CN102081094B - Method for detecting patulin and special enzyme linked immunosorbent assay kit thereof - Google Patents
Method for detecting patulin and special enzyme linked immunosorbent assay kit thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting patulin and a special enzyme linked immunosorbent assay kit thereof. The enzyme linked immunosorbent assay kit comprises patulin monoclonal antibodies. The patulin monoclonal antibodies are secreted by patulin monoclonal antibody hybridoma cell strains PAT with collection number CGMCC No.3392. The enzyme linked immunosorbent assay kit qualitatively or quantitatively detects the patulin content of fruits or juice by mainly adopting an indirect competitive ELISA method. The kit and the detection method have low pretreatment requirement for a sample and simple sample pretreatment process, and can quickly detect a large batch of samples at the same time; and by adopting the high-specificity patulin monoclonal antibodies, the detection method is convenient and feasible and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like. The kit plays a great role in the detection of the patulin.
Description
Technical field
The present invention relates to a kind of method and special ELISA reagent kit thereof that detects penicidin.
Background technology
Penicidin (Patulin, PAT) be mainly the fruit higher, nutritious by moisture content and vegetables are higher at storage temperature, under the storage requirement of improper ventilation, a kind of secondary metabolite produced by part fungi (expansion mould, Penicillium patulum, excellent type mould etc.).Molecular formula is C
7h
6o
4, 100 ℃ of fusing points, molecular weight is 154, is colourless needle crystal, can be water-soluble, ethanol, acetone, ethyl acetate and chloroform.At first Glister finds also separation and purification in Oxford University in nineteen forty-one.In early days, penicidin is considered to a kind of Broad spectrum antibiotics, can be used for the treatment of people's flu, but experimentation on animals is subsequently found, it has stronger acute and subacute toxicity, immunotoxicity, teratogenesis, mutagenesis to laboratory animal, from then on the research emphasis of penicidin is transferred in its toxicity and the pollution to food and feed.Penicidin mainly pollutes apple, hawthorn and juice product thereof, and has become an important indicator of judgement Sucus Mali pumilae quality.Therefore, in national regulation apple, the haw products such as Chinese and EU, the highest allowance of penicidin is 50 μ g/kg.
The detection method of penicidin mainly comprises tlc (TLC), high performance liquid chromatography (HPLC) and vapor-phase chromatography (GC) etc., but aforesaid method all is difficult to realize the rapid detection to batch sample.
Summary of the invention
An object of the present invention is to provide a kind of penicidin hapten compound.
Penicidin hapten compound provided by the present invention, its structural formula is suc as formula shown in I:
The penicidin haptens is that penicidin and the reaction of pentanedioic acid glycosides obtain, and this haptens contains pendant carboxylic group, can form coating antigen and immunogen with carrier protein couplet.
Shown in above-mentioned formula I, the conjugate of compound and carrier proteins also belongs to protection scope of the present invention.Wherein, described carrier proteins is bovine serum albumin (BSA), human serum albumin (HSA), mouse serum protein (MSA), thyroprotein (BCG), rabbit anteserum albumen (RSA), hemocyanin (KLH) or oralbumin (OVA).
The conjugate of described penicidin haptens and carrier proteins refers to the product that penicidin haptens and carrier proteins obtain by active ester method.
The concrete conjugate that there is following structural formula of the conjugate of above-mentioned arbitrary described penicidin haptens and carrier proteins:
Wherein, n is coupling ratio, is the arbitrary natural integer in 1-30.
Described for coated conjugate, described carrier proteins can be ovalbumin, bovine serum albumin, human serum albumin, mouse serum protein, thyroprotein (BCG), rabbit anteserum albumen or hemocyanin.
The application of conjugate in preparing the penicidin monoclonal antibody of compound and carrier proteins shown in compound shown in above-mentioned formula I and/or above-mentioned formula I also belongs to protection scope of the present invention.
Another object of the present invention is to provide a kind of penicidin monoclonal antibody.
Penicidin monoclonal antibody provided by the present invention is by deposit number, to be CGMCC No.3392 penicidin monoclonal antibody hybridoma cell strain PAT secretion produces.
The penicidin monoclonal antibody hybridoma cell strain PAT that deposit number is CGMCC No.3392 also belongs to protection scope of the present invention.
The application in the enzyme linked immunological kit of preparation detection penicidin of the conjugate of compound and carrier proteins shown in compound shown in above-mentioned formula I, above-mentioned formula I and/or above-mentioned penicidin monoclonal antibody also belongs to protection scope of the present invention.
