CN108107223A - Detect method and its enzyme linked immunological kit used of estradiol and/or oestradiol benzoate - Google Patents
Detect method and its enzyme linked immunological kit used of estradiol and/or oestradiol benzoate Download PDFInfo
- Publication number
- CN108107223A CN108107223A CN201611050507.3A CN201611050507A CN108107223A CN 108107223 A CN108107223 A CN 108107223A CN 201611050507 A CN201611050507 A CN 201611050507A CN 108107223 A CN108107223 A CN 108107223A
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- Prior art keywords
- estradiol
- carrier protein
- antigen
- antibody
- calf serum
- Prior art date
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Classifications
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Abstract
The invention discloses detection estradiol and/or method and its enzyme linked immunological kits used of oestradiol benzoate.The method of present invention detection estradiol and/or oestradiol benzoate, using the estradiol and/or the content of oestradiol benzoate detected containing the enzyme linked immunological kit of estradiol coating antigen and estradiol specific antibody in sample to be tested.Wherein, the estradiol coating antigen is the conjugate of estradiol haptens and carrier protein 1；The estradiol specific antibody is the polyclonal antibody or monoclonal antibody obtained as immunogene using the conjugate of the estradiol haptens and carrier protein 2；The carrier protein 1 and the carrier protein 2 are identical or different carrier proteins.The IC of the method for present invention detection estradiol50For 0.129ng/ml, IC20‑IC80The range of linearity is 0.0279ng/ml 0.597ng/ml.
Description
Technical field
The present invention relates to methods that estradiol and/or oestradiol benzoate are detected in enzyme linked immunosorbent detection field and its used
Enzyme linked immunological kit.
Background technology
Oestrone, estradiol, estriol are existing natural estrogens in animal body, wherein estradiol (Estradiol, E2)
One of natural sex hormone most active as animal reproduction correlation, the formation with the female immature animal sexual organ of promotion,
The functions such as the development and promotion dam heat of secondary sex characters, and promoting body growth and improving production performance etc. effect
Significantly.Estradiol and its derivative (oestradiol benzoate) are widely used in Animal husbandry production, however if violation medication
Or animal food is not produced according to the off-drug period, and it be easy to cause animal products -- the residual of estradiol in meat, egg, milk, and then
Health of people is endangered by food chain.The health such as children's sexual precocity, women breast cancer in recent years, the increase of mankind spermatozoon abnormal rate are asked
It inscribes in rising trend, is remained to a certain extent with the growth promotions such as estradiol in animal derived food, production sex hormone
It closes.The food-safety problem that the hormone medicines such as estradiol induce is more sharp, to ensure that animal food is safe and healthy, China
The Ministry of Agriculture announces No. 176 file clear stipulaties and forbids addition estradiol, diethylstilbestrol, benzoic acid female in feed and drinking water
The hormone medicines such as glycol；And announce No. 193 files in the Ministry of Agriculture《The veterinary drug of food animal disabling and other compounds are clear
It is single》Middle regulation, all edible animals are forbidden using oestradiol benzoate (mark residue is estradiol), Methyltestosterone, benzene
The sex hormone drugs such as testobolin[5], therefore detect the weight that the estradiol residual in edibility livestock products is to ensure that food security
Want measure.
Hormone medicine method for detecting residue mainly has high performance liquid chromatography (HPLC), liquid chromatography-tandem matter at present
The analysis methods such as spectrometry (LC/MS/MS) and gas chromatography-mass spectrography method (GC-MS).Although above-mentioned instrumental method sensitivity
Height, but accurate instrument is needed, it is difficult to reach accurate testing goal without extraction repeatedly and purification process, therefore is producing
It is difficult to popularize in practice.
The content of the invention
The technical problems to be solved by the invention are how quick, sensitive, inexpensive, specific, it is female to detect with high throughput
Glycol and/or oestradiol benzoate.
In order to solve the above technical problems, the present invention provides the examinations of the enzyme linked immunological of detection estradiol or oestradiol benzoate
Agent box.
The enzyme linked immunological kit of detection estradiol or oestradiol benzoate provided by the present invention, is coated with including estradiol
Former and estradiol specific antibody；The estradiol coating antigen is the conjugate of estradiol haptens and carrier protein 1；It is described female
Glycol specific antibody is the polyclonal antibody obtained using the conjugate of the estradiol haptens and carrier protein 2 as immunogene
Or monoclonal antibody；The carrier protein 1 and the carrier protein 2 are identical or different carrier proteins；
The estradiol haptens is the compound of formula 1：
In above-mentioned enzyme linked immunological kit, the estradiol coating antigen and the estradiol half are prepared using carbodlimide method
The conjugate of antigen and carrier protein 2.
In above-mentioned enzyme linked immunological kit, the coupling ratio of the estradiol haptens and the conjugate of carrier protein 2 is
11.8:1。
In above-mentioned enzyme linked immunological kit, the carrier protein 1 and the carrier protein 2 can be bovine serum albumin(BSA), blood
Azurin, human serum albumins, ovalbumin, mouse serum albumin, thyroglobulin or albumin rabbit serum；Specifically,
The carrier protein 1 can be chicken ovalbumin, and the carrier protein 2 can be bovine serum albumin(BSA).
In above-mentioned enzyme linked immunological kit, the estradiol specific antibody can be rabbit source, mouse source, Ma Yuan, Yang Yuan, pig
Source, rabbit source or cavy source antibody.The polyclonal antibody concretely rabbit polyclonal antibody.
