CN106442478A - Chemiluminescent ELISA (enzyme-linked immunosorbent assay) kit for ITeA (iso-tenuazonic acid) and use method of kit - Google Patents
Chemiluminescent ELISA (enzyme-linked immunosorbent assay) kit for ITeA (iso-tenuazonic acid) and use method of kit Download PDFInfo
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Abstract
The invention discloses a chemiluminescent ELISA (enzyme-linked immunosorbent assay) for ITeA (iso-tenuazonic acid) and a use method of the kit. The kit comprises a chemiluminescent ELISA plate coating an ITeA antigen, an ITeA antibody, an ELISA antibody, an ITeA standard solution, a chemiluminescent solution and a concentrated cleaning solution. The invention further discloses a method for assaying ITeA residues by using the kit. The kit for assaying ITeA adopts an indirect competition chemiluminescent ELISA adsorption analysis technology, the maximum assay range is 0.2-3.78 ng/mL, the sensitivity is 0.78 ng/mL, the detection limit is 0.032 ng/mL, the recovery rate is 82.4%-105.8%, and the kit is low in detection limit, high in sensitivity, good in stability, low in cost and very suitable for screening of a large number of samples and has important actual application and popularization significance.
Description
Technical field
A kind of the invention belongs to chemiluminescent enzyme-linked immunosorbent technical field of immunoassay, in particular it relates to different alternaria bacterium ketone
Sour chemical luminescence ELISA detection kit and its using method.
Background technology
Rod method is mould to be distributed widely in the agricultural product such as soil and corn, veterinary antibiotics, is important phytopathogen.
NSC 70328 (ITeA), as one of its main secondary metabolite, is belonged to together with tenuazonic acid (TeA)
In tetramino acid derivative, it is highly soluble in the organic solvents such as methanol, ethyl acetate, dimethyl sulfoxide, in the low polarity such as petroleum ether
Solvent in dissolubility poor.Research shows, NSC 70328 has cytotoxic effect, can suppress the knot of aminoacid
Close, stop protein and DNA synthesis.According to investigations, alternaric bacteria the most often pollution Semen Tritici aestivi, Sorghum vulgare Pers., Fructus Hordei Vulgaris, Oleum Helianthi, Semen Allii Tuberosi, kind
Eggplant, Fructus Mali pumilae, citruss and Fructus Canarii albi etc., content ITeA is detected in Sorghum vulgare Pers. infant cereal food reaches 75 ± 8 μ g/kg, Fructus Foeniculi
In tea, content reaches 21 ± 14 μ g/kg, and in Sorghum vulgare Pers. confection, content reaches 64 ± 5 μ g/kg.
The detection method of the mycotoxin of document report has thin layer chromatography, gas chromatography, high performance liquid chromatography at present
The instrumental methods such as method, chromatograph and mass spectrometric hyphenated technique.However, due to its complex operation, time-consuming, therefore more difficult satisfaction high-volume
The live screening requirements of sample, and immunoassay because of low cost, simple to operate, speed is fast, one-time detection sample size is big, instrument
Change low degree, being worth us to promote becomes conventional screening technique.
Chemiluminescence immune analysis method is that luminescent substance is tagged on antigen or antibody, with antibody or antigen, spy occurs
After specific immunological reaction, determine measured antibody or the content of antigen, its sensitivity by detecting the chemiluminescence intensity of label
Height, simple and efficient, can serve as the screening of live batch samples.
Content of the invention
The technical problem to be solved in the present invention is the defect and not overcoming existing NSC 70328 class detection technique
Foot, provides a kind of NSC 70328 chemical luminescence ELISA detection kit, described test kit is accurate, sensitive, fast
Speed, being capable of high throughput testing NSC 70328.
Another object of the present invention is to providing the using method of mentioned reagent box.
The above-mentioned purpose of the present invention is achieved by the following technical programs.
