CN102838555A - Hapten, artificial antigen, antibody and synthesis of trinal(2,3-dibromopropyl) triallyisocyanurate - Google Patents
Hapten, artificial antigen, antibody and synthesis of trinal(2,3-dibromopropyl) triallyisocyanurate Download PDFInfo
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Abstract
The invention relates to synthesis of haptens of trinal (2,3-dibromopropyl) triallyisocyanurate and a preparation method of artificial antigens, antibodies of the trinal(2,3-dibromopropyl) triallyisocyanurate. A constitutional formula is shown in the specification. The preparation method of the haptens mainly consists of the following four parts: (1) synthesizing the haptens of the trinal (2,3-dibromopropyl) triallyisocyanurate containing carboxyl functional groups; (2) conducting covalent binding on the haptens and carrier proteins to obtain the artificial antigen; and (3) conducting animal immunization on obtained artificial antigens, and obtaining the antibodies with specificity identification of the trinal(2,3-dibromopropyl) triallyisocyanurate after purification.
Description
Technical field
The invention belongs to organic synthesis, immunochemistry, biochemical analysis field; Relate to three (2, the 3-dibromopropyl) isocyanuric acid ester hapten, artificial antigen and antibody preparation and synthetic.
Background technology
Three (2; The 3-dibromopropyl) [Tris (2 for isocyanuric acid ester; 3-dibromopropyl) isocyanurate; Be called for short TBC] be a kind of novel and effective hexabromo heterocycle flame-retardant additive, be widely used in polyolefine, SE, foamed polyurethane, polyphenyl alkene, acrylonitrile-butadiene-styrene plastics, unsaturated polyester and the goods such as multiple viton and synthon.Its molecular structure is:
TBC has very strong hydrophobicity, is difficult to degraded at occurring in nature; In addition, it can also be accumulated in ecotope neutralizes some organisms and be detected for a long time.Find through experimental study; TBC has not only possessed the sustainability organic pollutant, and (Persistent Organic pollutants, all character POPs) are a kind of nervous tissue to be had toxic compound; Simultaneously also be a kind of potential noxious (Ting Yuan et al. that human survival procreation and Sustainable development are constituted a threat to; Environmental Science & Technology, 43:3080-3086,2009; Qu guangbo et al., Science China, 54:1651-1658,2011).
To the traditional detection method of TBC, rely on conventional instrumental analysis means mostly, at present like vapor-phase chromatography (GC), liquid chromatography (HPLC), mass spectrum (MS) or performance liquid mass spectrum logotype (HPLC-MS).These methods are not only operated too very complicated, and the technician is required height, and sample need carry out pre-treatment, and workload is big, instrument is expensive, the cycle is long.
(Enzyme Linked Immunosorbent Assay ELISA) is based on the immune identification action of immune complex and a kind of analysis determining method that enzyme catalysis amplification mechanism grows up to enzyme-linked immunosorbent assay.During mensuration; Sample to be checked and enzyme-labelled antigen or antibody antibody or the antigen by different step and surface of solid phase carriers absorption is reacted; Method with washing is separated immune complex and free composition; Under the katalysis of enzyme, make the substrate colour developing then, thereby can carry out qualitative or quantitative mensuration.Because enzyme immune reaction has high selectivity and susceptibility, therefore, the process that immunodetection can make venomous injurant analyze, particularly pre-treatment step is simplified greatly.Compare with conventional instrument detecting method; The ELISA method except have high specificity, easy and simple to handle, instrumentation degree and advantage such as analysis cost is low, sample pre-treatments is simple, detection time is short, sample capacity is big, also do not have HPLC or GC requirement to physico-chemical properties such as determinand vapour pressure, thermostability, chromophoric grouies.
Because TBC belongs to organic molecule, itself can not produce antibody by the induced animal cell, realizes that therefore it is the hapten molecule that must obtain with the TBC structural similitude that the TBC enzyme linked immunological is inhaled the prerequisite of analyzing.And the design not only need structure similar with synthetic TBC hapten molecule with TBC, and must have can with carrier protein bonded activity functional groups.
Summary of the invention
First purpose of the present invention is to be to provide three (2, the 3-dibromopropyl) isocyanuric acid ester hapten.
