CN106680516B - Detect enzyme linked immunological kit and its application of estriol - Google Patents
Detect enzyme linked immunological kit and its application of estriol Download PDFInfo
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- CN106680516B CN106680516B CN201611102613.1A CN201611102613A CN106680516B CN 106680516 B CN106680516 B CN 106680516B CN 201611102613 A CN201611102613 A CN 201611102613A CN 106680516 B CN106680516 B CN 106680516B
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- 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
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Abstract
The present invention provides a kind of enzyme linked immunological kits for detecting estriol, it includes：It is coated with the ELISA Plate of coating antigen, estriol standard solution, ELIAS secondary antibody, substrate developing solution, terminate liquid, cleaning solution, redissolves liquid, the coating antigen is estriol coupled antigen, and the ELIAS secondary antibody is the estriol secondary antibody of enzyme label.The invention also discloses a kind of methods using above-mentioned enzyme linked immunological kit detection estriol, it includes：Sample pre-treatments are carried out first, are then detected with kit, ultimate analysis testing result.Enzyme linked immunological kit provided by the invention can be used for detection animal derived food, in water quality sample estriol content, easy to operate, low-cost, high sensitivity on-site supervision and can be suitble to the screening of great amount of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technologies, and in particular to a kind of ELISA reagent for being used to detect estriol
Box, can in qualitative and quantitative analysis animal derived food, water quality estriol drug residual quantity.
Background technology
Estriol (estriol, E3) is the metabolite of estradiol and oestrone, its value of non-pregnancy is very low, mainly by liver
Dirty synthesis；The gestational period is mainly synthesized by fetal placenta, by parent blood circulation after synthesis, in liver metabolism and sulfuric acid or grape
Uronic acid combines and forms mating type E3, from urine ejection.E3 plays an important role during Fetus Intrauterine Growth is adjusted, and can shadow
Ring sensibility of the gravid uterus to oxytocins.
Animal derived food, water quality are one of mankind's important foodstuffs, rich in protein, vitamin, the calcareous and other mankind
Required nutrient has very high nutritive value.At present, there are the phenomenon that abuse of antibiotics, hormone during raising animal,
Cause to contain a certain amount of estriol hormone in animal derived food, water quality.Estriol occupies significant proportion in female hormone,
Female hormone content increases in animal derived food, water quality, easily makes one that hormone related disorders occur, such as mammary gland, ovary, preceding
Row adenoncus knurl, the relevant tumour of endocrine, inborn defect and birth defects etc., can also make baby and teen-age growth and development
Into seriously affecting.The female hormone in animal derived food is measured both at home and abroad using gas chromatography-mass spectrum (GC-MS), infrared light
The methods of spectrometry, enzyme immunoassay (EIA), radioimmunology, fluoroimmunoassay, high performance liquid chromatography.
The present invention measures the residual quantity of estriol drug in animal derived food, water quality, has inspection using enzyme-linked immunization
Survey limit is low, high specificity, easy to operate, detection speed is fast, testing cost is low, the advantages that being very easy to promote.
Invention content
Estriol drug residue in animal derived food, water quality can be detected the purpose of the present invention is to provide a kind of
Enzyme linked immunological kit, and a kind of efficient, accurate, simplicity, the qualitative and quantitative analysis side screened suitable for batch samples are provided
Method.
Kit of the present invention, it includes：It is coated with the ELISA Plate of coating antigen, estriol standard solution, ELIAS secondary antibody, bottom
Object developing solution, cleaning solution, redissolves liquid at terminate liquid, and the coating antigen is estriol coupled antigen, and the ELIAS secondary antibody is marked for enzyme
Estriol secondary antibody.
The estriol coupled antigen is to be obtained by estriol haptens with carrier protein couplet, the estriol haptens
It is to be obtained through chemical reaction, the carrier protein can be mouse haemocyanin, thyroprotein, bovine serum albumin(BSA), rabbit anteserum egg
In vain, human albumin, ovalbumin, hemocyanin or fibrinogen.
The estriol specific antibody is prepared using estriol coupled antigen as immunogene, and the estriol is special
Heterogenetic antibody can be estriol monoclonal antibody or estriol polyclonal antibody, wherein it is preferred that estriol monoclonal antibody.