Another object of the present invention is to provide a kind of enzyme linked immunological kit that detects penicidin.
The enzyme linked immunological kit of detection penicidin provided by the present invention is following 1), 2), 3) or 4) in arbitrary described enzyme linked immunological kit:
1) enzyme linked immunological kit comprises compound shown in above-mentioned arbitrary described formula I and the conjugate of carrier proteins, above-mentioned penicidin monoclonal antibody and enzyme labelling anti-antibody; Wherein, described conjugate is as coating antigen;
2) enzyme linked immunological kit comprises the enzyme labelling thing of compound shown in above-mentioned formula I, above-mentioned penicidin monoclonal antibody and anti-antibody; Wherein, described anti-antibody is as coating antigen;
3) enzyme linked immunological kit comprises the enzyme labelling thing of compound shown in above-mentioned formula I, above-mentioned penicidin monoclonal antibody; Wherein, described penicidin monoclonal antibody is as coating antigen;
4) enzyme linked immunological kit comprises compound shown in above-mentioned arbitrary described formula I and the conjugate of carrier proteins, the enzyme labelling thing of above-mentioned penicidin monoclonal antibody; Wherein, described conjugate is as coating antigen.
The detection principle of above-mentioned 4 kinds of test kits is as follows:
When the conjugate of pre-coated penicidin haptens and carrier proteins on the enzyme plate capillary strip, after adding sample solution or standard solution, add again the penicidin monoclonal antibody solution, coated penicidin haptens and the conjugate competition penicidin monoclonal antibody of carrier proteins on residual penicidin or penicidin standard substance and enzyme plate in sample, add the anti-amplification that carries out of enzyme labelling two, with nitrite ion, develop the color, the sample light absorption value becomes negative correlation with the content of penicidin in sample, relatively can draw the content of penicidin in sample with typical curve.Simultaneously also can be according to the depth of color on enzyme plate, with the comparison of the series concentration penicidin standard solution color concentration range of penicidin content in judgement sample roughly.
When on the enzyme plate capillary strip pre-coated two when anti-, after adding the penicidin monoclonal antibody to hatch, add sample solution or standard solution, add again enzyme labelling penicidin haptens solution, penicidin in sample or penicidin standard substance and enzyme labelling penicidin haptens competition penicidin specific antibody, with the nitrite ion colour developing, the sample absorbance becomes negative correlation with the content of penicidin in sample, with typical curve, relatively can draw the content of penicidin in sample.Simultaneously also can be according to the shade on enzyme plate, with the comparison of the series concentration penicidin standard solution color concentration range of penicidin content in judgement sample roughly.
When pre-coated penicidin monoclonal antibody on the enzyme plate capillary strip, after adding sample solution or standard solution, add again enzyme labelling penicidin haptens solution, in sample, residual penicidin or penicidin standard substance and enzyme-labelled antigen competition are coated on the penicidin monoclonal antibody on enzyme plate, with nitrite ion, develop the color, the sample light absorption value becomes negative correlation with the content of penicidin, with typical curve, relatively can draw the content of penicidin in sample.Simultaneously also can be according to the shade on enzyme plate, with the comparison of the penicidin standard solution color of the series concentration concentration range of penicidin content in judgement sample roughly.
When the conjugate of pre-coated penicidin haptens and carrier proteins on the enzyme plate capillary strip, after adding sample solution or standard solution, add again enzyme labelling penicidin monoclonal antibody solution, coated penicidin antigenic competition penicidin monoclonal antibody on penicidin in sample or penicidin standard substance and enzyme plate, with nitrite ion, develop the color, the sample light absorption value becomes negative correlation with the content of penicidin in sample, with typical curve, relatively can draw the content of penicidin in sample.Simultaneously also can be according to the shade on enzyme plate, with the comparison of the series concentration penicidin standard solution color concentration range of penicidin content in judgement sample roughly.
Also can comprise penicidin standard solution, nitrite ion, washings and/or sample diluting liquid in above-mentioned arbitrary described test kit.
In above-mentioned arbitrary described test kit, described anti-antibody is sheep anti mouse anti-antibody or goat-anti rabbit anti-antibody.
In above-mentioned arbitrary described test kit, the concentration of described penicidin standard solution is respectively 0 μ g/L, 1.0 μ g/L, 5.0 μ g/L, 20.0 μ g/L, 50.0 μ g/L and 100.0 μ g/L.