A kind of chemical luminescence ELISA detection kit of NSC 70328, comprises following component：
(1) it is coated with the chemiluminescence ELISA Plate of NSC 70328 antigen, described different alternaria bacterium ketone coating antigen
Concentration is 15.6ng/mL, and described chemiluminescence ELISA Plate is removable opaque white color luminous plaque；
(2) NSC 70328 antibody；
(3) enzyme mark anti antibody；
(4) NSC 70328 standard solution, concentration is 1mg/mL；
(5) chemical luminescence for liquid；
(6) concentrated cleaning solution；
Described NSC 70328 antigen is that (chemical entitled 3- (1- hydrazone group ethyl) -4- hydroxyl -5 is different for hapten ITeAHGA
Butyl -1H- pyrroles -2 (5H) -one) synthesize, with ovalbumin (OVA) covalent coupling, the conjugate obtaining；The structural formula of ITeAH is such as
Shown in lower：
Wherein the preparation method application reference number of ITeAHGA is 201310747570.2 patent.
Preferably, described chemiluminescence ELISA Plate is the removable opaque ELISA Plate in 96 holes or 40 holes, and material is polystyrene,
It is coated with the NSC 70328 antigen being combined with anti-NSC 70328 antibody specificity, and closed porosity table
The site of face unadsorbed NSC 70328 antigen.
The preparation method of the described chemiluminescence ELISA Plate being coated with NSC 70328 antigen is：Will with being coated liquid
NSC 70328 antigen dilutes on demand, adds and is coated liquid, put into 37 DEG C of environment and be incubated in luminous plaque micropore
Overnight, incline and be coated liquid, washed with cleaning mixture, in every hole, then add confining liquid, 37 DEG C of incubations, in the hole of inclining liquid, do
Aluminium film vacuum sealing is used to preserve after dry.
Furthermore it is possible to the material as fixing NSC 70328 antigen solid phase carrier is more, such as polystyrene,
Celluloid, polyethylene, polypropylene, polyacrylamide, cross-linked glucose, silicone rubber, agarose gel etc..The shape of this carrier
Formula can be shrinkage pool, the scraps of paper, globule etc..
Preferably, described be coated NSC 70328 antigen be coated liquid be in proportion by 1.69g sodium carbonate and
2.95g sodium bicarbonate is dissolved in 1L distilled water and obtaining, and the concentration that is coated of NSC 70328 antigen is 0.167ug/L.
Preferably, described confining liquid is that 0.1g BSA (bovine serum albumin), 5g glycine, 5g sucrose are dissolved in 100mL
PBS (0.01mol/L pH7.4) solution obtains.
Preferably, described NSC 70328 standard solution when using with the phosphoric acid of 0.01mol/L, pH5.4
Salt Tween buffer (PBST) by standard solution be diluted to concentration be 0.0 μ g/L, 0.00512 μ g/L, 0.0256 μ g/L,
0.128 μ g/L, 0.64 μ g/L, 3.2 μ g/L, a series of NSC 70328 standard solutions of 16 μ g/L.
Preferably, the formula of described phosphate Tween buffer (PBST) is：NaH2PO4·12H2O 2.9g、
NaCl8.5g、KCl 0.2g、KH2PO40.2g, 500 μ l Tween-20, are settled to 1L.
Preferably, described chemical luminescence for liquid is made up of A liquid and B liquid, and A liquid and B liquid are 1 by volume:1；The formula of A liquid
For：Deionized water will be dissolved in iodophenol, luminol, Tris, pH is 8.2～8.6；The formula of B liquid is：By volume fraction
0.40% H2O2, Tris be dissolved in deionized water, pH be 6.8～7.2.It is highly preferred that A liquid pH is 8.4, B liquid pH is 7.0.
Preferably, described concentrated cleaning solution is the phosphorus of pH 7.4 0.4mol/L containing volumetric concentration 0.5%Tween-20
Phthalate buffer；Described concentrated cleaning solution is 20 times of concentrated cleaning solutions, and during use, deionized water is diluted to 1 times of cleaning mixture.
The present invention also provides the user of described NSC 70328 chemical luminescence ELISA detection kit
Method, comprises the steps of：
S1. test kit is taken out from cold storage environment, be placed in 15～35 DEG C of balance 30min～45min；By described different alternaria
Bacterium keto acid standard solution phosphate buffer is diluted to a series of Concentraton gradient；
S2. take out chemiluminescence ELISA Plate, add the NSC 70328 solution of variable concentrations, sample well toward in gauge orifice
Middle addition testing sample, then every hole addition NSC 70328 antibody, covers the shaking of cover plate film and mixes, incubation；
S3. absorb the reactant liquor in plate hole, washed with cleaning mixture, ELISA Plate is patted dry；Described cleaning mixture is thickening and washing
Liquid dilutes 20 times and obtains；
S4. add enzyme mark anti antibody toward in each hole, pat mixing, incubation；
S5. every hole adds chemical luminescence for liquid, pats mixing, covers cover plate film, measures the luminous value RLU in each hole after 1～2min；
S6. the calculating of testing result and analysis：Determine the content of NSC 70328 in sample.