Second purpose of the present invention is to be a kind of lack step, three (2, the 3-dibromopropyl) haptenic compound method of isocyanuric acid ester simple to operate.
The 3rd purpose of the present invention provided a kind of artificial antigen that is come by above-mentioned haptin preparation.
The 4th purpose of the present invention be prepared a kind of can specific recognition TBC and have the polyclonal antibody albumen of three (2, the 3-dibromopropyl) isocyanuric acid ester that height tires.
The present invention synthesizes three (2, the 3-dibromopropyl) haptenic structure of isocyanuric acid ester:
Formula I
Wherein, the integer of n=0 ~ 10.
N among the said formula I is preferably 1,3 or 5.
The invention provides a kind of above-mentioned haptenic preparation process is: with 2,4,6-three (allyloxy)-1,3,5-triazines, copper chloride dihydrate, water and toluene reaction make 3,5-diallyl isocyanuric acid ester imines; With make 3, the reaction of 5-diallyl isocyanuric acid ester imines and aqueous sodium hydroxide solution obtains 3,5-diallyl isocyanuric acid ester imines sodium salt; Again with 3,5-diallyl isocyanuric acid ester imines sodium salt and bromo C
1～11After the acetoacetic ester generation substitution reaction,, under acidic conditions, carry out the hydrolysis reaction of ester again, promptly obtain three (2, the 3-dibromopropyl) isocyanuric acid ester hapten with the bromine addition.
In the described preparation process 2,4,6-three (allyloxy)-1,3,5-triazines, Copper dichloride dihydrate, water and toluene react 3 ~ 5h under 90 ~ 100 ° of C; 3,5-diallyl isocyanuric acid ester imines and aqueous sodium hydroxide solution are at 5 ~ 50 ° of C reaction 1h; 3,5-diallyl isocyanuric acid ester imines sodium salt and bromo C
1～11Acetoacetic ester is substitution reaction 8 ~ 20h under 30 ~ 70 ° of C; With the reaction conditions of bromine addition be 5 ~ 50 ° of C reaction 1 ~ 2h; The hydrolysis reaction condition of ester is 70 ~ 100 ° of C reaction 10 ~ 24h under the acidic conditions.
Three (2, the 3-dibromopropyl) isocyanuric acid ester artificial antigen that the present invention also provides a kind of above-mentioned haptin to make, said artificial antigen structural formula is:
Formula II
The integer of n=0 ~ 10 wherein; Protein representes carrier protein.
N among the said formula II is preferably 1.
Described carrier proteins comprises bovine serum albumin or ovalbumin.
The present invention also provides a kind of above-mentioned three (2; The 3-dibromopropyl) polyclonal antibody that obtains of isocyanuric acid ester artificial antigen; The Tegeline that described polyclonal antibody is obtained behind the specific immune response of animal by three (2, the 3-dibromopropyl) isocyanuric acid ester artificial antigen.
The reaction equation of the present invention's design is represented as follows:
3, the synthesis step of 5-diallyl isocyanuric acid ester imines sodium salt is:
The synthesis step of three (2, the 3-dibromopropyl) isocyanuric acid ester hapten 1a, 1b and 1c is:
The present invention further provides three types of haptenic synthesis steps of TBC, and is specific as follows said.
TBC haptin midbody 3, the synthesis step of 5-diallyl isocyanuric acid ester imines sodium salt (compound 3) is:
(1) successively with compound 2,4,6-three (allyloxy)-1,3,5-triazines, water, copper chloride dihydrate (CuCl
22H
2O) and toluene (mol ratio is 1:1:0.045:3.8) join in the reaction flask, under 90 ~ 100 ° of C the reaction 3 ~ 5h.Be cooled to room temperature then, remove water and toluene under reduced pressure, resistates with methylene dichloride after with the sherwood oil recrystallization after, suction filtration obtains light blue solid, obtains white solid 3 through Hydrogen chloride and water washing again, 5-diallyl isocyanuric acid ester imines (compound 2).
(2) with compound 2 and aqueous sodium hydroxide solution (mol ratio is 1:1) room temperature reaction 1h, remove water under reduced pressure and obtain 3,5-diallyl isocyanuric acid ester imines sodium salt (compound 3).