In above-mentioned arbitrary described test kit, described substrate nitrite ion is comprised of A liquid and B liquid, the aqueous solution that A liquid is the final concentration urea peroxide that is 1.5%-2.5%, and described final concentration is the concentration of urea peroxide in described A liquid; The aqueous solution that B liquid is the final concentration tetramethyl benzidine that is 0.5%-1.5%, described final concentration is the concentration of tetramethyl benzidine in described B liquid;
In above-mentioned arbitrary described test kit, the polysorbas20 that described washings is 0.8%～1.2% by final concentration, final concentration are forming of 0.5% sodiumazide, and solvent is the 0.01M phosphate buffered saline buffer; Described final concentration is the concentration of each material in washings; The pH value of described washings is 7.4.
In above-mentioned arbitrary described test kit, the phosphate buffered saline buffer that described sample diluting liquid is 20 * (0.03mol/L-0.05mol/L).
Marker enzyme in described enzyme labelling is horseradish peroxidase or alkaline phosphatase, wherein preferred horseradish peroxidase; Anti-or the goat-anti rabbit two of the sheep anti mouse two of enzyme labelling is anti-adopts glutaraldehyde method or sodium periodate method that marker enzyme and two is resisted and carries out coupling and obtain, horseradish peroxidase can adopt several different methods of the prior art by it and two anti-carry out coupling, as glutaraldehyde method, sodium periodate method etc., the present invention improves the sodium periodate method through long-term labor and creation, make it save time, reduce horseradish peroxidase (HRP) and two anti-concentration rates, saved starting material.
Another object of the present invention is to provide a kind of method that detects penicidin in sample.
The method of penicidin in detection sample provided by the present invention is following 1) or 2) described:
1) described testing sample is fruit juice; With above-mentioned arbitrary described test kit, described fruit juice is detected;
2) described testing sample is fruit; Described fruit is carried out to homogenized, obtain the homogenate tissue, more described homogenate is organized centrifugal, get supernatant liquor, after supernatant liquor is diluted, obtain the supernatant liquor diluent, with above-mentioned arbitrary described test kit, described supernatant liquor diluent is detected.
Kit test method provided by the invention is:
When coating antigen is the penicidin coupled antigen, in the enzyme plate micropore, add standard solution or sample solution to add again antibody, after incubation, washing pats dry, then adds ELIAS secondary antibody, and after incubation, washing pats dry, and colour developing, termination, measure absorbance by microplate reader;
When coating antigen is the penicidin coupled antigen, in the enzyme plate micropore, add standard solution or sample solution to add again enzymic-labelled antibody, after incubation, washing pats dry, and colour developing, termination, measure absorbance by microplate reader;
When coating antigen is the penicidin specific antibody, in the enzyme plate micropore, add standard solution or sample solution to add again enzyme labelling penicidin haptens, after incubation, washing pats dry, and colour developing, termination, measure absorbance by microplate reader;
When coating antigen is two when anti-, add penicidin antibody in the enzyme plate micropore, after incubation, washing pats dry, add enzyme mark penicidin haptens after adding again standard solution or sample solution, after incubation, washing pats dry, and colour developing, termination, measure absorbance by microplate reader.
Analysis of test results process provided by the invention is:
Absorbance (B with the absorbancy mean value (B) of the standard solution of each obtained concentration divided by first standardized solution (0 standard)
0) be multiplied by again 100%, i.e. percentage absorbance.Calculation formula is:
Percentage absorbance (%)=(B/B
0) * 100%
The semilog value of concentration (μ g/L) of penicidin standard solution of take is X-axis, and the percentage absorbance is Y-axis, the drawing standard graphic representation.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample can be read from typical curve the content of penicidin in sample.
In the present invention, the analysis of detected result also can adopt regression equation method, calculates sample solution concentration.
In the present invention, the analysis of detected result can also utilize computer professional software, the be more convenient for real-time analysis of a large amount of samples of this method, and whole testing process only needs the short period, in 1.5h, can complete.
Last purpose of the present invention is to provide a kind of a kind of method and method for preparing the conjugate of compound shown in above-mentioned formula I and carrier proteins for preparing compound shown in above-mentioned formula I.
The method of compound shown in the above-mentioned formula I of preparation provided by the present invention, comprise the steps: that the mol ratio with 1: 18: 5 is dissolved in anhydrous tetrahydro furan by penicidin, Pyroglutaric acid and DMAP, 37 ℃ of oscillatory reaction 3h, termination reaction, obtain described compound.