Suppression ratio (%)=B/B0× 100 (%), in formula：B is that the standard substance of variable concentrations NSC 70328 are molten
Fluid apertures or the luminous value in testing sample hole；B0For 0 concentration NSC 70328 standard solution luminous value；
With suppression ratio as vertical coordinate, the logarithm of the concentration of NSC 70328 draws standard curve for abscissa, thus really
In random sample product NSC 70328 content.
Preferably, the testing sample described in S2 is medicated beer, fruit juice, tomato sauce or rice.
Testing sample pre-treating method is as follows：
Medicated beer, fruit juice sample process：Measure 1mL sample in centrifuge tube, other groups directly add ITeA standard in addition to blank group
Product.Add 2mL × 2 chloroform oscillation extractions to be centrifuged 10min with 4000r/min, organic be added to 100 μ L 98% hydrazine hydrate, vibration
After reaction 30min, evaporated under reduced pressure solvent, residue l mL H2O redissolves.
Tomato sauce is processed：In centrifuge tube, in addition to blank group, other groups directly add the tomato sauce sample weighing 1g homogenate
Plus the ITeA of variable concentrations level, add 1mL saturation NaCl solution, after mixing, add 2mL × 2 chloroform oscillation extraction, with
4000r/min be centrifuged 10min, organic be added to 100uL 98% hydrazine hydrate, after oscillating reactionss 30min, evaporated under reduced pressure solvent, l
mL H2O redissolves.
The inventive method applies following principle：
The present invention adopts indirect competition chemiluminescence enzyme linked immunosorbent assay (ic-CLIA), devises and detects different thin friendship using the method
The test kit of pink mold keto acid, the shortcoming that compensate for prior art.
Chemiluminescence immune assay is that chemical reflective system is combined with immunoreation, for detecting trace antigen or anti-
A kind of novel markings immunoassay of body.Its Cleaning Principle is：(1) antigen or antibodies are made to certain solid phase carrier table
Face, and keep its immunocompetence.(2) antigen or antibody and certain enzyme is made to connect into enzyme-labelled antigen or antibody, this enzyme-labelled antigen
Or antibody had both retained its immunocompetence, retain the activity of enzyme again.(3) add luminous substrate after washing, enzymatic makes luminous substrate send out
Light, detects the quantity of photon, thus inverse goes out unknown antigen or the concentration of antibody by special instrument.The method prominent excellent
It is high that point shows as sensitivity, linear kineticss wide ranges, and the optical signal persistent period is long, and analysis method is easy to be quick, result is stable,
Error is little, and safety is good and validity period is long.Sensitive 100,000 times than absorption spectrometer of luminometer, at least cleverer than luminoscope
Quick 1,000 times.Compare ELISA immune analysis method, test limit at least order of magnitude lower is therefore sensitiveer.Compare instrument to divide
The Heterosis of analysis method exist：Enable mass detection, and more economical, save time.
Compared with prior art, the present invention has the beneficial effects that：The invention provides a kind of NSC 70328
Learn luminous enzyme-linked immunologic detecting kit and its using method, its maximum detection range is 0.2～3.78ng/mL, sensitivity is
0.78ng/mL, detection is limited to 0.032ng/mL, and the response rate is 82.4～105.8%,
The detection of this test kit is quick, substantially reduce detection time, does not consider the impact of testing staff's skilled operation degree, entirely
Detection process need only to 70min about can complete, and detection limit is lower, sensitivity is higher.Meanwhile, using envelope antigen
It is coated stability and the precision that plate greatly improves test kit, detection is more stable, more accurate.
In addition, test kit of the present invention adopts chemiluminescence method, do not use concentrated sulphuric acid etc. have severe corrosive reagent,
And have the substrate of strong carcinogenecity, more environment-friendly and safer.And simple to operate, low cost, it is especially suitable for a large amount of samples or scene sieve
Look into, there is important practical application dissemination.