The synthesis step of TBC haptin Ia ~ Ic is following, with the example that synthesizes of Ia:
(1) with N, dinethylformamide (DMF) is a solvent, and compound 3 and METHYL BROMOACETATE (mol ratio is 1:1) are reacted an evening under 50 ° of C.Reaction solution is cooled to room temperature, washs anhydrous Na with a large amount of water and salt solution behind the adding methylene dichloride
2SO
4Drying removes solvent again under reduced pressure, and resistates obtains compound 4a through silica gel column chromatography separating purification.
(2) bromine is added drop-wise in the dichloromethane solution of compound 4a, back flow reaction 2h, wherein compound 4 is 1:2.5 with the bromine mol ratio.After the adding saturated sodium sulfite aqueous solution takes off to the redness of reaction solution, use dichloromethane extraction, anhydrous sodium sulfate drying.Remove solvent under reduced pressure, resistates obtains compound 5a through silica gel column chromatography separating purification.
(3) compound 5a, concentrated hydrochloric acid (30%) are added in the reaction flask one night of back flow reaction.Suction filtration is got solid, and drying gets compound I a.
The synthesis step of TBC haptin Ib ~ Ic and last similar.
Effect of the present invention: the present invention makes several kinds of novel three (2, the 3-dibromopropyl) isocyanuric acid ester haptens through short step, simple operations, and productive rate all is no less than 63%; And prepared corresponding artificial antigen body, the binding ratio of haptin (Ia) and BSA reaches 30:1, reaches 22:1 with the binding ratio of OVA; Through the antibody polyclonal antibody albumen that the animal immune experiment obtains to have specific recognition, height is tired; Can be applicable to the detection of ELISA method; It is very low to detect lower limit, is 0.1ng/mL like the antibody I that obtains in the present embodiment to the lowest detection lower limit of eight chloro-styrenes (is that 10% concentration is represented with inhibiting rate).
Description of drawings
[Fig. 1] is that the ultraviolet of haptin of the present invention, artificial antigen and BSA compares spectrogram; The 1st, BSA; The 2nd, haptin; The 3rd, artificial antigen
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for the present invention and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after having read the content that the present invention tells about, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1:3,5-diallyl isocyanuric acid ester imines sodium salt (compound 3) synthetic
1) with compound 2,4,6-three (allyloxy)-1,3,5-triazines (24.90g, 0.10mol), water (1.80g, 0.10mol), CuCl
22H
2O (0.767g, 4.5mmol) and toluene (35.01g, 0.38mol) under 93 ° of C the reaction 5h.After removing water and toluene under reduced pressure, resistates obtains light blue solid with methylene dichloride and sherwood oil recrystallization, again with obtaining 14.5g white solid compound 2, productive rate 69% after Hydrogen chloride and the water washing.
2) successively with NaOH (400mg, 10mmol), (2.09g 10mmol) adds in the reaction flask room temperature reaction 1h for water (4.0mL) and compound 2.After removing water under reduced pressure, obtain 2.31g white crystal compound 3, productive rate 100%.
3: white solid, m.p.205 ° of C;
1H NMR (D
2O, 400MHz):: 4.41 (dd, J
1=6.00Hz&J
2=1.60Hz, 2H), 5.07 (dd, J=17.40Hz&J=1.20Hz, 2H), 5.15 (dd, J=10.40Hz&J=1.20Hz, 4H), 5.86 ~ 5.95 (m, 2H).
Embodiment 2: the compound method of compound I a
(1) with compound 3 (600mg, 2.60mmol), the 2-METHYL BROMOACETATE (435mg, 2.60mmol) and DMF (5mL) at room temperature react and spend the night.After adding methylene dichloride (30mL), organic phase is with big water gaging and saturated common salt water washing, anhydrous Na
2SO
4Dry.Filter, remove solvent under reduced pressure, resistates separates [V (sherwood oil): V (ETHYLE ACETATE)=5:1] through silica gel column chromatography, gets 613mg oily liquids shape compound 4a, yield 80%.