Above-mentioned enzyme linked immunological kit may also include carry out enzyme linked immunosorbent detection needed for coating buffer solution, cleaning solution, envelope
Close liquid, secondary antibody dilution and/or estradiol standard solution.
In above-mentioned enzyme linked immunological kit, the confining liquid contains the calf serum that volumn concentration is 10%.
In above-mentioned enzyme linked immunological kit, the secondary antibody dilution contains the calf serum that volumn concentration is 5%.
Above-mentioned enzyme linked immunological kit may also include to fix the solid phase carrier of the estradiol coating antigen.
In above-mentioned enzyme linked immunological kit, can as the solid phase carrier substance it is very much, as polystyrene, cellulose,
Polyacrylamide, polyethylene, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc..The form of the solid phase carrier
Can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc..
In order to solve the above technical problems, the method the present invention provides detection estradiol and/or oestradiol benzoate.
The method of detection estradiol and/or oestradiol benzoate provided by the present invention, using above-mentioned enzyme linked immunological
The content of estradiol and/or oestradiol benzoate in kit detection sample to be tested.
The above method includes：
1) antigen coat：The estradiol coating antigen is fixed on the surface of solid phase carriers, forms solid phase antigen；
2) wash：It is washed with cleaning solution；
3) close：It is closed with confining liquid；
4) sample to be tested is added：The estradiol specific antibody and sample to be tested are added in, reacts 1h at 4 DEG C；
5) ELIAS secondary antibody is added：The ELIAS secondary antibody with secondary antibody diluted is added in, is reacted；
6) develop the color：Carry out chromogenic reaction；
7) reaction is terminated, measures the content of the estradiol and/or oestradiol benzoate in sample to be tested.
In the above method, in the antigen coat, the estradiol coating antigen is carried with the concentration coating solid phase of 0.9 μ g/ml
Body.
In the above method, the confining liquid contains the calf serum that volumn concentration is 10%.
In the above method, the secondary antibody dilution contains the calf serum that volumn concentration is 5%.
In the above method, the chromogenic reaction time is 9 minutes.
In the above method, the sample to be tested be in vitro sample, such as animal food.
The application of the above method or above-mentioned enzyme linked immunological kit in detection estradiol and/or oestradiol benzoate also belongs to
In protection scope of the present invention.
The present invention by alkylation reaction using bromoacetic acid introduce active group carboxyl by estradiol (E2) transform as estradiol-
3- carboxymethyl esters (E2-3-CME) synthesize artificial antigen using carbodlimide method coupling carrier albumen (BSA or OVA), are immunized real
Test the estradiol polyclonal antibody that rabbit prepares high-titer, high specificity.Indirect competitive ELISA (icELISA) analog is handed over
Pitch reaction test the result shows that, the prepared estradiol polyclonal antibody obtained of the present invention and estriol, quinestrol, valeric acid female two
Alcohol, diethylstilbestrol, the cross reacting rate of nonyl phenol be respectively less than 0.1%, and intersects with oestradiol benzoate, oestrone, ethinyloestradiol
Reactivity is respectively up to 150%, 3.11%, 1.52%.By many reaction conditions in optimizing reaction system, as envelope antigen is dense
The reaction conditions such as degree, cleaning solution ionic strength, primary antibody reaction temperature and substrate TMB developing times, are obtained using prepared by the present invention
The estradiol polyclonal antibody and estradiol coating antigen obtained establishes indirect competitive ELISA method (icELISA), IC50For
0.129ng/ml, IC20-IC80The range of linearity is 0.0279ng/ml-0.597ng/ml.The detection estradiol and/or benzene of the present invention
The method of formic acid estradiol can not only mass detection sample, and with it is easy to operate, cheap, detection is rapid, specificity
By force, the advantages that high-throughput, the present invention are applied to livestock products estradiol residue detection.
Description of the drawings
Fig. 1 is estradiol haptens transformation process.
Fig. 2 is coupled BSA or OVA for carbodlimide method and synthesizes artificial antigen.
Fig. 3 transforms product mass spectra qualification figure for estradiol haptens.
Fig. 4 identifies for artificial antigen BSA-E2 UV scannings.Wherein, three curves from top to bottom be followed successively by E2, BSA and
BSA-E2。
Fig. 5 identifies for artificial antigen OVA-E2 UV scannings.
Wherein, three curves from top to bottom are followed successively by E2, OVA and OVA-E2.
The MALDI-TOF that Fig. 6 is artificial antigen BSA-E2 detects mass spectrogram.
Wherein A is BSA, B BSA-E2.
Fig. 7 is the standard curve that icELISA measures estradiol.
Wherein, abscissa is estradiol solution concentration (ng/ml).
Specific embodiment
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.Experimental method in following embodiments unless otherwise specified, is
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, indirect competitive ELISA method detection estradiol
1. experiment material
1.1 instrument：Thermo companies of U.S. Multiskan GO type all-wave lengths microplate reader, Eppendorf at a high speed freeze from
Scheming (model 5810R), Thermo companies CO2gas incubator etc..
