CN102659883A - DiHT-type universal hapten and artificial antigen of food radiation marker, and preparation method and application thereof - Google Patents
DiHT-type universal hapten and artificial antigen of food radiation marker, and preparation method and application thereof Download PDFInfo
- Publication number
- CN102659883A CN102659883A CN2012100478480A CN201210047848A CN102659883A CN 102659883 A CN102659883 A CN 102659883A CN 2012100478480 A CN2012100478480 A CN 2012100478480A CN 201210047848 A CN201210047848 A CN 201210047848A CN 102659883 A CN102659883 A CN 102659883A
- Authority
- CN
- China
- Prior art keywords
- diht
- solution
- antigen
- cooh
- haptin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- DQTNUZBYVJDTGD-QUBJWBRDSA-N CCCCCC1C(C)C2O[C@H]1C2 Chemical compound CCCCCC1C(C)C2O[C@H]1C2 DQTNUZBYVJDTGD-QUBJWBRDSA-N 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides a DiHT-type universal hapten and a DiHT-type artificial antigen of food radiation marker, and a preparation method and application thereof. The preparation method comprises the steps of: taking dihydrothymine (DiHT) as an initial reactant, synthesizing a derivative of dihydrothymine by using a succinic anhydride method, wherein the derivative is hapten DiHT-COOH; coupling the hapten with carrier proteins BSA, OVA by adopting a mixed anhydride method and an active ester method, preparing a dihydrothymine artificial antigen and a dihydrothymine coating antigen, preparing a polyclonal antibody serum through BALB/c mouse immunized by the artificial antigen. A titer of the polyclonal antibody serum reaches 1:2.56*10<4> assayed by an indirect uncompetitive ELISA method, and an IC50 of the polyclonal antibody serum for DiHT is 191.8669 ng/mL, thereby indicating that the obtained DiHT antibody serum has a high affinity for DiHT-type materials. According to the invention, the yield is high, the effect is good, the operation is convenient and quick, and a base for establishing a food radiation immunological identification technology based on DiHT, the specific products of irradiated food, is provided.
Description
Technical field
The invention belongs to immunodetection and technical field of biochemical industry, relate to a kind of Food irradiation mark DiHT class universal hapten, artificial antigen and preparation method thereof and application.
Background technology
Irradiation technique is through the research of decades, as a kind of efficient, ecological, safe new and high technology, to the hygienic safety of guaranteeing food, reduce post-harvest loss, give a impetus to trade and consumption plays irreplaceable effect.Its application is approved by more and more countries that according to the preliminary statistics, existing 38 countries and regions, the whole world have ratified 548 kinds of food and seasonings can be used radiation treatment.But singularity in view of irradiated food; The universal standard of CAC irradiated food is all stipulated will identify irradiated food with the rules of a lot of countries; Stipulate in the general rule of CAC irradiated food; The maximum absorbed dose of irradiated food should not surpass 10kGy, even the irradiation composition of the finished product constitutes less than 1%, must force on label, to indicate this product postdose yet.So far; CAC has issued the authentication method of ten kinds of irradiated foods; Release light (PSI) method, DNA comet method etc. like thermoluminescence (TL) method, ESR spectrum (ESR) method and light; For the supervision of irradiated food provides technical support, but the time that these authentication methods detect is long, pre-treatment is loaded down with trivial details, need expensive plant and instrument and need the professional to operate.Along with normally carrying out of irradiated food international trade; Various countries are urgent further to the requirement of irradiated food authenticate technology, in international trade, and especially customs and commodity inspection department; The sample that needs to identify gets more and more, and is badly in need of setting up easy, quick, special, sensitive authentication method.