The method of the conjugate of compound and carrier proteins shown in the above-mentioned formula I of preparation provided by the present invention, comprise the steps: compound shown in above-mentioned formula I, N-hydroxy-succinamide and dicyclohexylcarbodiimide are dissolved in to anhydrous tetrahydro furan with the mol ratio of 1: 3: 3, oscillatory reaction 5h, remove precipitation and tetrahydrofuran (THF), residue is dissolved in DMSO, splashes into wherein and is dissolved in 0.2mol/L NaHCO
3the solution of 6.0% (quality percentage composition) carrier proteins, under room temperature lucifuge condition, oscillatory reaction is spent the night, dialysis 3d obtains the conjugate of described compound and carrier proteins.
In the present invention, penicidin is carried out to structure of modification, add spacerarm to introduce carboxylic group, simultaneously outstanding penicidin characteristic group.Synthesized immunogen, coating antigen by active ester method, easy, the easy row of method, immune effect is good, and antibodies specific is high.Enzyme linked immunological kit of the present invention mainly adopts the content of penicidin in indirect competitive ELISA method qualitative or detection by quantitative fruit or fruit juice.Test kit of the present invention and detection method are low to the pre-treatment requirement of sample, and sample pretreatment process is simple, simultaneously the rapid detection batch samples; Adopt the penicidin monoclonal antibody of high specific, the method for inspection is convenient and easy, has that specificity is high, highly sensitive, tolerance range is high, the accuracy high.And enzyme linked immunological kit of the present invention, the screening that simple in structure, easy to use, low price, carrying convenience, detection method be efficient, accurate, easy, be suitable for on-the-spot batch samples.Test kit of the present invention will play a significant role in the detection of penicidin.
The accompanying drawing explanation
Fig. 1 is the penicidin typical curve.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
One, the haptenic structure of penicidin and preparation thereof
Penicidin is purchased from Sigma Aldrich company, and catalog number is 32759;
Penicidin, Pyroglutaric acid and DMAP (DMAP) (mol ratio 1: 18: 5) are dissolved in anhydrous tetrahydro furan, 37 ℃ of oscillatory reaction 3h, the adding distil water termination reaction, obtain formula I compound, is penicidin haptens (PAT-HG).
Two, penicidin antigen and preparation thereof
1, the penicidin immunizing antigen is synthetic
Penicidin is a kind of small molecules chemical substance, and non-immunogenicity will prepare anti-penicidin antibody, it must be coupled on relative molecular mass larger protein carrier (as BSA, OVA etc.).Because penicidin is simple in structure, pick out the characteristic group in more outstanding molecular structure after a spacerarm by the Pyroglutaric acid method, and introduced a carboxyl for being connected with the amino of carrier proteins, make the penicidin antibody of preparation very high to the specificity of penicidin.
PAT-HG, N-hydroxy-succinamide (NHS), dicyclohexylcarbodiimide (DCC) (mol ratio is 1: 3: 3) are dissolved in anhydrous tetrahydro furan, oscillatory reaction 5h under room temperature condition, the centrifugal precipitation of removing, vacuum is drained tetrahydrofuran (THF), residue is dissolved in DMSO, slowly splashes into and is dissolved in 0.2mol/L NaHCO
36.0% (quality percentage composition) ovoserum albumin (OVA) solution, under room temperature lucifuge condition, slowly oscillatory reaction is spent the night, dialysis 3d in 4 ℃ of 0.1mol/LpH 7.2 phosphate buffered saline buffers (PBS), vacuum freezedrying, obtain penicidin immunizing antigen (PAT-HG-OVA).
The structural formula of penicidin immunizing antigen is suc as formula shown in II.
Wherein, in formula II, the coupling ratio of haptens and carrier proteins is 1: 15.
2, the penicidin coating antigen is synthetic
Method is identical with immunogenic preparation method in above-mentioned experiment 1, and different is that carrier proteins used is hemocyanin (KLH), and the penicidin haptens obtained and the structural formula of carrier protein couplet thing are as shown in formula III.
(formula III)
Wherein, in formula III, the coupling ratio of haptens and carrier proteins is 1: 13.
Three, the preparation of penicidin monoclonal antibody
1, the preparation of monoclonal antibody
Using and test PAT-HG-OVA in two as immunogen.
A. animal immune
Immunogen PAT-HG-OVA is injected in the Balb/c Mice Body, and immunizing dose is 75 μ g/, makes it produce antiserum(antisera).