Brief description
Fig. 1 is the chemiluminescent standard curve of NSC 70328.
Specific embodiment
With reference to Figure of description and specific embodiment, the present invention is described in further details, but embodiment is not right
The present invention limits in any form.Unless stated otherwise, the reagent that the present invention adopts, method and apparatus are that the art is normal
Rule reagent, method and apparatus.
Embodiment 1
Used in embodiment, the preparation method of reagent is as follows：
It is coated liquid：1.69g sodium carbonate and 2.95g sodium bicarbonate are dissolved in 1L distilled water and obtain.
20 times of concentrated cleaning solutions：Include the phosphate-buffered of the pH7.4 0.4mol/L of volume fraction 0.5%Tween20
Liquid, during use, deionized water is diluted to 1 times.
Confining liquid：0.1g BSA (bovine serum albumin), 5g glycine, 5g sucrose is taken to be dissolved in 100mL PBS solution
(0.01mol/L pH7.4) obtains.
NSC 70328 standard solution：With hplc grade methanol by adjacent NSC 70328 standard solution
It is diluted to 1mg/mL standby；Again with the PBST of 0.01mol/L pH5.4 be diluted to concentration be respectively 50ng/mL, 12.5ng/mL,
The NSC 70328 of 3.125ng/mL, 0.7812ng/mL, 0.1953ng/mL, 0.0488ng/mL, 0.0122ng/mL
Standard solution, 4 DEG C of preservations.
Chemical luminescence for liquid：Chemical luminescence for liquid is made up of A liquid and B liquid, A liquid be by 20mg to iodophenol, 8mg luminol,
1.21gTris is dissolved in 100mL deionized water, adjusts pH to obtain to 8.2～8.6 with hydrochloric acid；B liquid is by volume fraction 0.40%
H2O2, the Tris of 1.21g be dissolved in 100mL deionized water, with hydrochloric acid adjust pH obtain to 6.8～7.2；During use, A liquid and B liquid are pressed
Volume ratio 1:1 ratio mixing.
NSC 70328 polyclonal antibody (2.5mg/mL)：Prepared by laboratory early stage.
The goat anti-rabbit igg (10mg/mL) of horseradish peroxidase labelling：Obtain biological engineering company limited purchased from Wuhan doctor.
Embodiment 1 NSC 70328 polyclonal antibody, it is coated the preparation of original antibody
(1) preparation of coating antigen
Coating antigen ITeAH-OVA is prepared using active ester method.
(2) preparation of NSC 70328 polyclonal antibody
Using immunogen ITeAH-BSA as immunogen immune 3 rabbits respectively, by the immunity assistant of the immunogen preparing and equivalent
Uniformly, immunity is dynamic for agent (first time immunity cannot be used up full Freund adjuvant, booster immunization is all with incomplete Freund's adjuvant later) emulsifying
Thing.The new zealand white rabbit of 2.5～3kg is respectively adopted dorsal sc, each position be subcutaneous, leg muscle and auricular vein are multiple
Injection system immunity, second immunity after 4 weeks, added at interval of 3 weeks later and exempt from once.After 4th booster immunization, 1 week ear edge is quiet
Arteries and veins takes blood, and measures serum titer using indirect competitive ELISA.When potency no longer rises it is, using auricular vein booster immunization.
Culling heart blood after one week, water-bath 0.5～1h, 4 DEG C of 10000 centrifugation 15min, take supernatant to be antiserum.Antiserum adopts sulphuric acid
Ammonium sedimentation method purification to polyclonal antibody, frozen with -20 DEG C standby.
The foundation of embodiment 2 chemiluminescence enzyme linked immunoassay method
(1) it is coated the preferred of original content and antibody concentration
1) by coating antigen according to 31.2,15.6,8.9,5.0,2.5ng/mL is with being coated liquid dilution and being longitudinally coated opaque white color
Luminous plaque, 100 μ g/mL, 37 DEG C of overnight incubation, washed twice with cleaning mixture, absorbent paper pats dry.
2) add the confining liquid 150 μ L/ hole preparing to be closed, 37 DEG C overnight, dry and put into baking oven.