(2) with compound 4a (400mg, 1.36mmol) with after methylene dichloride (20mL) dissolving, to system drip bromine (542mg, 3.39mmol), the about 2h of reflux.Add saturated NaHSO
3After solution to the reaction solution red color disappeared, use dichloromethane extraction.Organic phase is with saturated NaCl solution washing, anhydrous Na
2SO
4Dry.Filter, remove solvent under reduced pressure, resistates obtains 787mg white solid compound 5a, yield 94% through silica gel column chromatography separating purification [V (sherwood oil): V (ETHYLE ACETATE)=5:1].
(3) with compound 5a (350mg, 0.57mmol), concentrated hydrochloric acid (30%, 10mL) react a night in 110 ° of C refluxed.The TLC tracking monitor, when raw material reaction is complete, stopped reaction.After naturally cooling to room temperature, the adularescent solid generates, suction filtration, and filter cake washs with clear water, uses acetone solution, anhydrous Na then
2SO
4Dry.Filter, remove solvent under reduced pressure, get 210mg white solid compound 1a, yield 63%.
1a: white solid, m.p.:97 ° of C;
1H NMR (CDCl
3, 400MHz): 3.90 ~ 3.95 (m, 2H), 3.97 ~ 4.01 (m, 2H), 4.36 ~ 4.42 (m, 2H), 4.43 ~ 4.49 (m, 2H), 4.62 (s, 2H), 4.62 ~ 4.70 (m, 4H).
13C NMR (CDCl
3, 100MHz): 35.69 (CH
2), 43.91 (CH
2), 48.25 (CH), 49.11 (CH
2), 49.15 (CH
2), 149.61 (C=O), 149.87 (C=O), 168.47 (C=O) .MS (ES) m/z (%): 581.7 (M
-, 21), 583.6 (M
-+ 2,71), 585.6 (M
-+ 4,100), 587.6 (M
-+ 6,63), 589.6 (M
-+ 8,11).
Embodiment 3: the compound method of compounds ib
(1) with compound 3 (790mg, 3.42mmol), 4-bromo-butyric acid ethyl ester (667mg, 3.42mmol) and DMF (7m L) at room temperature react and spend the night.Add dichloromethane extraction (30mL), organic phase is with big water gaging and saturated common salt water washing, anhydrous Na
2SO
4Dry.Filter, remove solvent under reduced pressure, resistates separates [V (sherwood oil): V (ETHYLE ACETATE)=5:1] through silica gel column chromatography, gets 818mg oily liquids shape compound 4b, yield 74%.
(2) with compound 4b (760mg, 2.35mmol) with after methylene dichloride (20mL) dissolving, to system drip bromine (941mg, 5.88mmol), the about 2h of reflux.Add saturated NaHSO
3After solution to the reaction solution red color disappeared, use dichloromethane extraction.Organic phase is with saturated NaCl solution washing, anhydrous Na
2SO
4Dry.Filter, remove solvent under reduced pressure, resistates obtains 1.51g oily liquids shape compound 5b, yield 99% through silica gel column chromatography separating purification [V (sherwood oil): V (ETHYLE ACETATE)=5:1].
(3) with compound 5b (400mg, 0.62m mol), and concentrated hydrochloric acid (30%, 10mL) react a night in 110 ° of C refluxed.The TLC tracking monitor, when raw material reaction is complete, stopped reaction.After naturally cooling to room temperature, supernatant liquid is toppled over away.Dissolve insolubles with methylene dichloride, organic phase is with the saturated ammonium chloride solution washing being arranged once, anhydrous Na
2SO
4Dry.Filter, remove solvent under reduced pressure, get 328mg compound 1b, yield 86%.
1b: white solid, m.p.:65 ° of C;
1H NMR (CDCl
3, 400MHz): 1.99 ~ 2.02 (m, 2H), 2.43 ~ 2.48 (m, 2H), 3.70 ~ 3.76 (m, 2H), 3.84 ~ 3.89 (m, 2H), 4.03 (t, J=6.40Hz, 2H), 4.43 (d, J=7.20Hz, 4H), 4.56 ~ 4.70 (m, 2H).