1.2 reagent：The standard items such as estradiol, estriol, Estradiol Valerate, oestradiol benzoate are purchased from Chinese drug biology
Product examines and determine institute；The standard items such as oestrone, diethylstilbestrol, quinestrol are purchased from National Institute for Food and Drugs Control；Ethinyloestradiol, nonyl
Phenol is purchased from Dr.Ehrenstorfer GmbH companies of Germany；Bovine serum albumin(BSA) (BSA), chicken ovalbumin (OVA), N- hydroxyl ambers
Amber acid imide (NHS), 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine (EDC), N, N- dicyclohexylcarbodiimides
(DCC), dimethyl sulfoxide (DMSO) (DMSO), Freund's complete adjuvant (FCA) and incomplete Freund's adjuvant (FICA), casein
(casein), Proclin300, Triton X-100 and Tween-20 are purchased from Sigma companies；HRP mark goat anti-rabbit igg purchases
From Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；N, N-dimethylformamide (DMF), NaH2PO4·2H2O、Na2HPO4·
12H2O、NaCl、KCl、KH2PO4、Na2SO4、Na2HCO3、NaHCO3, dichloromethane, methanol and sucrose is purchased from Chinese medicines group chemistry
Reagent Co., Ltd；Calf serum is purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.；TMB one-components developing solution is purchased from north
Capital Solarbio bio tech ltd.
Related solution in the present embodiment is as follows：
Concentration is 0.01M, and pH value is the preparation of 7.4 PBS buffer solution：8.5g NaCl、0.2g KCl、2.9g
Na2HPO4·12H2O、0.59g NaH2PO4·2H2O, 1L deionized water.
It is coated with buffer solution：The sodium carbonate-bicarbonate buffer solution (pH9.6) of 0.05mol/L, solvent are water, solute and its
Concentration is as follows：Na2CO31.59g/L and NaHCO3 2.93g/L。
Cleaning solution:Three kinds of cleaning solutions involved in following embodiments are respectively designated as 0.1% tween cleaning solution, 0.5% tween
Cleaning solution and 1.0% tween cleaning solution.The content for differing only in tween of these three cleaning solutions.Every 1 liter of three kinds of cleaning solution are equal
It prepares as follows：It is 0.01M in concentration, adds in polysorbas20 and sodium azide in the PBS buffer solution that pH value is 7.4, obtain
To three kinds of cleaning solutions.In 0.1% tween cleaning solution, 0.5% tween cleaning solution and 1.0% tween cleaning solution, sodium azide contains
Amount is 5g/L, and the content of polysorbas20 is respectively 1mL/L, 5mL/L and 10mL/L.
Concentration is 0.02M, and pH value is the preparation of 7.4 PBS buffer solution：17g NaCl、0.4g KCl、5.8g
Na2HPO4·12H2O、1.18g NaH2PO4·2H2O, 1L deionized water.
Confining liquid：It is 0.02M in concentration, sucrose, casein and calf serum is added in the PBS buffer solution that pH value is 7.4,
Three kinds of different confining liquids of calf serum content are obtained, are respectively designated as 10% calf serum confining liquid, the closing of 5% calf serum
Liquid and 1% calf serum confining liquid.The content of sucrose is 5g/100mL in these three confining liquids, and the content of casein is
0.25g/100mL, the content of calf serum is different, and calf serum content is 10% (volume hundred in 10% calf serum confining liquid
Point content), calf serum content is 5% (volumn concentration) in 5% calf serum confining liquid, 1% calf serum confining liquid
Middle calf serum content is 1% (volumn concentration).
Secondary antibody dilution：It is 0.02M in concentration, pH value is to add in calf serum in 7.4 PBS buffer solution, obtains calf
Five kinds of different secondary antibody dilutions of serum content, are respectively designated as 1% calf serum secondary antibody dilution, 3% calf serum secondary antibody
Dilution, 5% calf serum secondary antibody dilution, 7% calf serum secondary antibody dilution and 10% calf serum secondary antibody dilution.
In 1% calf serum secondary antibody dilution calf serum content be 1% (volumn concentration), 3% calf serum secondary antibody dilution
Middle calf serum content is 3% (volumn concentration), and calf serum content is 5% (body in 5% calf serum secondary antibody dilution
Product percentage composition), calf serum content is 7% (volumn concentration) in 7% calf serum secondary antibody dilution, 10% small ox blood
Calf serum content is 10% (volumn concentration) in clear secondary antibody dilution.
1.3 experimental animals adult Japan large ear rabbit is purchased from Wenzhou Medical University's Experimental Animal Center.
2. test method
The preparation and identification of 2.1 artificial antigens (estradiol coating antigen and estradiol immunogene)
This experiment is prepared for estradiol coating antigen OVA-E2 and estradiol immunogene BSA-E2.
Estradiol coating antigen OVA-E2 is the conjugate (coupling in Fig. 2 of estradiol haptens and 1-OVA of carrier protein
Product)；Estradiol immunogene BSA-E2 is the conjugate (idol in Fig. 2 of the estradiol haptens and 2-BSA of carrier protein
Co-product)；
The estradiol haptens is the compound (estradiol -3- carboxymethyl esters (E2-3-CME)) of formula 1：
Specific method is as follows：
2.1.1 transformation estradiol obtains estradiol haptens
The inactive group of estradiol itself does not possess coupling condition, it is therefore desirable to carry out molecular modification.Utilize alkylation reaction
Estradiol molecules are introduced by pendant carboxylic group by bromoacetic acid, structural modification process is as shown in Figure 1.Specific method is as follows：This experiment
Using estradiol and the nucleophilic substitution of bromoacetic acid, and pendant carboxylic group is introduced, structural modification process is as shown in Figure 1.Specific side
Method is as follows：
(1) estradiol (1eq 1g) and sodium hydroxide (18eq 2.65g) are dissolved in dimethyl sulfoxide (100ml), are stirred at 30 DEG C
It mixes to dissolving；
(2) bromoacetic acid (2eq 1g) is added in reaction solution, is reacted overnight at 40 DEG C；
(3) reaction solution is poured into ice water (100ml), dilute hydrochloric acid tune pH to 6, ethyl acetate (common 100ml three times) extraction 3
It is secondary；
(4) extract liquor is washed 1 time with saturated salt solution (50ml), anhydrous sodium sulfate drying；
(5) after ethyl acetate layer is evaporated, with column chromatography (dichloromethane:Methanol=20:1) separate to get to female two
Alcohol transformation product (E2 transforms product).