3) the NSC 70328 standard solution of the different gradients that addition 50 μ L/ hole PBS have diluted
4) the NSC 70328 polyclonal antibody (1 of the different gradients that addition 50 μ L/ hole PBS dilute:32000、1:
64000、1:96000、1:128000), 37 DEG C of incubation 30min, wash plate 5 times, absorbent paper pat dry.
5) 100 μ L/ holes have diluted two are added to resist, 37 DEG C are incubated 30min, wash plate 5 times, absorbent paper pats dry.
6) add the chemical luminescence for liquid now joined, 100 μ L/ holes, measure luminous value with chemical illumination immunity analysis instrument.With luminous
Value has the envelope antigen concentration of significant change and antibody diluted concentration to carry out specific assay for optium concentration with being coated original content.
Obtaining coating antigen optium concentration is 15.6ng/ml, and antibody extension rate is 64000 times.
(2) mensure of antibody sensitivity
To be coated concentration for coating antigen optium concentration as 15.6ng/ml, antibody extension rate is 64000 times, carries out antibody sensitive
The mensure of degree.
1) 96 hole opaque white color luminous plaques are taken, by coating antigen diluted to 15.6ng/ml, every hole adds 100 μ
L, 37 DEG C of overnight incubation, washed twice with cleaning mixture, absorbent paper pats dry
2) add the confining liquid 150 μ L/ hole preparing to be closed, 37 DEG C overnight, dry and put into baking oven.
3) it is separately added into the NSC 70328 standard solution of different gradients that 50 μ L/ hole PBS have diluted and dilute
Release 64000 times of antibody, 37 DEG C of incubation 30min, wash plate 5 times, absorbent paper pat dry.
4) add 100 μ L/ holes to dilute 5000 times two resist, 37 DEG C of incubation 30min, wash plate 5 times, absorbent paper pats dry.
5) add the chemical luminescence for liquid now joined, 100 μ L/ holes, measure luminous value with chemical illumination immunity analysis instrument.
Result is calculated with suppression ratio, suppression ratio (%)=B/B0× 100 (%), B are the competitions of various criterion strength solution
Luminous value, B0The luminous value of Shi Bujia standard substance, calculates the concentration that 50% suppression ratio is standard substance and is different alternaria bacterium ketone
The sensitivity of sour polyclonal antibody, as 0.78ng/mL.
The preparation of embodiment 3 enzyme linked immunological kit
1st, prepare reagent
(1) it is coated with the ELISA Plate of NSC 70328 antigen：The removable ELISA Plate in 96 holes, NSC 70328 resist
Former and confining liquid, being coated concentration is 0.05mg/L.Described NSC 70328 is NSC 70328 hapten
The conjugate of ITeAH and OVA.
Being coated of ELISA Plate microwell plate：Envelope antigen is diluted to 0.05mg/L with being coated liquid, and every in the hole adds 100 μ L to be coated
Liquid, 37 DEG C of overnight incubation, in the hole of inclining liquid, cleaning mixture washs 2 times, pats dry.Then 120 μ L confining liquids are added in every hole, 37
DEG C incubation 3h, in the hole of inclining liquid, it is placed in after drying in 37 DEG C of baking ovens, with the preservation of 4 DEG C of aluminium foil bag vacuum sealing.
(2) preparation of NSC 70328 standard solution：Accurately weigh NSC 70328 standard substance molten
Liquid, is diluted to 1mg/mL with hplc grade methanol, then (formula is NaH with 0.01mol/L PBST buffer2PO4·12H2O2.9g、
NaCl 8.5g、KCl 0.2g、KH2PO40.2g, 500 μ LTween-20, are settled to 1L) prepare 50ng/mL, 12.5ng/ respectively
ML, 3.125ng/mL, 0.7812ng/mL, 0.1953ng/mL, 0.0488ng/mL, 0,0122ng/mL NSC 70328
Solution, 4 DEG C of preservations.
(3) NSC 70328 Anti-TNF-α bulk concentration is 1mg/mL；Its working concentration is 1:64000, entered with PBS
Row dilution.
(4) chemical luminescence for liquid：It is made up of A liquid and B liquid；By A liquid and B liquid by volume 1 during use:1 ratio mixing.