13C NMR (CDCl
3, 100MHz): 22.56 (CH
2), 31.07 (CH
2), 33.23 (CH
2), 42.58 (CH
2), 46.08 (CH), 46.13 (CH), 46.22 (CH), 46.55 (CH), 46.63 (CH), 46.78 (CH), 47.05 (CH
2), 47.10 (CH
2), 47.41 (CH
2), 47.48 (CH
2), 55.58 (CH
2), 148.63 (C=O), 148.66 (C=O), 148.73 (C=O) .MS (ES) m/z (%): 645.5 (M
-+ CH
3OH, 18), 647.5 (M
-+ 2+CH
3OH, 54), 649.5 (M
-+ 4+CH
3OH, 100), 651.5 (M
-+ 6+CH
3OH, 79), 653.5 (M
-+ 8+CH
3OH, 20).
Embodiment 4: compound I c's is synthetic
(1) with compound 3 (978mg, 4.23mmol), 6-bromocaproic acid ethyl ester (944mg, 4.23mmol) and DMF (7mL) at room temperature react and spend the night.Add dichloromethane extraction (30mL), organic phase is with big water gaging and saturated common salt water washing, anhydrous Na
2SO
4Dry.Filter, remove solvent under reduced pressure, resistates separates [V (sherwood oil): V (ETHYLE ACETATE)=5:1] through silica gel column chromatography, gets 1.04g oily liquids shape compound 4c, yield 70%.
(2) with compound 4c (964mg, 2.75mmol) with after methylene dichloride (20mL) dissolving, to system drip bromine (1.10g, 6.87mmol), the about 1.5h of reflux.Add saturated NaHSO
3After solution to the reaction solution red color disappeared, use dichloromethane extraction.Organic phase is with saturated NaCl solution washing, anhydrous Na
2SO
4Dry.Filter, remove solvent under reduced pressure, resistates obtains 1.50g oily liquids shape compound 5c, yield 81% through silica gel column chromatography separating purification [V (sherwood oil): V (ETHYLE ACETATE)=5:1].
(3) with compound 5c (450mg, 0.67mmol), concentrated hydrochloric acid (30%, 13mL) react a night in 110 ° of C refluxed.The TLC tracking monitor, when raw material reaction is complete, stopped reaction.After naturally cooling to room temperature, supernatant liquid is toppled over away.Dissolve insolubles with methylene dichloride, organic phase is with the saturated ammonium chloride solution washing being arranged once, anhydrous Na
2SO
4Dry.Filter, remove solvent under reduced pressure, get 336mg compound 1c, yield 78%.
1c: white solid, m.p.:52 ° of C;
1H NMR (CDCl
3, 400MHz): 1.36 ~ 1.44 (m, 2H), 1.64 ~ 1.73 (m, 4H), 2.37 (t, J=6.00Hz, 2H), 3.70 ~ 3.75 (m, 2H), 3.85 ~ 3.89 (m, 2H), 3.94 (t, J=7.20Hz, 2H), 4.43 (d, J=7.20Hz, 4H), 4.64 ~ 4.71 (m, 2H).
13C NMR (CDCl
3, 100MHz): 24.13 (CH
2), 25.92 (CH
2), 27.22 (CH
2), 33.17 (CH
2), 33.69 (CH
2), 43.16 (CH
2), 46.05 (CH), 46.10 (CH), 47.39 (CH
2), 47.46 (CH
2), 148.54 (C=O), 148.75 (C=O), 148.83 (C=O) .MS (ESI) m/z (%): 673.5 (M
-+ 2H
2O, 18), 675.5 (M
-+ 2+2H
2O, 52), 677.5 (M
-+ 4+2H
2O, 100), 679.5 (M
-+ 6+2H
2O, 72), 681.5 (M
-+ 8+2H
2O, 31).
Embodiment 5: preparation of artificial antigen and evaluation
5.1 artificial antigen is synthetic
Compound 1a (15mg, 25.6 μ mmol) with after DMF (0.8mL) dissolving, is added DCC (8mg, 38.8 μ mmol) and NHS (4.5mg, 39.1 μ mmol) successively, after reacting 4h under the room temperature, be positioned over 4 ° of C refrigerator overnight again.Next day the centrifuging and taking supernatant liquid, under 4 ° of C with its slowly be added drop-wise to BSA (57mg, 0.85 μ mmol) PBS solution (c 0.05mol/L, 10mL) in, rise to room temperature and continue reaction 2h, be positioned over 4 ° of C refrigerator overnight again.Then reaction solution is packed in the dialysis tubing, the PBS solution under 4 ° of C (c 0.01mol/L, pH=7.4) in dialysis, changed dialyzate one time in per 12 hours, dialyse altogether 6 times.After dialysis finishes conjugate BSA-1a in the dialysis tubing is carried out lyophilize, and preserve down in-20 ° of C.