Transform product utilization mass spectrography detection correctional effect.Mass Spectrometric Identification is carried out to transformation product, the results are shown in Figure 3.
E2 molecular weight is 272, and molecular weight becomes 330 after introducing pendant carboxylic group, along with Na+ molecular weight reaches 353, therefore transforms product
Molecular weight for 353, from scanning of the mass spectrum result, 353 molecular ion peak of appearance is target product peak.Show female two
Alcohol haptens, which is transformed, successfully, transforms the structure of product as the estradiol haptens shown in above-mentioned formula 1, available for next step carrier egg
White coupling.
2.1.2 carbodlimide method synthesis artificial antigen estradiol coating antigen OVA-E2 and estradiol immunogene BSA-E2
Respectively using two kinds of carbodiimides of EDC and DCC as dehydration agent, using NHS as linker (spacer) and carrier egg
(BSA/OVA) coupling synthesis estradiol artificial antigen, coupling process are as shown in Figure 2 in vain.Operating procedure is as follows：
(1) 15mg estradiol transformation product is weighed to be dissolved in 2ml DMF；
(2) 15mg NHS and 15mg EDC are added in thereto, and reaction 2h is stirred at room temperature；
(3) it (is 0.01M by 5.6ml concentration, the PBS that pH value is 7.4 is buffered 40mg BSA to be dissolved in 6.0ml lysates
Liquid and 0.4ml DMF compositions)；
(4) solution in (2) is slowly added drop-wise in (3) dropwise, treats drug and albumen after mixing, ice-water bath stirs
Mix 4h；
(5) using concentration as 0.01M, the PBS buffer solution that pH value is 7.4 is dialyzate, is dialysed under the conditions of 4 DEG C, small every 8
When change the liquid once, dialyse 72h；
(6) dialysis finishes, and 6 000rpm are centrifuged off sediment, and supernatant is taken to dispense, and in -20 DEG C of preservations, obtains estradiol
Immunogene BSA-E2.
(7) E2 coupling OVA albumen is prepared in the same fashion and obtains estradiol coating antigen OVA-E2, and it is former to make detection.
2.1.3 the analysis and identification of artificial antigen
(1) coupling of SDS-PAGE protein electrophoresis preliminary analysis coupled products is utilized using BSA and OVA as control respectively
Effect；Using ultraviolet spectra absorption process UV scanning in 200-340nm wave-length coverages, abosrption spectrogram is imitated for analyzing coupling
Fruit；Purity and structural analysis are carried out with infrared spectrum and mass spectrography；10mM PBS (PH=7.4) are set up as blank pair
According to coupled product progress photometric measurement, according to formula calculating protein concentration.
Protein concentration (mg/ml)=(1.55 × OD280nm-0.757×OD260nm)×(10/0.5)
The artificial antigen of uv scan method Preliminary Identification synthesis, comparison vehicle protein B SA (OVA) coupled product are maximum
Absworption peak generates offset, and estradiol has maximal ultraviolet absorption peak in 230nm, and coupling BSA/OVA maximum absorption bands are offset to
210nm (Figure 4 and 5) illustrates estradiol haptens and carrier protein couplet success, obtains estradiol coating antigen OVA-E2 and female two
Alcohol immunogene BSA-E2, structure are the coupled product in Fig. 2.
(2) Matrix-assisted laser desorption ionization method (Matrix-Assisted Laser are used
Desorption/Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS) to synthesis
Artificial antigen estradiol coating antigen OVA-E2 and estradiol immunogene BSA-E2 are measured, and judge whether haptens is even with albumen
Work(is unified into, and calculates coupling ratio.E2 artificial antigens (BSA-E2) MALDI-TOF detections are by Biology College, Chinese Agriculture Univ.'s agricultural
Biotechnology National Key Laboratory measures, and testing result is as shown in fig. 6, BSA molecule measurings are set to 65809.07, artificial antigen
It is 11.8 that molecule measuring, which is set to the coupling ratio of BSA and estradiol haptens in 69709.26, BSA-E2,:1.Illustrate that estradiol half is anti-
Former and OVA is also coupled success.
The preparation of 2.2 estradiol specific antibodies
The estradiol specific antibody is the polyclonal antibody that the BSA-E2 prepared using 2.1 is obtained as immunogen immune rabbit.
Specific method is as follows：
Using Japan large ear rabbit of growing up as immune animal, immune preceding ear edge vein exploitating blood simultaneously separates serum as negative blood
Clearly, the BSA-E2 prepared using 2.1 is as immunizing antigen, by the immunogene of corresponding dosage and equivalent adjuvant emulsion, immunization ways with
And immune programme is as shown in table 1, immune (head exempts from) carries out second immune (two exempt from) for the first time after 30 days, afterwards every 30 days into
Row primary immune response until the 8th time immune (eight exempt from), the 8th time it is 30 days immune after carry out first time auricular vein booster immunization (the
Once strengthen), carry out within 14 days after first time booster immunization second auricular vein booster immunization (second strengthen), second plus
Strong be immunized carries out third time auricular vein booster immunization (third time is strengthened) for latter 14 days, 14 days after third time booster immunization, heart
A large amount of blood samplings obtain serum and obtain estradiol specific antibody, the serum hereinafter referred to as positive serum.