The marker enzyme of the ELIAS secondary antibody extracts alkaline phosphatase for horseradish peroxidase or bacterium, wherein it is preferred that peppery
Root peroxidase；ELIAS secondary antibody is coupled to obtain by enzyme and estriol secondary antibody.
In order to be more convenient on-site supervision and great amount of samples screening, the kit further includes estriol standard solution, bottom
Object developing solution, cleaning solution, redissolves liquid at terminate liquid.
5 bottles of the estriol standard solution, concentration be respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L,
1.35μg/L。
When marker enzyme is horseradish peroxidase, the substrate developing solution is made of substrate solution A liquid and substrate solution B liquid, A
For hydrogen peroxide or urea peroxide, B liquid is o-phenylenediamine or tetramethyl benzidine, and the terminate liquid is the sulfuric acid of 1~2mol/L
Or salt solution acid buffer；When marker enzyme extracts alkaline phosphatase for bacterium, the substrate developing solution is to nitro phosphate
Buffer solution, the terminate liquid are 1~2mol/L sodium hydroxide solutions.
The cleaning solution is preferably that pH value is 7.4, contains 0.5%~1.0% Tween-20,0.01 ‰~0.03 ‰ nitrine
Change the phosphate buffer of sodium preservative, 0.1~0.3mol/L, the percentage is percent weight in volume.
The liquid that redissolves is preferably phosphate buffer of the pH value for 7.0,0.02mol/L, and the percentage is weighing body
Product percentage.
Used coating buffer solution is the carbonate that pH value is 9.6,0.05mol/L wherein in ELISA Plate preparation process
Buffer solution, confining liquid are that pH value is 7.1~7.5, the phosphate buffer containing 1%~3% casein, 0.1~0.3mol/L,
The percentage is percent weight in volume.
The preparation process of ELISA Plate is in the present invention：Coating antigen is diluted to 20 μ g/mL with coating buffer solution, is added in per hole
100 μ l, 25 DEG C are protected from light 2h or 4 DEG C of incubation overnight, and liquid in hole of inclining is washed 2 times with cleaning solution, and each 30s is patted dry, then
150~200 μ l confining liquids are added in every hole, 25 DEG C are protected from light 1~2h of incubation, and liquid pats dry in hole of inclining, and aluminium film is used after dry
Vacuum sealing preserves.
The present invention testing principle be：
This kit is using direct competive ELISA method, the pre-coated coupled antigen on ELISA Plate capillary strip, residual in sample
Pre-coated coupled antigen competes the ELIAS secondary antibody of anti-estriol on the estriol and ELISA Plate capillary strip stayed, is shown with tmb substrate
Color, sample absorbance value and the content of its contained residue estriol are negatively correlated, and compared with standard curve, are corresponded to multiplied by with it
Extension rate, you can obtain the residual quantity of estriol in sample.
The present invention also provides a kind of methods using above-mentioned enzyme linked immunological kit detection estriol, it includes step：
(1) sample pre-treatments；
(2) it is detected with kit；
(3) testing result is analyzed.
The enzyme linked immunological kit of present invention detection estriol is mainly qualitatively or quantitatively detected in sample using ELISA method
The content of estriol；Low to the pre-treatment requirement of sample, sample pretreatment process is simple, can quickly detect high-volume sample simultaneously
Product；Main agents are provided in the form of working solution, and the method for inspection is convenient and easy, have specific height, high sensitivity, accuracy
High, the features such as accuracy is high.The enzyme linked immunological kit of the present invention, simple in structure, easy to use, cheap, carrying convenience,
Detection method efficiently, it is accurate, easy, suitable for the qualitative, quantitative of batch samples screening.
Description of the drawings
Fig. 1：Estriol haptens structure synthetic route chart
Fig. 2：Kit standard curve graph
Specific embodiment
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not used to limit the scope of the invention.
The preparation of 1 reagent constituents of embodiment
1st, the preparation of estriol haptens
Estriol 1g is taken, trifluoroacetic acid 20ml is added to dissolve, adds methenamine 1.46g, is heated, back flow reaction 3h is cooled to
Room temperature adds 1mol/L dilute hydrochloric acid 30ml, stirs 2h.Stopping reaction, add water, sodium hydroxide adjusting pH value to 7 adds ethyl acetate,
Extraction, washing, anhydrous sodium sulfate drying are evaporated, and upper silicagel column, petrol ether/ethyl acetate (3/1, v/v) obtains haptens product
0.84g, yield 75.57%.