Compound 1a (15mg, 25.6 μ mmol) with after DMF (0.8mL) dissolving, is added DCC (8mg, 38.8 μ mmol) and NHS (4.5mg, 39.1 μ mmol) successively, be positioned over 4 ° of C refrigerator overnight again after reacting 4h under the room temperature.Next day the centrifuging and taking supernatant liquid, under 4 ° of C with its slowly be added drop-wise to OVA (65mg, 1.44 μ mmol) PBS solution (c 0.05mol/L, 10mL) in, rise to room temperature and continue reaction 2h, be positioned over 4 ° of C refrigerator overnight again.Then reaction solution is packed in the dialysis tubing, the PBS solution under 4 ° of C (c 0.01mol/L, pH=7.4) in dialysis, changed dialyzate one time in per 12 hours, dialyse altogether 6 times.After dialysis finishes conjugate OVA-1a in the dialysis tubing is carried out lyophilize, and preserve in-20 ° of C.
5.2 the evaluation of artificial antigen
(BSA, (200 ~ 400nm), the contrast three calculates its binding ratio in the absorbancy of 208m and 278nm OVA) to carry out UV scanning with coupled product through double antigen 1 a, carrier proteins.Contrast conjugate BSA-1a simultaneously and compare, considerable change occurs with haptin 1a, BSA and carrier proteins.(seeing accompanying drawing 1 for details)
Following through calculation result: the binding ratio of haptin 1a and BSA is 30:1; The binding ratio of haptin 1a and OVA is 22:1.As zooperal artificial antigen, the binding substances OVA-1a of haptin and Protalbinic acid (OVA) is as the coating antigen of immunoassay with the binding substances BSA-1a of haptin and bovine serum albumin (BSA).
Embodiment 6: the preparation of polyclonal antibody I
6.1 immune animal prepares serum I
Experiment is selected for use about half cycle year, body weight is about 2 kilograms, healthy new zealand white rabbit as the immune animal object.
With 400 μ L saline water with 0.4mg artificial antigen BSA-1a dissolved dilution after, add isopyknic Freund's complete adjuvant and carry out emulsification; Then the multiple spot subcutaneous injection is carried out in the vertebra both sides, back of new zealand white rabbit; After one week of first immunisation inoculation, replace Freund's complete adjuvant with 400 μ L Freund's incomplete adjuvants, immune strengthening on the useless fellow that generates after the immunization, later per two weeks are carried out immune strengthening once; Rabbit ear edge venous blood collection keeps as the negative control sample before the immunity, and a week is got blood l mL to rabbit ear edge vein after every immune strengthening, leaves standstill 2h under the room temperature, and after blood fully solidified, centrifuging and taking serum was checked antibody titer with indirect elisa method.
After treating that immunizing potency meets the requirements, White Rabbit is carried out carotid artery get blood, and getting Blood bottle is placed room temperature half a hour; Solidify the back and along the bottle inwall clot and glass are broken away from, be put into 4 ° of C refrigerator overnight again with inoculating needle; After treating blood clot retraction, with capillary pipet serum is sucked in the test tube, spinning goes out serum I under the 3000r/min rotating speed.
6.2 antibody purification
Through albumin A affinity chromatography column separating purification, obtain the antibody I of TBC.
6.3 the evaluation of antibody and application thereof
6.3.1 the detection of antibody titer
Begin for the first time from immune strengthening, after each immune strengthening the 7th day gathered rabbit auricular vein blood and also isolated serum, serum suitably diluted the back measure with indirect ELISA method and tire.When treating the 5th immunity, rabbit has obtained high antibody of tiring, and tiring of antiserum(antisera) I is 1:6400; Tiring after purified is 1:16000.