B. cytogamy and cloning
Get immune Balb/c mouse boosting cell, merge in 5: 1 (quantitative proportion) ratios and SP2/0 myeloma cell, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay to carry out cloning to positive hole, until screening obtains the hybridoma cell strain of stably excreting penicidin monoclonal antibody, this cell strain name is called PAT, Classification And Nomenclature is the strain of penicidin monoclonal antibody hybridoma cell, this cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 3rd, 2009 and (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.3392.
C. cell cryopreservation and recovery
Penicidin monoclonal antibody hybridoma cell strain PAT is made to 1 * 10 with frozen storing liquid
6the cell suspension of individual/ml is preserved for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal frozen storing liquid, move in culturing bottle and cultivate.
D. the preparation and purification of monoclonal antibody
Increment culture method: penicidin monoclonal antibody hybridoma cell strain PAT CGMCC No.3392 is placed in to cell culture medium, under 37 ℃ of conditions, cultivated, by sad-saturated ammonium sulphate method, the nutrient solution obtained is carried out to purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in the RPMI-1640 substratum, making the final concentration of calf serum in cell culture medium is 20% (quality percentage composition), and making the final concentration of sodium bicarbonate in cell culture medium is 0.2% (quality percentage composition); The pH of described cell culture medium is 7.4.
Said monoclonal antibody can also be taked the following methods preparation: the Balb/c mouse peritoneal is only injected to sterilizing paraffin oil 0.4mL/, 7 days pneumoretroperitoneum injection penicidin monoclonal antibody hybridoma cell strain PAT 5 * 10
5individual/as only, after 7 days, to gather ascites.Carry out purifying by sad-saturated ammonium sulphate method, the ascites after purifying is put into-20 ℃ of environment and is preserved.
Four, penicidin polyclonal antibody and preparation thereof
Adopt new zealand white rabbit as immune animal, the conjugate shown in formula II of take is immunogen, immunizing dose is 1.5mg/kg, when head exempts from, the Freund's complete adjuvant of immunogen and equivalent is mixed and made into to emulsifying agent, the subcutaneous multi-point injection of nape section, interval is got the same dose immunogen in 3～4 weeks and is added equivalent Freund's incomplete adjuvant mixing and emulsifying, and booster immunization is once, immunity is 5 times altogether, does not add for the last time adjuvant.Last immunity blood sampling afterwards in 10 days, measure serum antibody titer, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
Five, purposes and the effect of penicidin haptens, penicidin antigen and penicidin antibody
Utilize chessboard method to carry out the mensuration that monoclonal antibody is tired, result shows, tiring of penicidin monoclonal antibody is 2.6 * 10
6, lowest detectable limit (LOD value) is that 1.0 μ g/L are and half amount of suppression (IC
50) be 4.8 μ g/L).
Enzyme linked immunological kit and the preparation and application thereof of embodiment 2, detection penicidin
One, enzyme linked immunological kit is comprised of following substances:
1, the penicidin coating antigen shown in the formula III described in embodiment 1;
2, enzyme plate;
3, penicidin monoclonal antibody: the penicidin monoclonal antibody hybridoma cell strain PAT that is CGMCC No.3392 by deposit number secretion produces;
4, penicidin standard substance: standard solution concentration is respectively 0 μ g/L, 1.0 μ g/L, 5.0 μ g/L, 20.0 μ g/L, 50.0 μ g/L and 100.0 μ g/L; The penicidin standard substance are purchased from Sigma Aldrich company; Catalog number is 32759; Be diluted to above-mentioned each concentration with sample diluting liquid;
5, anti-with the sheep anti mouse two of horseradish peroxidase-labeled;
6, substrate nitrite ion: formed the aqueous solution that A liquid is 2% urea peroxide, the aqueous solution that B liquid is 1% tetramethyl benzidine by A liquid and B liquid;
7, stop buffer: the aqueous solution of the aqueous solution of 2mol/L sulfuric acid or 2mol/L hydrochloric acid;
8, washings: the polysorbas20 that is 0.8%～1.2% by final concentration, final concentration are forming of 0.5% sodiumazide, and solvent is the 0.01M phosphate buffered saline buffer, and described final concentration is the concentration of each material in washings; The pH value of washings is 7.4.
9, the phosphate buffered saline buffer of sample diluting liquid: 20 * 0.04mol/L.
