CN110470831A - A kind of chemiluminescence immune analysis method and kit directly detecting tenuazonic acid based on hunchbacked source antibody - Google Patents
A kind of chemiluminescence immune analysis method and kit directly detecting tenuazonic acid based on hunchbacked source antibody Download PDFInfo
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- CN110470831A CN110470831A CN201910616023.8A CN201910616023A CN110470831A CN 110470831 A CN110470831 A CN 110470831A CN 201910616023 A CN201910616023 A CN 201910616023A CN 110470831 A CN110470831 A CN 110470831A
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- tenuazonic acid
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
Abstract
The invention discloses a kind of chemiluminescence immune analysis method and kit for directly detecting tenuazonic acid based on hunchbacked source antibody, the present invention has the artificial antigen of the tenuazonic acid as shown in formula (I) using one kind,Wherein n=1~6.A kind of establish is established a kind of directly to detect the chemiluminescence immune analysis method of tenuazonic acid based on hunchbacked source antibody and develop chemiluminescence immune detection reagent kit, 0.2ng/mL is limited to the detection of tenuazonic acid, 503nhibiting concentration is 8.0ng/mL, and the range of linearity is 0.9~69.8ng/mL.The kit high specificity of method and exploitation that the present invention establishes, high sensitivity, operating method is easy, is with a wide range of applications in terms of the analysis detection of tenuazonic acid in food.
Description
Technical field
The present invention relates to technical field of food safety detection, are directly detected more particularly, to one kind based on hunchbacked source antibody
The chemiluminescence immune analysis method and kit of tenuazonic acid.
Background technique
Alternaric bacteria can generate more than 70 kinds of toxic secondary metabolites, cereal, peanut, jam, fresh vegetables fruit and
Residual in juice drinks etc. is by social concerns, and alternaric bacteria can infect oilseed, cause edible oil contamination, sternly
Ghost image rings the market value of agricultural product.Tenuazonic acid (Tenuazonic Aci d, TeA), as the mould toxin of rod method
The strongest secondary metabolites of Poisoning, are included in toxic chemical substance liber by U.S. Food and Drug Administration.Study table
Bright, TeA has a very strong acute or subacute toxic action to animals such as mouse, dogs, and with rod method phenol (Alternariol,
AOH), a variety of toxin such as rod method phenol methyl ether (Alt ernariol Methyl Ether, AME) generate synergistic effect, generate
Acute toxicity.
In recent years, there is detection Te A in the numerous foods such as baby food, beer, potato, fruit, pepper, this makes
The detection of TeA toxin also increasingly merits attention in food.Currently, to the detection method of tenuazonic acid TeA in food
Have very much, but mostly instrument analytical method, instrumental method equipment is expensive, and condition is harsh, is not particularly suited for the quick detection of TeA.
Minority report is only through the analysis of TeA derivative using immune rapid detection method to realize the indirect inspection of TeA
It surveys, and deriving method is complicated for operation, inefficient, product purification is difficult.At present and have no the change analyzed directly against TeA
Chemiluminescence immunoassay method and immunity detection reagent are learned, therefore is highly desirable to provide a kind of directly detection alternaria bacterium ketone
The chemiluminescence immune analysis method and kit of acid.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of directly detect based on hunchbacked source antibody carefully to hand over
The chemiluminescence immune analysis method and kit of pink mold ketone acid.
The first purpose of the invention is to provide a kind of artificial antigens of tenuazonic acid.
A second object of the present invention is to provide application of the artificial antigen in detection tenuazonic acid.
Third object of the present invention is to provide the antibody that the artificial antigen is prepared to detect alternaria bacterium
Application in ketone acid.
Fourth object of the present invention is to provide a kind of method for detecting tenuazonic acid.
Fifth object of the present invention is to provide a kind of chemiluminescence immunoassay detection reagents for detecting tenuazonic acid
Box.
To achieve the goals above, the present invention is achieved by the following technical programs:
A kind of artificial antigen of tenuazonic acid has as shown in formula (I),
(I)
Wherein n=1~6.
Preferably, n=1.
Preferably, the carrier protein is keyhole limpet hemocyanin, bovine serum albumin or ovalbumin.
Application of the artificial antigen in detection tenuazonic acid.
Application of the antibody that the artificial antigen is prepared in detection tenuazonic acid.