2nd, the preparation of antigen
It is prepared by immunogene --- and estriol haptens obtains immunogene with bovine serum albumin(BSA) (BSA) coupling.
12mg aldehyde radical estriol haptens is taken, is dissolved in 0.3mL DMF, obtains A liquid.BSA 50mg are weighed, it is fully molten
Reaction solution A is slowly dropped in protein solution by solution dropwise in 4mL CB (PH 9.5), and is stirred at room temperature for 24 hours.With
4 DEG C of dialysis 3d of 0.02mol/L PBS, change liquid 3 times, obtain immunizing antigen, dispense, saved backup in -20 DEG C daily.
It is prepared by coating antigen --- and estriol haptens obtains immunogene with ovalbumin (OVA) coupling.
5.3mg aldehyde radical estriol haptens is taken, 0.2mL ethyl alcohol is dissolved in, obtains A liquid.OVA50mg is weighed, is allowed to abundant
It is dissolved in 4mL CB (PH 9.5), A liquid is slowly dropped in protein solution dropwise, and stir 4h at room temperature；With
4 DEG C of dialysis 3d of 0.01mol/L PBS, change 3 dialyzates, obtain envelope antigen, dispense, saved backup in -20 DEG C daily.3、
The preparation of estriol monoclonal antibody
Animal immune：The immunogene that above-mentioned steps obtain is injected into Balb/c Mice Bodies, immunizing dose is 150 μ g/
Only, it is made to generate antiserum.
Cell fusion and cloning：After mice serum measurement result is higher, its splenocyte is taken, by 8:1 (quantitative proportion) compares
Example is merged with SP2/0 myeloma cell, and cell supernatant is measured using indirect competitive ELISA, screens positive hole.Using limited dilute
Interpretation of the law carries out cloning to positive hole, until obtaining the hybridoma cell strain of secretion estriol monoclonal antibody.
Cell cryopreservation and recovery：Monoclonal hybridoma strain is made 1 × 10 with frozen stock solution6The cell suspension of a/mL,
It is preserved for a long time in liquid nitrogen.Cryopreservation tube is taken out during recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, after centrifugation removal frozen stock solution, is moved
Enter to cultivate culture in glassware.
The production and purifying of monoclonal antibody：Balb/c mouse peritoneals are injected into sterilizing paraffin oil 0.5mL/ only, abdomen after 7 days
The stable monoclonal hybridoma strain 5 × 10 of chamber injection5A/only, acquire ascites after 7 days.With octanoic acid-saturated ammonium sulfate method into
Row ascites purifies, -20 DEG C of preservations.
4th, the preparation of ELIAS secondary antibody
Estriol secondary antibody and horseradish peroxidase (HRP) are coupled to obtain.
5th, the preparation of ELISA Plate
Coating antigen is diluted to 20 μ g/mL with coating buffer solution, 100 μ l are added in per hole, 25 DEG C are protected from light incubation 2h, hole of inclining
Middle liquid is washed 2 times with cleaning solution, and each 30s is patted dry, and 200 μ l confining liquids is then added in every hole, 25 DEG C are protected from light incubation
2h, liquid pats dry in hole of inclining, and is preserved after dry with aluminium film vacuum sealing.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of estriol
The enzyme linked immunological kit of detection estriol is set up, makes that it includes following components：
(1) it is coated with the ELISA Plate of estriol coupled antigen；
(2) 5 bottles of estriol standard solution, concentration be respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L,
1.35μg/L；
(3) with the estriol secondary antibody of horseradish peroxidase-labeled；
(4) substrate developing solution is made of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(5) terminate liquid is 2mol/L sulfuric acid；
(6) it is 7.4 that cleaning solution, which is pH value, is prevented containing 0.5%~1.0% Tween-20,0.01 ‰~0.03 ‰ sodium azide
Rotten agent, the phosphate buffer of 0.1~0.3mol/L, the percentage are percent weight in volume；
(7) redissolve liquid be pH value be 7.0, the phosphate buffer of 0.02mol/L, the percentage be weight volume basis
Than.