Two, the preparation of test kit
(1) the penicidin coating antigen is coated with to enzyme plate
With coated damping fluid, the penicidin coating antigen shown in formula III is diluted to 10.0 μ g/mL, every hole adds 100 μ L, 37 ℃ of incubation 2h, the coating buffer that inclines, wash 3 times with after 20 times of washings dilutions, each 30s, pat dry, then in every hole, add 200 μ L confining liquids, 37 ℃ of incubation 2h, the liquid in hole that inclines, preserve with the vacuum-sealing of aluminium film after dry.
The sodium carbonate buffer of coated damping fluid: pH 9.6,0.01～0.1mol/L;
Confining liquid: phosphate buffered saline buffer: contain 0.5% horse serum, 1 ‰ sodiumazide, 3% caseic phosphate buffered saline buffer.
(2) the anti-preparation of sheep anti mouse two of horseradish peroxidase-labeled
1, two anti-preparation process
Sheep anti mouse two is anti-: using goat as immune animal, the mouse source antibody of take carries out immunity as immunogen to the pathogen-free domestic goat, obtains sheep anti mouse two anti-;
Goat-anti rabbit two is anti-: using goat as immune animal, the rabbit source antibody of take carries out immunity as immunogen to the pathogen-free domestic goat, obtains goat-anti rabbit two anti-.
2, enzyme labelling sheep anti mouse two is anti-
Sheep anti mouse two is resisted with horseradish peroxidase (HRP) and carries out coupling, and the method for employing is the sodium periodate method of improvement, and method is as follows:
A, 8mg horseradish peroxidase are dissolved in 2mL distilled water.
B, add the 100mmol/L NaIO of existing preparation
4solution 0.4mL, stirring at room reaction 20min.
C, use 1mmol/L acetate buffer, in 4 ℃ of dialysed overnight, are removed unnecessary NaIO
4, make the enzyme reduction of self coupling simultaneously.
D, phosphate buffered saline buffer (pH 8.6, the 5mol/L) 2.0mL that adds phosphate buffered saline buffer (pH8.6,0.5mol/L) 40 μ L and contain IgG 16mg, stirring at room reaction 4 hours.
E, add the NaBH of existing preparation
4the aqueous solution (1mol/L) 0.1mL, 4 ℃ of reactions 4 hours, with reduction Schiff alkali.
F, purification storage.
Three, the application of test kit
(1) detection method
1, sample pre-treatments
A. fruit (as apple, hawthorn)
The fruit such as apple, hawthorn, use juice extractor homogenate.Accurately take the uniform homogenate sample of 10g in centrifuge tube, centrifugal, get supernatant 1mL, with 10 times of distilled water dilutings (1+9), for measuring.
B. fruit juice, fruit wine
The fruit product such as fruit juice, fruit wine is directly detected.
2, with test kit, detect
2.1 the making of typical curve
Add penicidin standard solution 50 μ L in the enzyme plate micropore that is coated with conjugate described in experiment one, add again penicidin monoclonal antibody working fluid 50 μ L, with cover plate film shrouding, react 20min in 37 ℃ of thermostat containers, pour out liquid in hole, every hole adds 250 μ L washingss, pours out liquid in hole after 30s, repeat operation and wash altogether plate 4 times, pat dry with thieving paper.The anti-working fluid 100 μ L of sheep anti mouse two that add horseradish peroxidase-labeled, react 20min in 37 ℃ of thermostat containers, pour out liquid in hole, the repeated washing step, every hole adds substrate nitrite ion A liquid urea peroxide, substrate nitrite ion B liquid tetramethyl benzidine (TMB), vibration mixes gently, 37 ℃ of thermostat container lucifuge colour developing 10min, every hole adds the aqueous solution 50 μ L of 2mol/L stop buffer sulfuric acid, vibration mixes gently, uses microplate reader, measures every hole absorbance.
Absorbance (B with the absorbancy mean value (B) of the standard solution of each concentration divided by first standard solution (0 standard)
0) be multiplied by again 100%, obtain the percentage absorbance.The semilog value of penicidin standard substance concentration (μ g/L) of take is X-axis, and the percentage absorbance is Y-axis, the drawing standard graphic representation.The typical curve obtained as shown in Figure 1.