Preferably, the antibody is the hunchbacked source antibody that artificial antigen is immunized that camellid is prepared.
It is furthermore preferred that the camellid is alpaca, two-humped camel or one-humped camel.
It is furthermore preferred that the antibody includes but is not limited to polyclonal antibody, genetic engineering antibody.
A kind of hunchbacked source antibody of anti-tenuazonic acid, preparation method the following steps are included:
S1. camellid is immunized with the tenuazonic acid artificial antigen；
S2. and then it is primary in the 15th, 29,43,57 day each booster immunization respectively；
S3. at the 64th day, whole blood is acquired, collects tenuazonic acid camel source antibody.
A method of detection tenuazonic acid, the artificial antigen carrier protein are keyhole limpet hemocyanin (TeA-
AOAA-KLH it) is used as immunogene, the artificial antigen carrier protein is that ovalbumin (TeA-AOAA-OVA) is used as coating antigen, into
The detection of row tenuazonic acid.
Preferably, the artificial antigen carrier protein is that keyhole limpet hemocyanin is prepared as immunogen immune camellid
Hunchbacked source antibody, carries out the detection of tenuazonic acid.
Preferably, the artificial antigen carrier protein is that keyhole limpet hemocyanin is prepared as immunogen immune camellid
Antibody is above-mentioned antibody.
Preferably, the method for detecting tenuazonic acid is chemiluminescence immune analysis method, comprising the following steps:
S1. there will be the artificial antigen of formula (I) structure as coating antigen, coating antigen is coated in the micropore of Chemiluminescent plate
It is interior, incubation, board-washing, closing, drying；
S2. the tenuazonic acid standard items or sample to be tested of gradient dilution are added in Chemiluminescent plate micropore, and
The hunchbacked source antibody is added in micropore of enzyme marker plate, is incubated for, board-washing；
S3. the anti-hunchbacked secondary antibody of enzyme mark is added in Chemiluminescent plate micropore, is incubated for, board-washing；
S4. luminescent solution is added in Chemiluminescent plate micropore, oscillation mixes, and measures luminous value under certain wavelength；
S5. using each standard concentration hole luminous value as ordinate, with the log of pharmaceutical standards liquid concentration10Value is abscissa, is drawn
The standard curve of immunologic detection method processed.
S6. detection limit LOD (drug concentration of 10% inhibiting rate, IC are calculated according to the standard curve of foundation10) and half
Amount of suppression (drug concentration of 50% inhibiting rate, IC50), tenuazonic acid content in quantitative analysis sample, inhibiting rate is pressed
Formula calculates:
Wherein, RLU0Luminous value when for tenuazonic acid drug concentration being 0, RLUx is that tenuazonic acid is
Luminous value when x, RLUmin are the luminous value of blank control wells.
Preferably, primary concentration of envelope 31.25ng/mL, the hunchbacked source antibody being prepared dilute 2000 times of uses.
Preferably, in step S2, it is incubated for 40min.
Preferably, in step S3, it is incubated for 40min.
Preferably, in step S3, pH7.4Tris-HCl is selected to dilute hunchbacked source antibody.
Preferably, in step S2, the drug dilution liquid of the tenuazonic acid standard items of gradient dilution is H2O。
A kind of chemiluminescence immune detection reagent kit detecting tenuazonic acid, the kit contain described artificial
Hunchbacked source antibody that antigen vectors albumen is keyhole limpet hemocyanin (TeA-AOAA-KLH) as immunogene and is prepared and described
Artificial antigen carrier protein is that ovalbumin (TeA-AOAA-OVA) is used as the coated Chemiluminescent plate of coating antigen.
Preferably, the artificial antigen carrier protein is that keyhole limpet hemocyanin (TeA-AOAA-KLH) is prepared as immunogene
Obtained hunchbacked source antibody is the hunchbacked source antibody.
It preferably, further include the secondary antibody of horseradish peroxidase-labeled, tenuazonic acid standard solution, sample redissolution
Liquid, sample diluting liquid, concentrated cleaning solution, luminous substrate liquid, substrate buffer solution, cover board film, valve bag, gloves, operation instructions.