Enzyme-linked immunosorbent assay (Enzyme-linked immunsorbent assay; Be called for short ELISA) be the method for quick of a kind of binding immunoassay specific reaction and enzymatic reaction; It is based on the specific reaction of antigen and antibody; Have high specificity, highly sensitive, amount of analysis waits advantage greatly, safely and fast, be used widely at aspects such as hazardous material such as detecting the residual and mycotoxins of food Chinese traditional medicine.Enzyme immunoassay is explored more existing reports in the irradiated food application in identification; (Christopher T.Elliott such as study on determination method like the special product 2-of Food irradiation dodecyl cyclobutanone; Lynne Hamilton.Detection of Irradiated Chicken Meat by Analysis of Lipid Extracts for 2-Substituted Cyclobutanones Using an Enzyme Linked Immunosorbent [J] .Analyst; 1995,120:2337-2341; Lynne Hamilton; M.Hilary Stevenson; Derek R.Boyd.Detection of 2-substituted cyclobutanes as irradiation products of lipid-containing foods:synthesis and applications of cis-and trans-2-(tetradec-5 '-enyl) cyclobutanones and11-(2 '-oxocyclobutyl) undecanoic acid [J] .J.Chem.Soc.; Perkin Trans.1; 1996,139-146; Anne L.Tyreman; Graham A.Bonwick; Christopher J.Smith.Detection of irradiated food by immunoassay-development and optimization of an ELISA for dihydrothymidine in irradiated prawns [J] .International Journal of Food Science&Technology.2004,39:533-540; Williams; J.HH.Tyreman; AL.Immunological detection of modified DNA bases in irradiated food [J] .In:Detection Methods for Irradiated Food:Current Status, 1996.367-374.).
The synthetic of artificial antigen is Antibody Preparation and the step of setting up the immune analysis method most critical in the food immunodetection, and haptenic synthesizing is artificial antigen synthetic basis.Most of haptin are molecular weight less than 1000 micromolecular compound, and itself does not possess immunogenicity, therefore, must first small molecules and the sub-coupling of carrier proteins prepare complete antigen.If the small molecules haptin can not directly pass through bi-functional cross-linking agent and carrier proteins covalent coupling, then need on small molecules, produce active function group through derivatization process earlier, with this as haptin, again with the carrier protein coupling.DiHT is a kind of small molecules, and relative molecular weight is 244.24, is haptin; Can't not possess immunogenicity for immune t-cell provides recognition site, so before the preparation artificial antigen; To transform its end group; Since DiHT originally on one's body less than and protein link coupled reactive group, must modify suitable connecting arm in the modification to haptin.The haptin key for design is the feature structure that keeps former test substance as much as possible, and introduce in position suitable connecting arm and with the reactive group of carrier protein couplet.The introducing position of the connecting arm of different structure, different coupling methods or connecting arm all influences sensitivity and specificity that the ELISA of follow-up foundation detects.At present, be that haptin prepares synthetic antibody and artificial antigen and carries out enzyme immunoassay (ELISA) and rarely have report with dihydro deoxythymidine (DiHT) verivate.
Summary of the invention
The present invention provides a kind of Food irradiation mark DiHT universal hapten, artificial antigen and preparation method thereof and application according to the demand and the blank in above-mentioned field.The present invention is an initial reactant with dihydro deoxythymidine (DiHT), uses the verivate of succinyl oxide method synthesizing dihydro deoxythymidine, i.e. haptin DiHT-COOH; Adopt mixed anhydride method and active ester method respectively haptin and carrier proteins BSA, OVA to be carried out coupling; The artificial antigen and the coating antigen of preparation dihydro deoxythymidine; This artificial antigen immunity BALB/c mouse prepares polyvalent antibody, indirect non-competing ELISA method detect tire reach 1: 2.56 * 10
4, polyvalent antibody is to the IC of DiHT
50Be 191.8669ng/mL, show that the DiHT antiserum(antisera) that obtains has higher affinity to DiHT class material, and productive rate height of the present invention is effective, simple and efficient, for the Food irradiation immunity authenticate technology of setting up based on the special auxilliary hydrolysis products DiHT of food provides the basis.
A kind of dihydro deoxythymidine class universal hapten has following molecular structure:
A kind of dihydro deoxythymidine class artificial antigen; It is characterized in that: the molecule that uses following structure; The molecule that
and carrier protein couplet obtain
Said carrier proteins refers to bovine serum albumin BSA or Protalbinic acid OVA, and the antigen for preparing is respectively immunogen and coating antigen.