The compound method of A liquid is：20mg is dissolved in 100mL deionized water to iodophenol, 8mg luminol, 1.21gTris,
PH is adjusted to obtain to 8.2～8.6 with hydrochloric acid；
The compound method of B liquid is：By volume fraction 0.40%H2O2, 1.21gTris be dissolved in 100mL deionized water, adjusted with hydrochloric acid
PH obtains to 6.8～7.2.
(5) 20 times of concentrated cleaning solutions；During use, deionized water is diluted to 1 times of cleaning mixture.
2nd, reagent subpackage
Each reagent is measured qualified rear aseptic subpackaged, to have diluted NSC 70328 standard solution l mL/ bottle, different
Tenuazonic acid polyclonal antibody 7mL/ bottle, A liquid 7mL/ bottle, B liquid 7mL/ bottle, 20 times of concentrated cleaning solution 50mL/ bottles.Subpackage
After label, dated lot number and effect duration, 4 DEG C of preservations.
3rd, the assembling of test kit
Respectively by above-mentioned 1 piece of chemiluminescence ELISA Plate being coated with NSC 70328 antigen, NSC 70328
Antibody, A liquid, B liquid, each 1 bottle of 20 times of concentrated cleaning solutions, 6 bottles of NSC 70328 standard solution and 1 part of operation instructions
It is placed in specified location in test kit, test kit encapsulates after the assay was approved, 4 DEG C of preservations.
The application of embodiment 4 NSC 70328 chemical luminescence ELISA detection kit
1st, detected using test kit of the present invention
(1) take out test kit, be placed in (20～24 DEG C) balance more than 30min of room temperature, take out chemiluminescence ELISA Plate, use
NSC 70328 standard solution is diluted to a series of different thin friendship of variable concentrations by 0.01mol/L PBST buffer
Pink mold keto acid standard solution (0.0 μ g/L, 0.00512 μ g/L, 0.0256 μ g/L, 0.128 μ g/L, 0.64 μ g/L, 3.2 μ g/
L、16μg/L).
(2) add the NSC 70328 standard solution of 50 μ L variable concentrations in gauge orifice, sample well adds 50 μ
L testing sample, the NSC 70328 labeling antibody that then every hole addition 50 μ L have been diluted with 0.01mol/L PBST, cover
After cover plate film shakes 10min on micro oscillator, it is placed in 37 DEG C of incubation 30min.
(3) absorb the reactant liquor in plate hole, each hole adds cleaning mixture about 300 μ L, stands 20 seconds about, removes wherein liquid
Body, so washes altogether 5 times, pats dry plate for the last time；Also plate 5 times can be washed with automatic washer, after washing, micropore frame is upside down in
(often wheel is washed plate and patted 3 times) is patted on absorbent paper, to ensure to completely remove the liquid in hole.
(4) every hole adds 100 μ L chemical luminescence for liquid, pats mixing, covers cover plate film, is exempted from chemiluminescence after 1～2min
The luminous value RLU in each hole of epidemic disease analysis-e/or determining, preserves data.
2nd, testing result calculates and analysis
Suppression ratio (%)=B/B0× 100 (%),
In formula：B is the luminous value in variable concentrations standard solution hole (or testing sample hole)；B0Send out for 0 concentration standards solution
Light value.
With suppression ratio as vertical coordinate, the logarithm of NSC 70328 standard solution concentration draws standard for abscissa
Curve, is substituted in above-mentioned standard curve with the light absorption value of NSC 70328 and obtains different alternaria bacterium ketone in testing sample
The content of acid.Do not consider the impact of testing staff's skilled operation degree, whole detection process need only to 80min about can be complete
Become.The analysis of testing result can also be calculated using computer professional software and be analyzed.
3rd, standard curve
By NSC 70328 canonical plotting is obtained (as accompanying drawing 1 institute to the Analysis of test results of standard solution
Show), indicating test kit of the present invention to the linear detection range of NSC 70328 is 0.2～3.78ng/ml, sensitivity
0.78ng/ml, detection limit 0.032ng/mL.
The application of embodiment 4 NSC 70328 chemical luminescence ELISA detection kit and precision, accurately
Degree test
Detection method is with embodiment 3.