Compared with prior art, the invention has the following beneficial effects:
(1) it using tenuazonic acid artificial antigen as immunogene, prepares directly against alternaria bacterium ketone
The high specific camel source antibody of acid, potency 1:128000；(2) a kind of chemistry hair of directly detection tenuazonic acid is provided
Light immunoassay method；(3) a kind of chemiluminescence immune detection reagent kit of directly detection tenuazonic acid is provided；(4)
The invention have the characteristics that it is easy quickly, it is high specificity, with higher sensitivity, 0.2ng/ is limited to the detection of tenuazonic acid
ML, 503nhibiting concentration 8.0ng/mL, the range of linearity are 0.9~69.8ng/mL.
Detailed description of the invention
Fig. 1 is tenuazonic acid haptens TeA-AOAA (n=1) and the ultraviolet qualification figure of artificial antigen.
Fig. 2 is the RLU/IC of different primary concentration of envelope and hunchbacked source antibody extension rate50、IC50, RLU curve.
Fig. 3 is the RLU/IC of different ELIAS secondary antibody extension rates50、IC50, RLU curve.
Fig. 4 is the RLU/IC of different primary antibody competitive reaction times50、IC50, RLU curve.
Fig. 5 is the RLU/IC in different ELIAS secondary antibody reaction time50、IC50, RLU curve.
Fig. 6 is the RLU/IC of different antibodies dilution50、IC50, RLU curve.
Fig. 7 is the RLU/IC of different antibodies dilution pH50、IC50, RLU curve.
Fig. 8 is the RLU/IC of different pharmaceutical dilution50、IC50, RLU curve.
Fig. 9 is with hunchbacked source antibody that TeA-AOAA (n=1) is haptens preparation to the suppression curve of tenuazonic acid.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
The preparation and identification of 1 tenuazonic acid camel source antibody of embodiment
One, experimental method
The artificial antigen obtained to haptens and carrier protein couplet carries out ultraviolet identification, and tenuazonic acid is immunized
Former TeA-AOAA-KLH with Freund's adjuvant (not exclusively helped with Freund by the 1st immune Freund's complete adjuvant, later booster immunization
Agent) Freund's complete adjuvant emulsification, 3 years old healthy male Bactrian camel is immunized using the subcutaneous multiple spot of neck.Steps are as follows: taking 0.5mg thin
The incomplete Freund's adjuvant of Alternariaspp ketone acid immunogene TeA-AOAA-KLH and 500 μ L emulsification, respectively the 15th, 29,43,57
Its booster immunization acquired whole blood 50mL from camel neck, blood is placed 30 minutes, 4 DEG C in 37 DEG C of water-baths at the 64th day
12000rpm is centrifuged 30 minutes, and collecting supernatant is tenuazonic acid camel source antibody, packing, -20 DEG C of preservations.Between
Connect Inhibition ELISA measurement antibody titer.
Two, experimental result
Fig. 1 is tenuazonic acid haptens TeA-AOAA (n=1) and the ultraviolet qualification figure of artificial antigen, indirect competition
It is 1:128000, inhibiting rate 69% that ELISA method, which measures tenuazonic acid camel source antibody titer,.
A kind of tenuazonic acid chemiluminescence immune analysis method of embodiment 2
One, the foundation of tenuazonic acid chemiluminescence immune analysis method
(1) a certain concentration coating antigen (TeA-AOAA-OVA) is coated in the micropore of Chemiluminescent plate, is coated with through 37 DEG C
After overnight, 120 μ L, 5% skimmed milk power solution is added in 37 DEG C of closing 3h in every hole.It dries in hole after liquid, 37 DEG C of baking 1h are standby
With；
(2) by a series of concentration tenuazonic acid titers of 50 μ L or analyte sample fluid additionization through pre-treatment
In the micropore for learning luminescent screen, the antiserum of the 50 certain extension rates of μ L is added into the micropore of Chemiluminescent plate, 37 DEG C are incubated for one
The section time, liquid in hole is poured out, is washed 5 times with cleaning solution, is patted dry on blotting paper；
(3) the HRP label goat-anti camel two corresponding anti-solution of the 100 certain extension rates of μ L is added into the micropore of Chemiluminescent plate,
It 37 DEG C of incubation a period of times, is washed 5 times, is patted dry with cleaning solution；
(4) 100 μ L luminescent solutions are added into the micropore of Chemiluminescent plate, oscillation mixes.Each hole is measured at wavelength 425nm
Luminous value.Using each standard concentration hole luminous value as ordinate, with the log of pharmaceutical standards liquid concentration10Value is abscissa, draws mark
Directrix curve figure.