The preparation method of above-mentioned artificial antigen comprises being prepared as follows step:
(1) synthetic universal hapten;
(2) said universal hapten and carrier protein couplet;
The synthetic route of said synthetic universal hapten is:
Said haptin DiHT-COOH and carrier protein BSA link coupled synthetic route are:
Said haptin DiHT-COOH and carrier protein OVA link coupled synthetic route are:
The preparation process of said universal hapten is:
(1) take by weighing DiHT 5mg and succinyl oxide 16.4mg, be dissolved in the 5mL anhydrous pyridine, backflow 10h is stirred in 60 ℃ of heating;
(2) after reaction finishes, in solution, add the 10mL methylene dichloride, collect lower floor's mixture, use 10mL 5% (v/v) salt pickling 3 times then, discard dichloromethane layer, residue is used the 10mL ether dissolution, and with 10mL de-ionized washing 3 times, purifying gets haptin,
Said universal hapten prepares immunogen with carrier proteins BSA and OVA coupling and coating antigen adopts mixed anhydride method and active ester method preparation respectively, and concrete steps are following:
(1) mixed acid anhydride prepares artificial antigen DiHT-COOH-BSA
A, HT-COOH powder 6.68mg are dissolved in the 3mL dry DMF, and vibration is stirred, and adds 40 μ L tri-n-butylamines, and ice bath adds isobutyl chlorocarbonate 40 μ L down, 4 ℃ of stirring reaction 1h, after rise to room temperature 1h, solution 1;
B, title 120mg BSA are dissolved in the borate buffer solution (pH=8.7) of 12mL 0.2mol/L, get solution 2;
C, solution 1 dropwise joined in the solution 2 under stirring at room go, in 15 ℃ of following stirred overnight.4 ℃ of zero(ppm) water of mixed solution stir dialysis, and 4h changes water once, changes altogether 6 times, in dialyzate, can not survey till the haptin uv-absorbing, and lyophilize gets DiHT-COOH-BSA,
(2) active ester method prepares coating antigen DiHT-COOH-OVA
A, take by weighing DiHT-COOH powder 6.68mg and be dissolved in the 1mL dry DMF, then with 80mg DCC heating for dissolving in the 3mL dry DMF.The DCC dissolving is cooled to room temperature is added in the MBC solution, add 28mg NHS, stirring reaction 5h under the room temperature.Put then in 4 ℃ of refrigerators more than the airtight 2h of leaving standstill, the centrifuging and taking supernatant, solution 3;
B, take by weighing OVA 40mg, be dissolved in respectively among the PB of 4mL, 0.05mol/L, pH=8.0, solution 4;
C, solution 3 mixed be added to solution 4, rise to stirring at room reaction 1h then gradually, in 4 ℃ down slow stirring reactions spend the night.Be respectively charged into reaction solution in the dialysis tubing next day, under the PBS of 0.01mol/L, pH=7.4 and 4 ℃ of conditions, dialyses, and every 6h changes dialyzate one time, changes altogether 6 times, in dialyzate, can't check till the haptin uv-absorbing, DiHT-COOH-OVA solution.
Above-mentioned artificial antigen is used for detecting the application of dihydro deoxythymidine antibody-like or antiserum(antisera) in preparation as immunogen.
Above-mentioned artificial antigen is adopting indirect Elisa method to detect in the dihydro deoxythymidine class application as coating antigen.
The polyvalent antibody that described artificial antigen prepares as the immunogen immune animal, polyclonal antibody, antibody or hybridoma.
The key point of enzyme-linked immunoassay method is that preparation has an immunogenic artificial antigen, and the height antigenic preparation of tiring is the important prerequisite that obtains specific antibody and guarantee the immunodetection susceptibility, and wherein haptenic preparation is again the key of antigen prepd.It is a kind of small molecules that the Food irradiation specificity is assisted hydrolysis products-dihydro deoxythymidine DiHT, and relative molecular weight is 244.24, is haptin; Can't not possess immunogenicity for immune t-cell provides recognition site, so before the preparation artificial antigen; To transform its end group,, must modify haptin because DiHT does not originally have and protein link coupled reactive group on one's body; Analyze, study, explored several different methods in this test and modify connecting arm, and adopt TLC and NMR to characterize during the course, find to adopt the succinyl oxide method that the terminal hydroxyl of DiHT is modified; Effect is better, can reach requirement of experiment.The haptin of the present invention's preparation has the common trait of the auxilliary hydrolysis products of Food irradiation specificity-dihydro deoxythymidine DiHT quasi-molecule structure; The antiserum(antisera) for preparing as the immunogen immune animal can combine the material specificity with this molecular characterization, i.e. specific recognition Food irradiation specificity is assisted hydrolysis products-dihydro deoxythymidine DiHT quasi-molecule.For can with the coupling of carrier proteins molecule; Need to introduce connecting arm and carboxyl; The synthetic route that antigen of the present invention adopted is based on has considered that connecting arm and carboxyl introduce the influence of position choice to the three-D space structure of synthetic product; Avoid having influenced the space conformation of the molecular structure characteristic of synthetic product owing to the introducing of connecting arm as far as possible, unaffected to guarantee as the structure in antigen determining site.