The present invention adopts indirect ELISA method that the polyclonal antibody serum I is carried out titration, and concrete steps are following:
1) adopt indirect ELISA method to draw the best effort concentration of antigen and test serum: encapsulating material OVA-1a concentration is 10 μ Lg/mL, and the extension rate of antibody is 1:16000, and two anti-extension rates are 1:4000.
2) encapsulate: with the 0.05mol/L carbonate buffer solution (CBS, pH=9.6) gradient dilution coating antigen OVA-1a is added on the enzyme plate to 10 μ g/mL, and every hole drips 100 μ L/ holes on enzyme plate; After 37 ° of C place 1h, spend the night under 4 ° of C; Inferior daily washings (the PBS solution that contains 0.05%TWeen-20) repetitive scrubbing 3 times, each 3 minutes.
3) sealing: (PBS pH=7.4) dilutes OVA to 1% (massfraction), and every hole drips 300 μ L on by enzyme plate with the 0.01mol/L phosphate buffer soln; 37 ° of C incubation 1h; It is inferior to give a baby a bath on the third day after its birth repeatedly with washings.
4) application of sample: on pressing enzyme plate, in every hole, add target substance reference liquid 100 μ L and antibody 50 μ L with the different concns of PBS gradient dilution successively, every group of concentration repeats 2 holes, and makes blank with PBS; 37 ° of C incubation 2h; It is inferior to give a baby a bath on the third day after its birth repeatedly with washings.
5) add ELIAS secondary antibody: with 4000 times of goat-anti rabbit HRP-IgG dilutions, be added drop-wise in each hole of enzyme plate by 100 μ L/ holes with the 0.01mol/L phosphate buffer soln; 37 ° of C place 1h; It is inferior to give a baby a bath on the third day after its birth repeatedly with washings, uses the zero(ppm) water repetitive scrubbing again 2 times, each 3 minutes.
6) colour developing and termination: in each hole of enzyme plate, add the adding substrate developer (3,3', 5,5'-TMB+ydrogen peroxide 50) of 100 μ L, behind the room temperature lucifuge 5-10min, 50 μ L add stop buffer (2mol/L sulphuric acid soln) in each hole of enzyme plate again; Measuring wavelength through ELIASA is the OD value (optical density, OD value) of 450nm.
6.3.2 the mensuration of antibody inhibiting rate
The present invention adopts the indirect competitive ELISA method to measure the inhibiting rate of antibody, and concrete steps are following:
1) encapsulate: (pH=9.6) gradient dilution coating antigen OVA-1a is to 10 μ g/mL, and every hole drips 100 μ L/ holes on enzyme plate for CBS, 0.05mol/L with carbonate buffer solution; 37 ° of C place 1h, spend the night under 4 ° of C afterwards; Inferior daily washings (the PBS solution that contains 0.05%TWeen-20) repetitive scrubbing three times, each 3 minutes.
2) sealing: (pH=7.4) dilution OVA to 1% (massfraction) is added in each hole of enzyme plate by 300 μ L/ holes for PBS solution, 0.01mol/L with phosphate buffer soln; Behind 37 ° of C incubation 1h, with washings washing three times.
3) application of sample: in each hole of enzyme plate, add the eight chloro-styrene solution and the 50 μ L antibody (with 64000 times of PBS dilutions) of the different weaker concns of 100 μ L, every group of concentration repeats twice; Make blank with PBS, behind 37 ° of C incubation 2h, with washings washing three times.
4) add ELIAS secondary antibody:, be added in each hole of enzyme plate by 100 μ L/ holes with 4000 times of PBS solution goat-anti rabbit HRP-IgG dilutions; After 37 ° of C place 1h,, use distilled water wash twice again, each 3 minutes with washings washing three times.
5) colour developing and termination: in each hole of enzyme plate, add the adding substrate developer (3,3', 5,5'-TMB+ydrogen peroxide 50) of 100 μ L, behind the room temperature lucifuge 5-10min, 50 μ L add stop buffer (2mol/L sulphuric acid soln) in each hole of enzyme plate again; Measuring wavelength through ELIASA is the OD value (OD value) of 450nm.
Adopt indirect ELISA successively different concns to be detected, obtained following data.