(5) according to the luminous value of canonical plotting and sample to be tested, tenuazonic acid in sample to be tested can be calculated
Content.
Various response parameters have very big influence to the sensitivity of method in chemiluminescence immune analysis method, therefore, choosing
It selects the following conditions and carries out parameter optimization.
Two, the concentration of coating antigen and hunchbacked source antibody
1, experimental method
Optimize primary concentration of envelope and polyclonal antibody extension rate by Checkerboard titration method, setting primary concentration of envelope (1000,
500,250,125,62.5,31.25ng/mL) and hunchbacked source antibody extension rate (2000,4000,8000,16000,32000,
64000,128000,256000 times), relative luminous intensity (RLU) is detected according to methodology above, makees competition inhibition curve, is led to
It crosses and compares RLU/IC50、IC50, RLU size select optimal parameter.
2, experimental result
As a result as shown in Fig. 2, optimal primary concentration of envelope and two-humped camel polyclonal antibody extension rate are 31.25ng/mL-
2000 times.
Three, ELIAS secondary antibody extension rate
1, experimental method
Setting ELIAS secondary antibody extension rate is respectively 6000,8000,10000,12000,14000 times, according to side above
Method detects relative luminous intensity (RLU), makees competition inhibition curve, by comparing RLU/IC50、IC50, the size selection of RLU it is best
Parameter.
2, experimental result
As a result as shown in figure 3, optimal ELIAS secondary antibody extension rate is 10000 times.
Four, the primary antibody competitive reaction time
1, experimental method
The primary antibody competitive reaction time is set as 30,40,50min, detects relative luminous intensity according to methodology above
(RLU), make competition inhibition curve, by comparing RLU/IC50、IC50, RLU size select optimal parameter.
2, experimental result
As a result as shown in figure 4, the optimal primary antibody competitive reaction time is 40min.
Five, the ELIAS secondary antibody reaction time
1, experimental method
The ELIAS secondary antibody reaction time is set as 30,40,50min, detects relative luminous intensity according to methodology above
(RLU), make competition inhibition curve, by comparing RLU/IC50、IC50, RLU size select optimal parameter.
2, experimental result
As a result as shown in figure 5, the optimal ELIAS secondary antibody reaction time is 40min.
Six, antibody diluent
1, experimental method
Antibody diluent is set as PBS, PBST, Tris-HCl, detects relative luminous intensity according to methodology above
(RLU), make competition inhibition curve, by comparing RLU/IC50、IC50, RLU size select optimal parameter.
2, experimental result
As a result as shown in fig. 6, optimum antibody dilution is Tris-HCl.
Seven, antibody diluent pH
1, experimental method
The pH of antibody diluent Tris-HCl is set as 6.5,7.0,7.4,8.0,8.5, detects phase according to methodology above
To luminous intensity (RLU), make competition inhibition curve, by comparing RLU/IC50、IC50, RLU size select optimal parameter.
2, experimental result
As a result as shown in fig. 7, the pH of optimum antibody dilution Tris-HCl is 7.4.
Eight, drug dilution liquid
1, experimental method
Drug dilution liquid is set as PBS, PBST, H2O detects relative luminous intensity (RLU) according to methodology above, makees competing
Suppression curve is striven, selects optimal parameter by comparing the size of RLU/IC50, IC50, RLU.
2, experimental result
As a result as shown in figure 8, optimal drug dilution is H2O。
Nine, a kind of tenuazonic acid chemiluminescence immune analysis method
(1) 31.25ng/mL coating antigen (TeA-AOAA-OVA) is coated in the micropore of Chemiluminescent plate, is wrapped through 37 DEG C
After overnight, 120 μ L, 5% skimmed milk power solution is added in 37 DEG C of closing 3h in every hole.It dries in hole after liquid, 37 DEG C of baking 1h are standby
With；
(2) by a series of concentration tenuazonic acid titer (H of 50 μ L2O is as dilution) or through pre-treatment to
Sample liquid is added in the micropore of Chemiluminescent plate, and the antiserum that 50 μ L dilute 2000 times is added into the micropore of Chemiluminescent plate
(Tris-HCl of pH 7.4 is as dilution), 37 DEG C of incubation 40min, pours out liquid in hole, is washed 5 times, inhaled with cleaning solution
It is patted dry on water paper；
(3) the HRP label goat-anti camel two corresponding anti-solution that 100 μ L dilute 2000 multiples is added into the micropore of Chemiluminescent plate,
37 DEG C of incubation 40min, are washed 5 times with cleaning solution, are patted dry；
(4) 100 μ L luminescent solutions are added into the micropore of Chemiluminescent plate, oscillation mixes.Each hole is measured at wavelength 425nm
Luminous value.Using each standard concentration hole luminous value as ordinate, using the log10 value of pharmaceutical standards liquid concentration as abscissa, establish
Ic-CLEIA standard curve.