The detection of estriol in 3 animal derived food of embodiment, water quality
1st, sample pre-treatments
Animal tissue (chicken, pork, the flesh of fish, shrimp) Sample pretreatment method：With homogenizer homogeneous structure sample；It weighs
In sample to 50ml polystyrene centrifuge tubes after 1.0 ± 0.05g homogeneous, 5ml ethyl acetate is added in, is vibrated with oscillator
1min, 3000g room temperature (20-25 DEG C/68-77 ℉) centrifuge 5min；1ml upper organic phases are pipetted to 10ml clean dried glass tubes
In, the drying addition 1ml n-hexanes under 50-60 DEG C of (122-140 ℉) water-bath nitrogen stream, 1ml redissolves working solution, with vortex instrument whirlpool
Dynamic 1min, mixing, 3000g room temperatures (20-25 DEG C/68-77 ℉) centrifugation 3min；Upper organic phase is removed, removes 50 μ l of layer water phase
For analyzing.
Milk sample：It pipettes in 1ml milk sample to 50ml polystyrene centrifuge tubes, 5ml ethyl acetate is added in, with oscillation
Device vibrates 1min, 3000g room temperatures (20-25 DEG C/68-77 ℉) centrifugation 5min；1ml upper organic phases are pipetted to 10ml clean drieds
In glass tube, drying addition 1ml n-hexanes, 1ml redissolve working solution, use whirlpool under 50-60 DEG C of (122-140 ℉) water-bath nitrogen stream
Revolve instrument whirling motion 1min, mixing, 3000g room temperatures (20-25 DEG C/68-77 ℉) centrifugation 3min；Upper organic phase is removed, takes lower water
50 μ l of phase are used to analyze.
Water quality sample：It adds in redissolution working solution to be handled, takes 50 μ l for analyzing.
2nd, it is detected with kit
It adds in 50 μ l to corresponding micropore of standard items/sample, antibody working solution and ELIAS secondary antibody concentrate is pressed 10:1
Volume ratio mixing (such as 1ml antibody working solution adds in 100 μ l ELIAS secondary antibodies concentrates), adds antibody working solution and ELIAS secondary antibody dense
The 50 μ l/ holes of mixed liquor of contracting liquid, gently vibrate mixing, are reacted in 25 DEG C of (77 ℉) light protected environments of cover board membrane cover plate postposition
30min；Cover board film carefully is opened, liquid in hole is dried, adds in 250 μ l/ holes of wash operating solution, fully washing 4-5 times, every time
10s is spaced, cleaning solution in board falling hole is sprinkled, pats dry that (bubble not being eliminated after patting dry can use original pipette tips with blotting paper
It pokes)；50 μ l/ holes of substrate solution A liquid are added in, 50 μ l/ holes of substrate solution B liquid is added, mixing is gently vibrated, after cover board membrane cover plate
It puts in 25 DEG C of (77 ℉) light protected environments and reacts 15min；Add in terminate liquid 50 μ l/ holes, gently vibrate mixing, setting microplate reader in
(it is recommended that being detected with dual wavelength 450/630nm, data are please run through in 5min) at 450nm, measured per hole OD values.
3rd, Analysis of test results
The percentage absorptance of standard items or sample be equal to the absorbance value of standard items or sample average value (diplopore) divided by
The average value of the absorbance value of first standard items (0 standard) multiplied by with 100%, obtains the percentage extinction of standard items or sample
Angle value.Using standard items percentage absorptance as ordinate, using the logarithm of estriol standard concentration (μ g/L) as abscissa, mark is drawn
Directrix curve figure.The percentage absorptance of sample is substituted into standard curve, the concentration corresponding to sample is read from standard curve, is multiplied
It is the actual concentrations of estriol in sample with its corresponding extension rate.
The determining experiment of 4 estriol technical parameter of embodiment
1st, kit sensitivity and detection limit
Conventionally assay kit sensitivity, ranging from 0~1.35 the μ g/L, IC of standard curve50(50% inhibits
Concentration) domain of walker be 0.16~0.3 μ g/L；20 parts of samples are detected, are found from standard curve corresponding to each percentage
The concentration of absorbance value represents detection limit, the results show that this method with the average value of 20 parts of concentration of specimens plus 3 times of standard deviations
Detection limit to animal derived food, water quality is 0.25 μ g/kg.
2nd, sample preci-sion and accuracy is tested
Using the rate of recovery as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication
(RSD%) as precision evaluation index.Calculation formula is：The rate of recovery (%)=actual measured value/theoretical value × 100%,
The addition concentration of middle theoretical value sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD are standard deviation, and X is
The average value of determination data.