Wherein inhibiting rate is to obtain through computes:
In the formula: OD
MaxAbsorbancy when not adding eight chloro-styrenes, OD
xAbsorbancy when adding eight chloro-styrenes, OD
MinAbsorbancy for the blank hole.
Can be learnt by last form: when the concentration of eight chloro-styrenes was 0.001,0.01,0.1,1.0,10.0 (ng/mL), the antibody inhibiting rate was respectively 5.5%, 8.0%, 14%, 31%, 66%.Therefore, antibody I is 0.1ng/mL to the lowest detection lower limit of eight chloro-styrenes (is that 10% concentration is represented with inhibiting rate).
Claims (8)
1. one kind three (2, the 3-dibromopropyl) isocyanuric acid ester hapten is characterized in that having structure shown in the formula I:
Formula I
Wherein, the integer of n=0 ~ 10.
2. three (2, the 3-dibromopropyl) according to claim 1 isocyanuric acid ester hapten is characterized in that the n among the said formula I is 1,3 or 5.
3. three (2, the 3-dibromopropyl) as claimed in claim 1 haptenic preparation method of isocyanuric acid ester is characterized in that; The preparation process is: with 2,4, and 6-three (allyloxy)-1; 3; 5-triazine, copper chloride dihydrate, water and toluene generation rearrangement reaction make 3,5-diallyl isocyanuric acid ester imines; With make 3, the reaction of 5-diallyl isocyanuric acid ester imines and aqueous sodium hydroxide solution obtains 3,5-diallyl isocyanuric acid ester imines sodium salt; Again with 3,5-diallyl isocyanuric acid ester imines sodium salt and bromo C
1～11After the acetoacetic ester generation substitution reaction,, under acidic conditions, carry out the hydrolysis reaction of ester again, promptly obtain containing three (2, the 3-dibromopropyl) isocyanuric acid ester hapten of fat carboxyl with the bromine addition.
4. preparation process according to claim 3 is characterized in that, in the described preparation process 2,4,6-three (allyloxy)-1,3,5-triazines, copper chloride dihydrate, water and toluene react 3 ~ 5h under 90 ~ 100 ° of C; 3,5-diallyl isocyanuric acid ester imines and aqueous sodium hydroxide solution are at 5 ~ 50 ° of C reaction 1h; 3,5-diallyl isocyanuric acid ester imines sodium salt and bromo C
1～11Acetoacetic ester is substitution reaction 8 ~ 20h under 30 ~ 70 ° of C; With the reaction conditions of bromine addition be 5 ~ 50 ° of C reaction 1 ~ 2h; The hydrolysis reaction condition of ester is 70 ~ 100 ° of C reaction 10 ~ 24h under the acidic conditions.
5. three (2, the 3-dibromopropyl) isocyanuric acid ester artificial antigen is characterized in that having structure shown in the formula II:
Formula II
The integer of n=0 ~ 10 wherein; Protein representes carrier proteins.
6. three (2, the 3-dibromopropyl) according to claim 5 isocyanuric acid ester artificial antigen is characterized in that the n among the said formula II is 1.
7. according to claim 5 or 6 described three (2, the 3-dibromopropyl) isocyanuric acid ester artificial antigen, it is characterized in that described carrier proteins comprises bovine serum albumin or ovalbumin.
8. specific recognition three (2; The 3-dibromopropyl) polyclonal antibody of isocyanuric acid ester; It is characterized in that the Tegeline that adopts claim 5 ~ 7 described three (2, the 3-dibromopropyl) isocyanuric acid ester artificial antigen behind the specific immune response of animal, to obtain.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105061343A (en) * | 2015-08-07 | 2015-11-18 | 上海交通大学 | Tri(2,3-dibromopropyl)triallyisocyanurate hapten, and preparation and application |
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| 余宇燕: "环境中微量氯苯酚类环境激素的免疫分析方法研究", 《东华大学博士学位论文》 * |
| 戴光坤: "八氯苯乙烯羧酸类半抗原的设计与合成", 《湖南大学硕士学位论文》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105061343A (en) * | 2015-08-07 | 2015-11-18 | 上海交通大学 | Tri(2,3-dibromopropyl)triallyisocyanurate hapten, and preparation and application |
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