The present invention has prepared immunogen DiHT-COOH-BSA and coating antigen DiHT-COOH-OVA through make haptin and protein coupling with mixed anhydride method, active ester method; Coupling ratio is between 5: 1～30: 1; Meet small molecules immunity requirement, the inventive method is easy and simple to handle, and side reaction is few.Mainly adopt evaluation of UV UV spectrum and SDS-PAGE method to carry out to immunogen and the antigenic authentication method of detection.Because carrier proteins and haptin all have distinctive uv absorption spectrum; So conjugate and carrier proteins are compared analysis through UV scanning; Can observe the variation of the uv-spectrogram of generation very intuitively; The maximum absorption band of DiHT-COOH-BSA and DiHT-COOH-OVA is respectively in 260nm and 287nm wavelength, and synthetic artificial antigen characteristic peak does not overlap with haptin and carrier, thereby can identify the coupling success through uv scan.SDS-PAGE also is the authentication method of using always, because the molecular weight of conjugate is greater than the molecular weight of carrier, its rate of migration when electrophoresis is different, and after dyed and the decolouring, two bands will be in different positions, therefore also can prove out whether success of coupling.
Efficient antigenic preparation is the important prerequisite that obtains high-affinity antibody, selects at the antigenic carrier proteins of hapten conjugation artificial immunization, and rabbit anteserum albumen (RSA), human serum protein (HSA), Thyroprotein (TG) etc. are arranged.The selection of protein carrier has heterology usually with by the animal of immunity, and the immunne response of animal body generation is stronger like this, thereby obtains high-titer antibody easily.But also the someone thinks that oneself protein also can be used as carrier, and immune effect is superior to heterologous protein.Select the carrier proteins of BSA as immunizing antigen among the present invention for use, OVA is as the carrier proteins of envelope antigen.These two kinds of albumen physicochemical property are stable, and contain higher lysine content and free amino group group, and economical and practical, draw materials conveniently.The animal immune test-results shows, dosage, the number of times of immunity, and the selection of adjuvant all can exert an influence to the result of immunity.Immunizing dose is 150 μ g/ for the first time; Employing tail vein injection immunity, severe side effect inboth behind the mouse immune as a result, back scheme is adjusted into the subcutaneous multi-point injection in back; Immunizing dose only is reduced to 100 μ g/; The result shows that immune effect is better, successful acquisition height tire, specificity polyvalent antibody preferably, for later on further development ELISA detect the irradiated food test kit and lay a good foundation.
DiHT and DiHT-COOH haptin white powder solution are carried out the TLC detection; Developping agent is a methyl alcohol: methylene dichloride: acetate=1: 9: 0.1 (v/v/v); The phosphor dot that obtains white solid obviously is different from the raw material point; Rf is 0.48, and tentatively obtaining white solid is title product DiHT-COOH.
Can know that from haptin DiHT nucleus magnetic resonance (1H-NMR) spectrum detected result the peak of target substance occurs, prove that product is exactly a haptin, can carry out the coupling preparation of artificial antigen.
The evaluation of artificial antigen is mainly identified through UV UV scanning and two kinds of methods of SDS-PAGE gel electrophoresis.
Close additive theory and can know according to what uv absorption spectrum and UV absorbed; Characteristic peak after the integration does not overlap with haptin and carrier; Skew has appearred; And be respectively 9.8: 1 and 12: 1 with the coupling ratio of BSA and OVA according to lambert's one Beer's law calculating DiHT-COOH, it is generally acknowledged that coupling ratio can produce specific antibody preferably at 5: 1～30: 1 in the scope, the coupling ratio explanation haptin that the present invention obtained is successful with carrier B SA and OVA coupling respectively.
The DiHT-COOH-BSA conjugate is carried out the SDS-PAGE gel electrophoresis to be identified; The mobility of its BSA of mobility ratio that can know synthetic artificial antigen DiHT-COOH-BSA band according to SDS-PAGE gel electrophoresis qualification result is smaller, and the molecular mass that shows artificial antigen increases than the molecular mass of its respective carrier.This is because carrier connects the haptenic result gone up certain number, electrophoresis result show DiHT-COOH with BSA success coupling.Traction phenomenon has in various degree all appearred in the synthetic antigen of 4 and 5 bands among the SDS-PAGE gel electrophoresis qualification result figure in the corresponding position, because carrier protein and inconsistent the causing of small-molecule substance bonded molecule number.Randomness when the just micromolecular average number of each carrier protein bonded that the UV scanning method calculates, the result of SDS-PAGE then link to carrier and haptin and disparity have more really reacts.