Percentage absorbance (%)=(B/B
0) * 100%
2.2 the mensuration of penicidin concentration in sample
Add test sample solution 50 μ L in the enzyme plate micropore that is coated with conjugate described in experiment one, add again penicidin monoclonal antibody working fluid 50 μ L, with cover plate film shrouding, react 20min in 37 ℃ of thermostat containers, pour out liquid in hole, every hole adds 250 μ L washingss, pours out liquid in hole after 30s, repeat operation and wash altogether plate 4 times, pat dry with thieving paper.The anti-working fluid 100 μ L of sheep anti mouse two that add horseradish peroxidase-labeled, react 20min in 37 ℃ of thermostat containers, pour out liquid in hole, the repeated washing step, every hole adds substrate nitrite ion A liquid urea peroxide, substrate nitrite ion B liquid tetramethyl benzidine (TMB), vibration mixes gently, 37 ℃ of thermostat container lucifuge colour developing 10min, every hole adds the aqueous solution 50 μ L of 2mol/L stop buffer sulfuric acid, vibration mixes gently, uses microplate reader, measures every hole absorbance.
Absorbance (B with the absorbancy mean value (B) of each test sample solution divided by first standard solution (0 standard)
0) be multiplied by again 100%, obtain the percentage absorbance.The percentage absorbance of corresponding each test sample solution, can read from typical curve the residual quantity of the penicidin in test sample solution.
(2) detection of enzyme linked immunological kit effect
1, standard substance precision test
Prepare test kit according to method described in experiment one, respectively extract 10 test kits (i.e. 10 enzyme plates) and tested from the test kit of 3 different batches, each enzyme plate is extracted 20 micropores out, measures the absorbance of 20 μ g/L standardized solution, calculate the variation coefficient, the results are shown in Table 1.
The method of calculation of the variation coefficient: the variation coefficient (the CV)=standard deviation of measurement result and the per-cent of its mean value.
Table 1 standard substance Precision test result (CV%)
By above-mentioned test-results, can draw, every batch of test kit measured 10 standard substance variation coefficient between 6.7% ~ 13.9%, meets precision and is less than or equal to 20% regulation.
2, sample precision test
To not containing adding penicidin in the sample (apple and Sucus Mali pumilae) of penicidin, make the final concentration of penicidin in sample be respectively 50 μ g/kg, 20 μ g/L; Sample after adding is carried out to pre-treatment according to method described in (two) respectively, obtain test sample solution.
Respectively extract 3 test kits and detected from the test kit of three different batches, each experiment repeats 5 times, calculates respectively the variation coefficient.Result respectively in Table 2, table 3.
The method of calculation of plate within variance coefficient: the variation coefficient of certain sample (being generally medium level) replication number of times acquired results in plate within variance coefficient=same same plate of once measuring.
The method of calculation of variation within batch coefficient: the variation coefficient of each parallel samples in variation within batch coefficient=same once mensuration.
The method of calculation of interassay coefficient of variation: interassay coefficient of variation=same sample, in the variation coefficient of different batches measurement result, is got its mean value.
The Precision test result of table 2 apple (adding concentration 50 μ g/kg)
Table 3 samples of juice precision test (adding concentration 20 μ g/L)
Result shows, this test kit adds samples to above 5 kinds, and the plate within variance coefficient is less than 10%, and the variation within batch coefficient is less than 15%, and interassay coefficient of variation is less than 20%, meets the regulation of test kit precision.
3, sample recovery test
To not containing the penicidin standard substance that add respectively different concns in the sample (apple and Sucus Mali pumilae) of penicidin, making the concentration of penicidin in sample is respectively 20 μ g/kg apples, 50 μ g/kg apples, 20 μ g/L fruit juice, 50 μ g/L fruit juice; Sample after adding is carried out to pre-treatment according to method described in (two) respectively, obtain sample solution, then detected.Each concentration do 5 parallel, calculate recovery rate (rate of recovery=measured value/interpolation value) respectively.
The results are shown in Table 4.
The sample determination of recovery rates (1) of table 4 test kit
As can be seen from the table, the interpolation rate of recovery of apple sample is between 79.2% ~ 98.2%; The interpolation rate of recovery of samples of juice is 79.8% ~ 98.5%, meets the bioassay standard of accuracy.
4, cross reacting rate test:
Select the veterinary drug that represents with the compound of penicidin similar structures and clinical use, measure cross reacting rate.Obtain respectively its 50% inhibition concentration by various typical curves.Calculate the cross reacting rate of test kit to other analogue with following formula.
Result is as shown in table 5.
