then the diazotizing furantoin solution described in above-mentioned steps is dropwise joined in ovalbumin solution, obtain mixed solution E; Then with the NaOH of 1mol/L by the pH of mixed solution E to being adjusted to 7.5, be then placed at 0-4 ℃, lucifuge is reacted 18 hours;

(4) the reacted reactant liquor of above-mentioned steps lucifuge is transferred in bag filter, with 0.01mol/L, the PBS damping fluid that pH value is 7.4 is dialysed five days at 0-4 ℃, within every 12 hours, changes a PBS damping fluid; Then by centrifugal 15 minutes of 3000 revs/min of dislysates, freeze-drying supernatant, obtained faint yellow solid powder and is envelope antigen furantoin-OVA, is placed on-20 ℃ of preservations, standby;

Step 2, coated:

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

(2), by the above-mentioned coated polystyrene board of lock solution sealing, 250 μ L/ holes, hatch washing after 1 hour for 37 ℃, obtain being coated with the luminous plaque of furantoin.

2. utilize furantoin chemiluminescence detection kit described in claim 1 to detect the method for Nitrofurantoin residue, it is characterized in that: its concrete detecting step is:

Step 1, application of sample: by furantoin standard solution and the sample solution of each concentration, addition with 50 μ L/ holes joins in the reacting hole of the luminous plaque that is coated with furantoin, then every hole adds 100 μ L to adopt coating buffer with the furantoin specific antibody of the horseradish peroxidase-labeled of 1:7000 dilution proportion, then hatches 0.5 hour for 37 ℃;

Step 2, washing: liquid in the hole of the luminous plaque that coated furantoin of inclining, every hole adds wash solution 250 μ L, washs 3 times, pats dry;

Step 3, add luminous substrate liquid: will the A liquid of luminous substrate and B liquid after mixing potpourri, in the luminous plaque of coated furantoin, every hole adds 100 μ L luminous substrate liquid mixtures,

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

Step 5, result judgement: get standard items concentration logarithm and do horizontal ordinate, standard items detect luminous value logarithm and do ordinate, do typical curve, and the concentration of each sample can be calculated from typical curve.

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