(5) according to the luminous value of canonical plotting and sample to be tested, tenuazonic acid in sample to be tested can be calculated
Content.
With hunchbacked source antibody that TeA-AOAA (n=1) is haptens preparation to suppression curve such as Fig. 9 of tenuazonic acid
It is shown.
A kind of tenuazonic acid chemiluminescence immune analysis method of embodiment 3
One, experimental method
Coating antigen (TeA-AOAA-OVA) is coated in the micropore of Chemiluminescent plate, incubation, board-washing, closing, drying；It will
The hunchbacked source antibody of the tenuazonic acid standard items of 50 μ L series of concentrations and the 50 appropriate extension rates of μ L, which is added to, to be coated with
In Chemiluminescent plate difference micropore, 40min is incubated at 37 DEG C, board-washing 5 times, the goat-anti camel secondary antibody for adding 100 μ L HRP enzymes to mark, 37
40min is incubated at DEG C, board-washing 5 times, adds 100 μ L luminescent solutions, and oscillation mixes, and each hole luminous value is measured at wavelength 425nm.With hair
Light value is ordinate, and respective standard product log concentration value is abscissa, using 8.5 software of Origin, four parameter logistic function into
Row curve matching.Several analogs of tenuazonic acid are subjected to gradient dilution, are then reacted with hunchbacked source antibody, are made
Make standard curve.The IC of each analog is calculated separately out by curve50, the chemiluminescence immune analysis method of foundation is calculated to thin
The cross reacting rate of Alternariaspp ketone acid analog: cross reacting rate=IC50(TeA)/IC50(analog) × 100%.
Curve matching is y=(A-D)/[1+ (x/C)B]+D,
Wherein, luminous value when A and D respectively represents tenuazonic acid standard concentration minimum and maximum, C is midpoint
Concentration；Light absorption value value when standard concentration is equal to C is (A+D)/2, is at point of inflexion on a curve, half amount of suppression concentration
For IC50, the steep of B expression curve, title slope factor；With IC10It is limited for detection, with IC20~IC80For detection range.With ladder
The tenuazonic acid solution of degree is standard items, establishes the standard curve of chemiluminescence immune assay.
Two, experimental result
It the results are shown in Table 1.Interpretation of result it is found that the chemiluminescence immune analysis method established of the present invention to alternaria bacterium ketone
The detection of acid is limited to 0.2ng/mL, 503nhibiting concentration 8.0ng/mL, and the range of linearity is 0.9~69.8ng/mL, inhibiting rate and thin
The logarithm of Alternariaspp ketone acid concentration is at significant S type curved line relation, coefficient R2It is 0.996.Therefore, this method can be with
The content of tenuazonic acid in food is directly detected, and has the advantages that highly sensitive and high specific.
Cross reacting rate of the detection method that 1 present invention of table establishes to tenuazonic acid analog:
4 tenuazonic acid chemiluminescence immune detection reagent kit of embodiment
One, the preparation of tenuazonic acid chemiluminescence immune detection reagent kit each component
(1) it is coated with the Chemiluminescent plate of tenuazonic acid coating antigen (TeA-AOAA-OVA): with pH's 9.6
Artificial antigen TeA-AOAA-OVA is diluted to 31.25ng/mL by 0.5mol/L carbonate buffer solution, in Chemiluminescent plate micropore
Every hole adds 100 μ L, and 37 DEG C of coatings overnight, are washed 2 times with cleaning solution, and 120 μ L, 5% skimmed milk power solution is added in 37 DEG C in every hole
Close 3h.It dries in hole after liquid, the Chemiluminescent plate being coated with is stored in dry aluminium foil bag by 37 DEG C of baking 1h.