Serum antibody titer is detected using indirect elisa method.Wherein, the first immunologic adjuvant exempted from is FCA, and two exempt to eight to exempt from
Immunologic adjuvant is FICA, and the immunologic adjuvant of reinforcement to third time reinforcement is physiological saline (NS) for the first time.
1 immunization ways of table and immune programme
NS:Physiological saline.Immunizing dose is in terms of the quality for the immunogene that every rabbit is injected.
IcELISA experimental methods before 2.3 optimizations
2.3.1 antigen coat concentration and serum dilution are groped
Chessboard method determines indirect elisa method antigen coat concentration and serum diluted concentration.With coating buffer solution dilution 2.1
Estradiol coating antigen OVA-E2 (original liquid concentration 1.8mg/ml) to 500 times, 1 000 times, 1 500 times, 2 000 times, 2 500
Again, 3 000 times, 5 000 times and 10 000 times, with its 8 dilution factors difference coated elisa plates.It will with the dilution of coating buffer solution
2.2 positive serums prepared are diluted to 200,000 times, 300,000 times, 400,000 times, 500,000 times of 4 dilution factors, while use rabbit feminine gender blood
(1 000 times dilution) as control clearly, more than be respectively coated under the conditions of concentration and different positive serum dilution factors, measure positive serum
(PC, positive control) OD values/negative serum (NC, negative control) OD values, with PC/NC ratios most your writing
It determines most preferably to be coated with concentration and serum diluted concentration for decision condition.
Specific method is as follows：
(1) it is coated with:With coating buffer solution dilution 2.1 estradiol coating antigen OVA-E2 (original liquid concentration 1.8mg/ml) extremely
The concentration of estradiol coating antigen OVA-E2 is 0.9 μ g/ml, obtains coating original solution, and experimental port is coated with the coating original solution,
100 μ L/ holes add to ELISA Plate, 37 DEG C of incubation 2h.
(2) wash：Incline hole endoperidium original solution, is washed 3 times with 0.5% tween cleaning solution, each 3min；It pats dry.
(3) close：Add in 10% calf serum confining liquid, 100ul/ holes, 37 DEG C of incubation 1h.
(4) it is loaded：It is added in by the test serum being serially diluted in enzyme mark hole, 100ul/ holes, 37 DEG C of incubation 1h.
(5) ELIAS secondary antibody is added：It adds in and carries out 1 with 10% calf serum secondary antibody dilution:5 000 diluted HRP marks sheep
Anti-rabbit IgG, 100ul/ holes, 37 DEG C of 50min.
(6) develop the color：TMB is added in, 10min is reacted in 100ul/ holes.
(7) terminate：It adds in 1M hydrochloric acid and terminates reaction, 100ul/ holes.
(8) measure：Each hole OD is read with microplate reader450nmNumerical value.
Checkerboard titration method determines the most suitable working concentration of antigen and serum.The results are shown in Table 2, and coating antigen (is examined through OD280nm
Survey concentration is 1.8mg/ml) 1：PC/NC ratios are maximum when 2 000 dilutions and serum are diluted to 300,000 times, determine coating antigen most
Good coating concentration is 2 000 times, and serum working concentration is dilution 1：300 000.
2 indirect ELISA of table detects the coating antigen of different dilution factors and serum combination
2.3.2 basic icELISA experimental methods
With coating buffer solution dilution estradiol standard items be prepared into concentration be respectively 0ng/ml, 0.312ng/ml,
0.625ng/ml, 1.250ng/ml, 2.500ng/ml, 5.000ng/ml, 10.000ng/ml, 20.000ng/ml and
40.000ng/ml estradiol standard solution.Four parameter logistic equations are calculated according to determination data result, according to equation model
Standard curve calculates IC50；With IC20-IC80As linear detection range.
Basic icELISA experimental methods are as follows：
2.3.2.1 coating：With estradiol coating antigen OVA-E2 (the original liquid concentration 1.8mg/ of coating buffer solution dilution 2.1
Ml it is) 0.9 μ g/ml to the concentration of estradiol coating antigen OVA-E2, obtains coating original solution, be coated with and tested with the coating original solution
Hole, 100 μ L/ holes add to ELISA Plate, 37 DEG C of incubation 2h.
2.3.2.2 washing：Incline hole endoperidium original solution, is washed 3 times with 0.5% tween cleaning solution, each 3min；It claps
It is dry.
2.3.2.3 closing：Add in 10% calf serum confining liquid, 100ul/ holes, 37 DEG C of incubation 1h.
2.3.2.4 sample-adding：
2.3.2.4.1 standard sample wells
The positive serum that 2.2 prepare is diluted 300,000 times with coating buffer solution dilution, it is dilute to obtain estradiol specific antibody
Release liquid.The estradiol standard solution of 50 μ L estradiol specific antibody dilutions and a kind of above-mentioned concentration of 50 μ L is added to enzyme mark
On plate, then 37 DEG C of reaction 1h, liquid in hole of inclining is washed 3 times, each 3min with cleaning solution；
2.3.2.4.2 negative control hole
Estradiol standard solution is replaced with to isometric high purity water, Qi Tabu with differing only in for 2.3.2.4.1
It is rapid constant.
2.3.2.4.3 blank control wells
The estradiol specific antibody dilution of addition is replaced with into high purity water with differing only in for 2.3.2.4.1,
Its step is constant.