1st, testing sample pre-treatment
(1) medicated beer, Sucus Mali pumilae sample process
Medicated beer, Sucus Mali pumilae sample process：Measure 1mL sample in centrifuge tube, other groups directly add ITeA mark in addition to blank group
Quasi- product.Add 2mL × 2 chloroform oscillation extractions to be centrifuged 10min with 4000r/min, organic be added to 100 μ L 98% hydrazine hydrate, shake
After swinging reaction 30min, evaporated under reduced pressure solvent, residue l mL H2O redissolves for analyzing.
(2) tomato sauce is processed
In centrifuge tube, in addition to blank group, other groups directly add variable concentrations level to the tomato sauce sample weighing 1g homogenate
ITeA, adds 1mL saturation NaCl solution, adds 2mL × 2 chloroform oscillation extraction, be centrifuged 10min with 4000r/min, have after mixing
Machine is added to 100uL 98% hydrazine hydrate, after oscillating reactionss 30min, evaporated under reduced pressure solvent, and l mL H2O redissolves for analyzing.
2nd, the replica test of NSC 70328 standard solution
From 3 batches of ELISA Plate according to the method preparation embodiment 2,20 micropores of each random extraction, according to embodiment 3 pilot scale
The detection method of agent box measures the luminous value of 0.8 μ g/L NSC 70328 standard solution, is repeated 20 times, and calculates variation
Coefficient CV%, result is as shown in table 1.
Table 1 NSC 70328 standard solution replica test
Result shows, the variation within batch coefficient range of kit standard product solution detection of the present invention, between 4.75～8.5%, is criticized
Between the coefficient of variation be 7.6%.
2nd, sample repeatability and accuracy test
Accuracy refers to the matching degree of measured value and true value, and in enzyme-linked immunoassay, accuracy is often represented with the response rate, essence
Density often to be represented with the coefficient of variation.In blank sample, add NSC 70328 extremely final concentration of 0.2,0.6,1.2
μ g/L, each 10 parallel, the mensure 3 batches of each concentration.Calculate meansigma methodss, TIANZHU XINGNAO Capsul and batch interior and interassay coefficient of variation.Knot
Fruit is shown in Table 2.
Table 2 sample repeatability and accuracy test result
Result shows, soy sauce, fruit juice, Wine Sample TIANZHU XINGNAO Capsul between 80.3～108.9%, variation within batch coefficient exists
3.6～9.3%, between interassay coefficient of variation 3.2～8.2%, meet the standard for test kit indices for the country.
Embodiment 5 NSC 70328 chemical luminescence ELISA detection kit storage life is tested
1st, the test kit of embodiment 2 is positioned over 2～8 DEG C, takes the examination storing 0,2,4,6,8,9,10,11 and 12 months respectively
Agent box, to the absorbance of NSC 70328 standard solution (0.8 μ g/L), 50% inhibition concentration, TIANZHU XINGNAO Capsul,
The each parameter of variation within batch coefficient is measured.
2nd, place 12 days under conditions of test kit being preserved at 37 DEG C, daily to NSC 70328 standard solution
The absorbance of (0.8 μ g/L), 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient are measured.
3rd, by test kit in -20 DEG C of Refrigerator stores 12 days, daily to NSC 70328 standard solution (0.8 μ
G/L absorbance), 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient are measured.
Result shows, through three kinds of condition food preservation test, the suction of NSC 70328 standard solution (0.8 μ g/L)
Shading value is dropped by less than 10%, and indices all conform to quality requirements, and therefore, test kit can preserve 12 months at 2～8 DEG C.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not subject to above-described embodiment
Limit, other any spirit without departing from the present invention and the change made under principle, modification, replacement, combine, simplify,
All should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (5)
1. a kind of chemical luminescence ELISA detection kit of NSC 70328 is it is characterised in that comprise following one-tenth
Point：
（1）It is coated with the chemiluminescence ELISA Plate of NSC 70328 antigen, described different alternaria bacterium ketone coating antigen
Concentration is 15.6ng/ mL, and described chemiluminescence ELISA Plate is removable opaque white color luminous plaque；
（2）NSC 70328 antibody；
（3）Enzyme mark anti antibody；
（4）NSC 70328 standard solution, concentration is 1mg/mL；
（5）Chemical luminescence for liquid；
（6）Concentrated cleaning solution；
Described NSC 70328 antigen is 3-（1- hydrazone group ethyl）- 4- hydroxyl -5- isobutyl group -1H- pyrroles -2（5H-
Ketone）Conjugate with ovalbumin.