(2) tenuazonic acid camel source antibody working solution: diluting 1:2000 for tenuazonic acid camel source antibody, will
Dilution is stored in 6mL brown bottle；
(3) two corresponding anti-solution of horseradish peroxidase-labeled: goat-anti camel ELIAS secondary antibody is diluted into 1:10000, by dilution
It is stored in 10mL brown bottle；
(4) tenuazonic acid standard solution: by tenuazonic acid titer be diluted to a series of concentration (0,
0.128,0.64,3.2,16,80,400,2000ng/mL), a series of dilutions are stored in respectively in 8 10mL brown bottles,
And it marks respectively；
(5) luminous substrate liquid: 2.0mg luminol, 0.8mg are dissolved in the Tris-HCl buffer (pH of 10mL to iodophenol
9.0) in, dilution is stored in 6mL brown bottle；
(6) substrate buffer solution: 20ul hydrogen peroxide (30%) is dissolved in 10ml Tris-HCl buffer (pH 7.0), will
Dilution is stored in 6mL brown bottle；
(7) concentrated cleaning solution: contain 1.0% polysorbas20 and 0.1mol/mL phosphate buffer, solution is stored in 10mL
In brown bottle, 20 times of dilutions are used.
(8) sample redissolves liquid: phosphate buffer (pH7.4PBS) KH containing 1% methanol2PO40.2g, Na2HPO4·
12H2O 2.9g, NaCl 8.5g, KCl 0.2g is added 10mL anhydrous methanol, adds distilled water to 1000mL.
(9) sample diluting liquid: phosphate buffer (pH7.4PBS) KH2PO40.2g, Na2HPO4·12H2O2.9g, NaCl
8.5g, KCl 0.2g, add distilled water to 1000mL.
Two, the assembling of tenuazonic acid chemiluminescence immune detection reagent kit
Tenuazonic acid chemiluminescence immune detection reagent kit is assembled, includes following component:
(1) it is coated with the Chemiluminescent plate of tenuazonic acid coating antigen: 1 piece；
(2) tenuazonic acid two-humped camel polyclonal antibody working solution: 1 bottle, 6mL/ bottles；
(3) two corresponding anti-solution of horseradish peroxidase-labeled: 1 bottle, 10mL/ bottles；
(4) tenuazonic acid standard solution: be respectively 0ng/mL, 0.128ng/mL, 0.64ng/mL, 3.2ng/mL,
16ng/mL, 80ng/mL, 400ng/mL, 2000ng/mL, each 1 bottle, 6mL/ bottles；
(5) sample redissolution liquid: 1 bottle, 10mL/ bottles；
(6) sample diluting liquid: 1 bottle, 10mL/ bottles；
(7) luminous substrate liquid: 1 bottle, 6mL/ bottles；
(8) substrate buffer solution: 1 bottle, 6mL/ bottles；
(9) concentrated cleaning solution, 1 bottle, 10mL/ bottles；
(10) cover board film: 2；
(11) valve bag: 6；
(12) gloves, 6；
(13) operation instructions: 1 part；
Three, application method
1, sample to be tested pre-treating method:
Samples of juice: it directly can be used to measure with sample diluting liquid dilution certain multiple.
Grain sample: taking grain sample 1g, is extracted 2 times after crushing uniformly with methanol/PBS mixed liquor, 8000rpm centrifugation
10min, the drying of supernatant liquid nitrogen, redissolving dilution certain multiple after liquid redissolves can be used to measure.
2, tenuazonic acid chemiluminescence immunoassay detects:
(1) kit is being taken out from cold storage environment, is being placed in and balances under room temperature；
(2) kit is added in 50 μ L tenuazonic acid standard solutions or testing sample solution through pre-treatment
In Chemiluminescent plate micropore, then 50 μ L tenuazonic acid two-humped camel polyclonal antibodies are added in every hole, and slight concussion mixes,
40min is incubated in 37 DEG C of environment；
(3) washing 5 times is carried out to Chemiluminescent plate micropore with cleaning solution, patted dry；
(4) the goat-anti camel secondary antibody of 100 μ L horseradish peroxidase-labeleds is added in every hole, and slight concussion mixes, 37 DEG C of environment
Middle incubation 30min；
(5) washing 5 times is carried out to Chemiluminescent plate micropore with cleaning solution, patted dry；
(6) 100 μ L luminescent solutions (mixing substrate buffer solution in equal volume with substrate solution) is added into every hole, slight concussion is mixed
It is even；The luminous value of each micropore of Chemiluminescent plate is measured at wavelength 425nm；
(7) using each standard concentration hole luminous value and zero standard hole luminous value as ordinate, with pharmaceutical standards liquid concentration
Log10 value is abscissa, canonical plotting is drawn, to calculate alternaria bacterium in sample according to the luminous value of sample to be tested
The content of ketone acid.