Plus ELIAS secondary antibody 2.3.2.5：It adds in and carries out 1 with 10% calf serum secondary antibody dilution:5 000 diluted HRP marks
Remember goat anti-rabbit igg, 100ul/ holes, 37 DEG C of 50min.
2.3.2.6 colour developing：TMB is added in, 10min is reacted in 100ul/ holes.
2.3.2.7 it terminates：It adds in 1M hydrochloric acid and terminates reaction, 100ul/ holes.
2.3.2.8 it measures：Each hole OD is read with microplate reader450nmNumerical value.
With OD450nmIt is worth for ordinate, with the 1og of estradiol standard solution concentration10It is worth for abscissa, drafting semilog
Canonical plotting.Standard curve has complete reverse-s shape shape, and with upper mounting plate and lower platform, the parallel determination of standard curve
Number 6 times, experimental repeatability is good, and relative standard deviation (coefficient of variation) is within 20%.It is drawn according to standard curve
50% amount of suppression (IC50)。
The foundation of the icELISA methods of 2.4 optimizations
2.4.1 protein blocking condition optimizing
2.3.2.3 closings in 2.3.2 are set to following three Seal treatment, other experimental implementations and 2.3.2 are complete
It is exactly the same：
10% calf serum is closed：Add in 10% calf serum confining liquid, 100ul/ holes, 37 DEG C of incubation 1h.
5% calf serum is closed：Add in 5% calf serum confining liquid, 100ul/ holes, 37 DEG C of incubation 1h.
1% calf serum is closed：Add in 1% calf serum confining liquid, 100ul/ holes, 37 DEG C of incubation 1h.
Compare IC50The relative mistake opposite sex, determines optimal sealing condition.The results are shown in Table 3, compares through overtesting in confining liquid
Containing 1%, 5% and 10% calf serum, increasing the confining liquid containing 10% calf serum and can reduce the moon with serum percentage
The reaction of property serum nonspecific proteins and antigen.
The IC of the different confining liquid icELISA of table 350Comparative test
| Standard items content (ng/ml) | 1% calf serum confining liquid | 5% calf serum confining liquid | 10% calf serum confining liquid |
| 0 | 3.527 | 3.506 | 3.316 |
| 0.312 | 3.019 | 2.688 | 2.459 |
| 0.625 | 2.907 | 2.509 | 2.033 |
| 1.250 | 2.293 | 1.998 | 1.699 |
| 2.500 | 1.715 | 1.507 | 1.307 |
| 5.000 | 1.231 | 1.135 | 0.962 |
| 10.000 | 0.924 | 0.873 | 0.736 |
| 20.000 | 0.631 | 0.455 | 0.523 |
| 40.000 | 0.483 | 0.411 | 0.354 |
| It is negative | 0.617 | 0.331 | 0.215 |
2.4.2 wash conditions optimize
2.3.2.2 washings in 2.3.2 are set to following three carrying out washing treatment, other experimental implementations and 2.3.2 are complete
It is exactly the same：
0.1% tween washs：Incline hole endoperidium original solution, is washed 3 times with 0.1% tween cleaning solution, each 3min；It claps
It is dry.
0.5% tween washs：Incline hole endoperidium original solution, is washed 3 times with 0.5% tween cleaning solution, each 3min；It claps
It is dry.
1.0% tween washs：Incline hole endoperidium original solution, is washed 3 times with 1.0% tween cleaning solution, each 3min；It claps
It is dry.
Compare IC50The relative mistake opposite sex, determines optimal wash conditions.
The results are shown in Table 4, and Tween-20 contents remove non-specific in ELISA systems between 0.1-1% in cleaning solution
Property albumen effect unobvious.
The IC of the different cleaning solution icELISA of table 450Comparative test
| Standard items content (ng/ml) | 0.1% tween cleaning solution | 0.5% tween cleaning solution | 1% tween cleaning solution |
| 0 | 3.561 | 3.455 | 3.334 |
| 0.312 | 3.154 | 2.679 | 2.597 |
| 0.625 | 1.915 | 1.623 | 1.601 |
| 1.250 | 1.734 | 1.576 | 1.523 |
| 2.500 | 1.527 | 1.255 | 1.132 |
| 5.000 | 1.063 | 0.963 | 0.865 |
| 10.000 | 0.738 | 0.638 | 0.624 |
| 20.000 | 0.636 | 0.458 | 0.476 |
| 40.000 | 0.423 | 0.312 | 0.310 |
| It is negative | 0.326 | 0.313 | 0.316 |
2.4.3 primary antibody reaction condition optimization
Primary antibody reaction condition in 2.3.2.4 sample-addings in 2.3.2 is replaced with into 37 DEG C of reactions respectively by 37 DEG C of reaction 1h
1h and 4 DEG C is reacted the two processing of 1h, other operations are identical with 2.3.2.Compare IC50The relative mistake opposite sex, determines optimal one
Anti- reaction condition.
1h is incubated as primary antibody incubation temperature at 4 DEG C and 37 DEG C respectively, as a result as 5,4 DEG C of incubation 1h of table can further drop
Nonspecific proteins and antigen are combined in low rabbit negative serum, and detection reaction sensitivity greatly improves.
The IC of 5 two kinds of primary antibody reaction condition icELISA of table50Comparative test
2.4.4 secondary antibody sealing condition optimizes
2.3.2.5 in 2.3.2 is added 10% calf serum secondary antibody dilution in ELIAS secondary antibody replace with 7% respectively small
Cow's serum secondary antibody dilution, 5% calf serum secondary antibody dilution, 3% calf serum secondary antibody dilution and 1% calf serum secondary antibody
This 4 processing of dilution, other operations are identical with 2.3.2.