By 0.25 μ g/kg, 0.5 two concentration of μ g/kg estriol respectively to chicken, pork, the flesh of fish, shrimp, milk, water
Quality sample is added recycling and measures, each sample do 4 it is parallel, be measured with three batches of different reagents, calculate the flat of sample
The equal rate of recovery and precision result see the table below.
1 precision of table and accuracy test
With 0.25 μ g/kg, 0.5 two concentration of μ g/kg estriol respectively to chicken, pork, the flesh of fish, shrimp, milk, water
Matter is added, chicken, pork, the flesh of fish, shrimp average recovery rate between 81.1%~113.5%, milk, water quality it is flat
The equal rate of recovery is between 73.8%~105.4%；Relative standard deviation is respectively less than 10% in batch, and relative standard deviation is small between batch
In 14%.3rd, stabilization of kit is tested
Kit preservation condition is 2~8 DEG C, by the measure of 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, estriol add actual measured value within normal range (NR).Consider during transport and use, have
Improper preservation condition occurs, and kit is placed 7 days under 37 DEG C of preservation conditions, carries out accelerated aging tests, the results showed that
The kit indices comply fully with requirement.It is happened in view of kit freezing, it is cold that kit is put into -20 DEG C of refrigerators
Freeze 7 days, measurement result also indicates that kit indices are completely normal.It can show that kit can be at 2~8 DEG C from result above
At least preserve 12 months or more.
Claims (2)
1. a kind of estriol enzyme-linked immunologic detecting kit, it is characterised in that：ELISA Plate, estriol including being coated with coating antigen
Standard solution, substrate developing solution, terminate liquid, cleaning solution, redissolves liquid at ELIAS secondary antibody, and the coating antigen is anti-for estriol coupling
Original, the ELIAS secondary antibody are the estriol secondary antibody of enzyme label, and the estriol coupled antigen is estriol haptens and carrier egg
White conjugate, the preparation method of the estriol haptens mainly include the following steps：
Estriol 1g is taken, trifluoroacetic acid 20ml is added to dissolve, adds methenamine 1.46g, is heated, back flow reaction 3h is cooled to room temperature,
Add 1mol/L dilute hydrochloric acid 30ml, stir 2h；Stopping reaction, add water, sodium hydroxide adjusting pH value to 7 adds ethyl acetate, extracts,
Washing, anhydrous sodium sulfate drying are evaporated, upper silicagel column, and the volume ratio of petroleum ether and ethyl acetate is 3:1, obtain haptens product
0.84g, yield 75.57%；
The molecular structure of the estriol haptens is：
2. the remaining method of estriol in estriol enzyme-linked immunologic detecting kit detection sample as described in claim 1, main
Step is wanted to include：
1) sample to be tested is subjected to pre-treatment, obtains sample to be tested solution；
2) sample to be tested solution is detected with estriol enzyme-linked immunologic detecting kit described in claim 1；
3) testing result is analyzed.
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Citations (3)
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|---|---|---|---|---|
| CN1227349A (en) * | 1998-11-25 | 1999-09-01 | 郭大智 | Enzyme linked immunoassay test kit for detecting estriol |
| CN101324590A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Immune analysis method for determining estriol content |
| CN105838680A (en) * | 2016-05-17 | 2016-08-10 | 江南大学 | Estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100737186B1 (en) * | 2005-10-27 | 2007-07-10 | 한국해양연구원 | Biosensor for Detection of estriol Having Anti-estriol Antiserum and Detecting Process of estriol |
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2016
- 2016-12-05 CN CN201611102613.1A patent/CN106680516B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1227349A (en) * | 1998-11-25 | 1999-09-01 | 郭大智 | Enzyme linked immunoassay test kit for detecting estriol |
| CN101324590A (en) * | 2007-06-13 | 2008-12-17 | 清华大学 | Immune analysis method for determining estriol content |
| CN105838680A (en) * | 2016-05-17 | 2016-08-10 | 江南大学 | Estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 and application thereof |
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| Title |
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| 抗雌三醇单克隆抗体的研制;秦鸿志 等;《生殖与避孕》;19891231;第9卷(第1期);第57-59页 * |
| 雌三醇单克隆抗体的制备与表征;王永成 等;《北京大学学报(自然科学版)》;20020731;第38卷(第4期);第459-465页 * |
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