5 tenuazonic acid chemiluminescence immune detection reagent kit of embodiment
One, experimental method
It is added to the fruit of 20,200 and 500ng/mL tenuazonic acid respectively using the kit detection in embodiment 4
Juice sample, three repetitions of each concentration.
Two, experimental result
The results are shown in Table 2, the height of the rate of recovery of samples of juice, is able to reflect the accuracy of this method, sample recycling
Rate is high, then this method is accurate and reliable.The rate of recovery of the ic-CLEIA detection samples of juice of this method is 82.6%~106.5%,
CV is both less than 15%, and illustration method accuracy is high, can be used for detecting the TeA in actual sample.
The measurement of 2 samples of juice TIANZHU XINGNAO Capsul of table:
Claims (9)
1. a kind of artificial antigen of tenuazonic acid has as shown in formula (I),
Wherein n=1~6.
2. application of the artificial antigen described in claim 1 in detection tenuazonic acid.
3. application of the antibody that artificial antigen described in claim 1 is prepared in detection tenuazonic acid.
4. a kind of method for detecting tenuazonic acid, which is characterized in that artificial antigen carrier protein described in claim 1 is
For keyhole limpet hemocyanin as immunogene, artificial antigen carrier protein described in claim 1 is that ovalbumin is used as coating antigen, progress
The detection of tenuazonic acid.
5. according to the method described in claim 4, it is characterized in that, artificial antigen carrier protein described in claim 1 is keyhole
Hemocyanin prepares hunchbacked source antibody as immunogen immune camellid, carries out the detection of tenuazonic acid.
6. according to the method described in claim 5, it is characterized in that, artificial antigen carrier protein described in claim 1 is keyhole
Hemocyanin as immunogen immune camellid prepare antibody the following steps are included:
S1. camellid is immunized with the tenuazonic acid artificial antigen；
S2. and then it is primary in the 15th, 29,43,57 day each booster immunization respectively；
S3. at the 64th day, whole blood is acquired, collects tenuazonic acid camel source antibody.
7. according to the method described in claim 6, it is characterized in that, the method for detection tenuazonic acid is exempted from for chemiluminescence
Epidemic disease analysis method, comprising the following steps:
S1. there will be the artificial antigen of formula (I) structure as coating antigen, coating antigen will be coated in the micropore of Chemiluminescent plate,
Incubation, board-washing, closing, drying；
S2. the tenuazonic acid standard items or sample to be tested of gradient dilution are added in Chemiluminescent plate micropore, and will power
Benefit requires the 4 hunchbacked source antibody to be added in micropore of enzyme marker plate, is incubated for, board-washing；
S3. the anti-hunchbacked secondary antibody of enzyme mark is added in Chemiluminescent plate micropore, is incubated for, board-washing；
S4. luminescent solution is added in Chemiluminescent plate micropore, oscillation mixes, and measures luminous value under certain wavelength；
S5. using each standard concentration hole luminous value as ordinate, with the log of pharmaceutical standards liquid concentration10Value is abscissa, and drafting is exempted from
The standard curve of epidemic disease detection method.
S6. according to tenuazonic acid content in the standard curve quantitative analysis sample of foundation.
8. a kind of chemiluminescence immune detection reagent kit for detecting tenuazonic acid, which is characterized in that the kit contains
Have and is coated with the hunchbacked source that artificial antigen carrier protein described in claim 1 is keyhole limpet hemocyanin as immunogene and is prepared and resists
Artificial antigen carrier protein described in body and claim 1 is ovalbumin as the coated Chemiluminescent plate of coating antigen.
9. chemiluminescence immune detection reagent kit according to claim 8, which is characterized in that further include horseradish peroxidase
The secondary antibody of enzyme label, tenuazonic acid standard solution, sample redissolve liquid, sample diluting liquid, concentrated cleaning solution, luminous substrate
Liquid, substrate buffer solution, cover board film, valve bag, gloves, operation instructions.
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