Compare IC50The relative mistake opposite sex, determines most secondary antibody sealing condition.Under more than optimal conditions, using containing 1%, 3%,
5%th, the secondary antibody diluted secondary antibody of 7% calf serum, as a result such as table 6, the secondary antibody dilution containing 5% calf serum can make
Positive, negative serum OD values are presented on zone of reasonableness.
The IC of the different secondary antibody sealing condition icELISA of table 650Comparative test
2.4.5 color condition optimizes
By in 2.3.2 2.3.2.6 colour developing in reaction 10min replace with respectively 3min, 6min, 9min and 12min this
Four processing, other operations are identical with 2.3.2.Compare IC50The relative mistake opposite sex, determines optimal color condition.By comparing
Different TMB developing times find, 9min to 12min reaction fully generation abundant in 3min to 6min unreacteds, so as to
It is optimum reacting time (table 7) to determine 9min.
The IC of the different color condition icELISA of table 750Comparative test
2.4.6 icELISA methods and its sensitivity are optimized
With coating buffer solution dilution estradiol standard items be prepared into concentration be respectively 0.000ng/ml, 0.033ng/ml,
The estradiol standard solution of 0.100ng/ml, 0.300ng/ml, 0.900ng/ml, 2.700ng/ml, 8.100ng/ml.According to
Determination data result calculates four parameter logistic equations, according to equation model standard curve, calculates IC50；With IC20-IC80As line
Property detection range.
1) it is coated with：With coating buffer solution dilution 2.1 estradiol coating antigen OVA-E2 (original liquid concentration 1.8mg/ml) extremely
The concentration of estradiol coating antigen OVA-E2 is 0.9 μ g/ml, obtains coating original solution, and experimental port is coated with the coating original solution,
100 μ L/ holes add to ELISA Plate, 37 DEG C of incubation 2h.
2) wash：Incline hole endoperidium original solution, is washed 3 times with 0.1% tween cleaning solution, each 3min；It pats dry.
3) close：Add in 10% calf serum confining liquid, 100ul/ holes, 37 DEG C of incubation 1h.
4) it is loaded：
A, standard sample wells
The positive serum that 2.2 prepare is diluted 300,000 times with coating buffer solution dilution, it is dilute to obtain estradiol specific antibody
Release liquid.The estradiol standard solution of 50 μ L estradiol specific antibody dilutions and a kind of above-mentioned concentration of 50 μ L is added to enzyme mark
On plate, then 4 DEG C of reaction 1h, liquid in hole of inclining is washed 3 times, each 3min with cleaning solution；
B, negative control hole
Estradiol standard solution is replaced with to isometric high purity water with differing only in for A, other steps are constant.
C, blank control wells
The estradiol specific antibody dilution of addition is replaced with into high purity water with differing only in for A, other steps are not
Become.
5) ELIAS secondary antibody is added：It adds in and carries out 1 with 5% calf serum secondary antibody dilution:5 000 diluted HRP marks goat-antis
Rabbit igg, 100ul/ holes, 37 DEG C of 50min.
6) develop the color：TMB is added in, 9min is reacted in 100ul/ holes.
7) terminate：It adds in 1M hydrochloric acid and terminates reaction, 100ul/ holes.
8) measure：Each hole OD is read with microplate reader450nmNumerical value.
Established standards curve measures estradiol IC with icELISA50As a result, four parameter logistic equations are calculatedCoefficient of determination R2=0.9952 represents that the goodness of fit is high, and E is drawn according to equation model2Mostly anti-mark
Directrix curve is as shown in Figure 7.It can be calculated IC50For 0.129ng/ml, IC20-IC80The range of linearity is 0.0279ng/ml-
0.597ng/ml。
2.4.7 the specific detection of the icELISA methods optimized
By measuring the estradiol mostly anti-cross reacting rate with estradiol analog, the mostly anti-specificity of identification estradiol.
Estradiol IC50(IC50(E2)) the same 2.4.6 of measure.The standard items of 2.4.6 are replaced with into difference respectively by estradiol
8 kinds of oestrone, estriol, oestradiol benzoate, ethinyloestradiol, quinestrol, Estradiol Valerate, diethylstilbestrol, nonyl phenol of concentration etc.
Analog is reacted, and is drawn standard curve, using estradiol as reference standard, is calculated the IC of each analog50, the intersection of competitor
Reactivity is shown in formula：
Result of the test is shown in Table the optimization icELISA methods of 8,2.4.6 and oestradiol benzoate, oestrone, ethinyloestradiol intersect
Reactivity respectively up to 150%, 3.11%, 1.52%, and with estriol, quinestrol, Estradiol Valerate, diethylstilbestrol, nonyl phenol
Cross reacting rate be respectively less than 0.1%.Showing the optimization icELISA methods of 2.4.6 has estradiol and oestradiol benzoate
High degree of specificity.
The optimization icELISA methods of 8 2.4.6 of table and the cross reacting rate of estradiol analog
To sum up, the estradiol standard curve I C of the optimization icELISA methods of 2.4.650It is worth for 0.129ng/ml, linearity test
Scope is 0.0279ng/ml-0.597ng/ml, reaches 150% with oestradiol benzoate cross reacting rate, female with estriol, alkynes
Ether, Estradiol Valerate, diethylstilbestrol, the cross reacting rate of nonyl phenol are respectively less than 0.1%.