The specificity of table 5, test kit
| Title | Cross reacting rate (%) |
| Penicidin | 100.0 |
| Vomitoxin | ＜0.1 |
| Brown aspertoxin | ＜0.1 |
| The T2 toxin | ＜0.1 |
| Aflatoxin | ＜0.1 |
| Penicillin | ＜0.1 |
Experiment shows, test kit of the present invention is good to the specificity of penicidin, and test kit of the present invention can detect penicidin.
5, test kit preservation period test
The test kit preservation condition is 2-8 ℃, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of test kit, 50% inhibition concentration, penicidin added the practical measurement value all within normal range.Consider in transportation and use procedure, have improper preservation condition and occur, test kit is placed 8 days under the condition of 37 ℃ of preservations, carry out the accelerated deterioration experiment, result shows that the indices of this test kit meets the requirements fully.Consider that the freezing situation of test kit occurs, test kit is put into to-20 ℃ of refrigerator freezings 8 days, measurement result also shows that the test kit indices is fully normal.Can show that from above result test kit can at least can preserve more than 12 months at 2-8 ℃.
Claims (5)
1. a penicidin monoclonal antibody is by deposit number, to be CGMCC No.3392 penicidin monoclonal antibody hybridoma cell strain PAT secretion produces.
2. the penicidin monoclonal antibody hybridoma cell strain PAT that deposit number is CGMCC No.3392.
3. the application of the monoclonal antibody of penicidin described in claim 1 in the enzyme linked immunological kit of preparation detection penicidin.
4. an enzyme linked immunological kit that detects penicidin, be following 1), 2), 3) or 4) in arbitrary described enzyme linked immunological kit:
1) enzyme linked immunological kit comprises penicidin monoclonal antibody and enzyme labelling anti-antibody described in the conjugate, claim 1 of compound shown in formula I and carrier proteins; Wherein, described conjugate is as coating antigen;
2) enzyme linked immunological kit comprises penicidin monoclonal antibody and anti-antibody described in the enzyme labelling thing, claim 1 of compound shown in formula I; Wherein, described anti-antibody is as coating antigen;
3) enzyme linked immunological kit comprises penicidin monoclonal antibody described in the enzyme labelling thing, claim 1 of compound shown in formula I; Wherein, described penicidin monoclonal antibody is as coating antigen;
4) enzyme linked immunological kit comprises the enzyme labelling thing of penicidin monoclonal antibody described in the conjugate, claim 1 of compound shown in formula I and carrier proteins; Wherein, described conjugate is as coating antigen;
Described carrier proteins is mouse serum protein, thyroprotein, bovine serum albumin, albumin rabbit serum, human serum albumin, ovalbumin or hemocyanin.
5. a method that detects penicidin in sample, be following 1) or 2) described:
1) described testing sample is fruit juice; With the described test kit of claim 4, described fruit juice is detected;
2) described testing sample is fruit; Described fruit is carried out to homogenized, obtain the homogenate tissue, more described homogenate is organized centrifugal, get supernatant liquor, after supernatant liquor is diluted, obtain the supernatant liquor diluent, with the described test kit of claim 4, described supernatant liquor diluent is detected.
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| ES2849398A1 (en) * | 2020-02-17 | 2021-08-17 | Consejo Superior Investigacion | BIOCONJUGATES AND ANTIBODIES FOR THE DERIVATIZATION-ASSISTED IMMUNODETECTION OF PATULIN MYCOTOXIN (Machine-translation by Google Translate, not legally binding) |
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| CN106317009B (en) * | 2016-08-16 | 2019-03-26 | 华南农业大学 | A kind of xanthene polyurethane haptens, artificial antigen and its application |
| CN107904210B (en) * | 2017-12-27 | 2020-02-07 | 江南大学 | Procaine monoclonal antibody hybridoma cell strain SS0718 and application thereof |
| CN111812327A (en) * | 2020-06-05 | 2020-10-23 | 武汉华美生物工程有限公司 | Screening method of monoclonal antibody cells expressing secretion recognition natural samples |
| CN114716535B (en) * | 2022-06-10 | 2022-08-26 | 北京纳百生物科技有限公司 | Synthetic method of patulin artificial antigen and preparation and application of monoclonal antibody thereof |
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| ES2849398A1 (en) * | 2020-02-17 | 2021-08-17 | Consejo Superior Investigacion | BIOCONJUGATES AND ANTIBODIES FOR THE DERIVATIZATION-ASSISTED IMMUNODETECTION OF PATULIN MYCOTOXIN (Machine-translation by Google Translate, not legally binding) |
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