2. chemical luminescence ELISA detection kit according to claim 1 is it is characterised in that described chemical luminescence for liquid
It is made up of A liquid and B liquid, A liquid and B liquid are 1 by volume：1；The formula of A liquid is：Iodophenol, luminol, Tris will be dissolved in
Ionized water, pH is 8.2～8.6；The formula of B liquid is：H by volume fraction 0.40%2O2, Tris be dissolved in deionized water, pH is
6.8～7.2.
3. chemical luminescence ELISA detection kit according to claim 1 is it is characterised in that described thickening and washing
Liquid is the phosphate buffer of the pH7.4 0.4mol/L containing volumetric concentration 0.5% Tween-20.
4. the making of the NSC 70328 chemical luminescence ELISA detection kit described in any one of claims 1 to 3
With method it is characterised in that comprising the steps of：
S1. test kit is taken out from cold storage environment, be placed in 15～35 DEG C of balance 30 min～45min；
S2. take out chemiluminescence ELISA Plate, add the NSC 70328 solution of variable concentrations, sample toward in gauge orifice
Add testing sample in hole, then every hole adds NSC 70328 antibody, cover the shaking of cover plate film and mix, incubation；Institute
State NSC 70328 solution and variable concentrations are become by phosphate buffer dilution NSC 70328 standard solution
Obtain；
S3. absorb the reactant liquor in plate hole, washed with cleaning mixture, ELISA Plate is patted dry；
S4. add enzyme mark anti antibody toward in each hole, pat mixing, incubation；
S5. every hole adds chemical luminescence for liquid, pats mixing, covers cover plate film, measures the luminous value RLU in each hole after 1～2min；
S6. the calculating of testing result and analysis：Determine the content of NSC 70328 in sample.
5. using method according to claim 4 is it is characterised in that the testing sample described in S2 is medicated beer, fruit juice, Fructus Lycopersici esculenti
Beans or rice.
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| CN201610315788.4A CN106442478A (en) | 2016-05-12 | 2016-05-12 | Chemiluminescent ELISA (enzyme-linked immunosorbent assay) kit for ITeA (iso-tenuazonic acid) and use method of kit |
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| CN201610315788.4A CN106442478A (en) | 2016-05-12 | 2016-05-12 | Chemiluminescent ELISA (enzyme-linked immunosorbent assay) kit for ITeA (iso-tenuazonic acid) and use method of kit |
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|---|---|---|---|---|
| CN102516148A (en) * | 2011-11-23 | 2012-06-27 | 华南农业大学 | Half antigen and antigen of tenuazonic acid and preparation method and application thereof |
| CN103487575A (en) * | 2013-09-02 | 2014-01-01 | 华南农业大学 | Chemiluminescent enzyme-linked immunosorbent assay kit of tenuazonic acid and application method thereof |
| CN103787946A (en) * | 2013-12-31 | 2014-05-14 | 华南农业大学 | Isokwas tenuazonowy artificial antigen and antibody as well as preparation method and application thereof |
| CN105486822A (en) * | 2014-10-11 | 2016-04-13 | 江苏维赛科技生物发展有限公司 | Immunoassay kit used for detecting heavy metal ion arsenic content in detection sample |
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2016
- 2016-05-12 CN CN201610315788.4A patent/CN106442478A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102516148A (en) * | 2011-11-23 | 2012-06-27 | 华南农业大学 | Half antigen and antigen of tenuazonic acid and preparation method and application thereof |
| CN103487575A (en) * | 2013-09-02 | 2014-01-01 | 华南农业大学 | Chemiluminescent enzyme-linked immunosorbent assay kit of tenuazonic acid and application method thereof |
| CN103787946A (en) * | 2013-12-31 | 2014-05-14 | 华南农业大学 | Isokwas tenuazonowy artificial antigen and antibody as well as preparation method and application thereof |
| CN105486822A (en) * | 2014-10-11 | 2016-04-13 | 江苏维赛科技生物发展有限公司 | Immunoassay kit used for detecting heavy metal ion arsenic content in detection sample |
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