Claims (10)
1. detect the method for estradiol and/or oestradiol benzoate, described enzyme-linked exempt from using any in claim 7-9
The content of estradiol and/or oestradiol benzoate in epidemic disease kit detection sample to be tested.
2. according to the method described in claim 1, it is characterized in that：The described method includes：
1) antigen coat：The estradiol coating antigen is fixed on surface of solid phase carriers, forms solid phase antigen；
2) wash：It is washed with cleaning solution；
3) close：It is closed with confining liquid；
4) sample to be tested is added：The estradiol specific antibody and sample to be tested are added in, reacts 1h at 4 DEG C；
5) ELIAS secondary antibody is added：The ELIAS secondary antibody with secondary antibody diluted is added in, is reacted；
6) develop the color：Carry out chromogenic reaction；
7) reaction is terminated, measures the content of the estradiol and/or oestradiol benzoate in sample to be tested.
3. method according to claim 1 or 2, it is characterised in that：In the antigen coat, the estradiol coating antigen with
The concentration coating solid phase carrier of 0.9 μ g/ml.
4. according to the method described in claim 1 or 2 or 3, it is characterised in that：The confining liquid contains volumn concentration
10% calf serum.
5. according to any method in Claims 1-4, it is characterised in that：The secondary antibody dilution contains volume basis
Content is 5% calf serum.
6. according to any method in claim 1 to 5, it is characterised in that：The chromogenic reaction time is 9 minutes.
7. the enzyme linked immunological kit of estradiol or oestradiol benzoate is detected, including estradiol coating antigen and estradiol specificity
Antibody；The estradiol coating antigen is the conjugate of estradiol haptens and carrier protein 1；The estradiol specific antibody is
The polyclonal antibody or monoclonal antibody obtained using the conjugate of the estradiol haptens and carrier protein 2 as immunogene；Institute
It is identical or different carrier protein to state carrier protein 1 and the carrier protein 2；
The estradiol haptens is the compound of formula 1：
8. enzyme linked immunological kit according to claim 7, it is characterised in that：The estradiol haptens and carrier protein
The coupling ratio of 2 conjugate is 11.8:1.
9. the enzyme linked immunological kit according to claim 7 or 8, it is characterised in that：The estradiol specific antibody is
Rabbit polyclonal antibody.
10. any enzyme linked immunological kit exists in any method or claim 7 to 9 in claim 1 to 6
Detect the application in estradiol and/or oestradiol benzoate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116284383A (en) * | 2023-03-01 | 2023-06-23 | 宜兴食品与生物技术研究院有限公司 | Method for rapidly obtaining 17 beta-estradiol murine IgG full-length antibody and Fab antibody |
| CN116836277A (en) * | 2022-03-23 | 2023-10-03 | 东莞市朋志生物科技有限公司 | Anti-estradiol antibody, reagent for detecting estradiol and kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004108811A (en) * | 2002-09-13 | 2004-04-08 | Matsushita Electric Ind Co Ltd | Monitoring device |
| WO2008007359A2 (en) * | 2006-07-09 | 2008-01-17 | Infigo Diagnostics Ltd. | Rapid diagnostic devices based on molecular imprinted polymers |
| CN102253222A (en) * | 2011-04-21 | 2011-11-23 | 中国农业科学院农业质量标准与检测技术研究所 | Method and special test paper for estrogen detection |
| CN102721811A (en) * | 2012-05-28 | 2012-10-10 | 许昌学院 | Enzyme-linked immunoassay kit for detecting estradiol benzonate |
| US9482678B1 (en) * | 2014-03-18 | 2016-11-01 | Rebecca Caffrey | Method for measurement of bioavailable testosterone |
-
2016
- 2016-11-24 CN CN201611050507.3A patent/CN108107223A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004108811A (en) * | 2002-09-13 | 2004-04-08 | Matsushita Electric Ind Co Ltd | Monitoring device |
| WO2008007359A2 (en) * | 2006-07-09 | 2008-01-17 | Infigo Diagnostics Ltd. | Rapid diagnostic devices based on molecular imprinted polymers |
| CN102253222A (en) * | 2011-04-21 | 2011-11-23 | 中国农业科学院农业质量标准与检测技术研究所 | Method and special test paper for estrogen detection |
| CN102721811A (en) * | 2012-05-28 | 2012-10-10 | 许昌学院 | Enzyme-linked immunoassay kit for detecting estradiol benzonate |
| US9482678B1 (en) * | 2014-03-18 | 2016-11-01 | Rebecca Caffrey | Method for measurement of bioavailable testosterone |
Non-Patent Citations (1)
| Title |
|---|
| 戴小华: "雌二醇单克隆抗体的制备与酶联免疫检测试剂盒的初步研究", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116836277A (en) * | 2022-03-23 | 2023-10-03 | 东莞市朋志生物科技有限公司 | Anti-estradiol antibody, reagent for detecting estradiol and kit |
| CN116836277B (en) * | 2022-03-23 | 2024-04-05 | 东莞市朋志生物科技有限公司 | Anti-estradiol antibody, reagent for detecting estradiol and kit |
| CN116284383A (en) * | 2023-03-01 | 2023-06-23 | 宜兴食品与生物技术研究院有限公司 | Method for rapidly obtaining 17 beta-estradiol murine IgG full-length antibody and Fab antibody |
| CN116284383B (en) * | 2023-03-01 | 2023-11-03 | 宜兴食品与生物技术研究院有限公司 | Method for rapidly obtaining 17 beta-estradiol murine IgG full-length antibody and Fab antibody |
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