Immunity back mice serum is tired and can be known through indirect non-competing ELISA detected result; Mouse all can produce higher positive antiserum(antisera) of tiring; Show that synthetic artificial antigen Hapten-BSA can stimulate animal to produce antibody well; But because the difference of animal individual, tiring that each mouse produces is slightly different.
Affinity qualification result by DiHT can know that in the 156.25-10000ng/mL scope, 3 mouse have all produced inhibition.With the haptin is that research object is set up regression equation, shows that the DiHT antiserum(antisera) that obtains has higher affinity to DiHT.
Description of drawings
Fig. 1 is a dihydro deoxythymidine molecular structure
Fig. 2 is a DiHT haptin synthetic route chart
Fig. 3 is haptin DiHT-COOH and carrier protein BSA link coupled synthetic route chart
Fig. 4 is haptin DiHT-COOH and carrier protein OVA link coupled synthetic route chart
Fig. 5 is Hapten TLC detected result figure
A:DiHT wherein; B: haptin
Fig. 6 be haptin DiHT nucleus magnetic resonance (
1H-NMR) spectrogram
Fig. 7 is the uv absorption spectra of haptin, BSA and immunizing antigen conjugate
Fig. 8 is the uv absorption spectra of haptin, OVA and coating antigen conjugate
Fig. 9 is the SDS-PAGE electrophoresis qualification result figure of DiHT-COOH-BSA conjugate
Wherein swimming lane 1: protein marker; Swimming lane 2:20 μ L BSA standard substance; Swimming lane 3:40 μ L BSA standard substance; Swimming lane 4:20 μ L synthetics DiHT-COOH-BSA; Swimming lane 5:40 μ L synthetics DiHT-COOH-BSA.
Figure 10 is that the sero-fast competition of DiHT suppresses graphic representation
Embodiment
It is in order further to understand the present invention better that the present invention provides following embodiment; Be not limited to said preferred forms; Content of the present invention and protection domain are not constituted restriction; Anyone all drops within protection scope of the present invention under enlightenment of the present invention and the identical or akin product with the present invention that draws with the combination of any prior art.
Preparation of embodiment 1. haptin and evaluation
Take by weighing DiHT 5mg and succinyl oxide 16.4mg, be dissolved in the 5mL anhydrous pyridine.Backflow 10h is stirred in 60 ℃ of heating.After reaction finishes, in solution, add the 10mL methylene dichloride, collect lower floor's mixture; Use 10mL 5% (v/v) salt pickling 3 times then, discard dichloromethane layer, residue is used the 10mL ether dissolution; With 10mL de-ionized washing 3 times, ether layer is used anhydrous magnesium sulfate drying, vacuum filtration; Behind purification by silica gel column chromatography, get oily matter after the ether volatilization.In reaction process, adopt thin-layer chromatography (TLC) to follow the tracks of, and to product with nuclear magnetic resonance map (
1H-NMR) characterize, process is seen Fig. 2.
Synthesizing of embodiment 2.DiHT artificial antigen
Adopt mixed anhydride method and active ester method respectively haptin DiHT and carrier proteins BSA, OVA to be carried out coupling.
(1) mixed acid anhydride prepares artificial antigen DiHT-COOH-BSA method
Take by weighing DiHT-COOH powder 6.68mg and be dissolved in the 3mL dry DMF, vibration is stirred, and adds 40 μ L tri-n-butylamines, and ice bath adds isobutyl chlorocarbonate 40 μ L down, 4 ℃ of stirring reaction 1h, after rise to room temperature 1h, this be called reaction solution 1.Claim that 120mg BSA is dissolved in the borate buffer solution (pH=8.7) of 12mL 0.2mol/L, this is a solution 2.Reaction solution 1 dropwise joined in the solution 2 under stirring at room go, in 15 ℃ of following stirred overnight.4 ℃ of zero(ppm) water of mixed solution stir dialysis, and 4h changes water once, changes altogether 6 times, in dialyzate, can not survey till the haptin uv-absorbing, and lyophilize gets DiHT-COOH-BSA (Fig. 3), and packing is subsequent use.
(2) active ester method prepares coating antigen DiHT-COOH-OVA
Take by weighing DiHT-COOH powder 6.68mg and be dissolved in the 1mL dry DMF, then with 80mg DCC heating for dissolving in the 3mL dry DMF.The DCC dissolving is cooled to room temperature is added in the MBC solution, add 28mg NHS, stirring reaction 5h under the room temperature.Put then in 4 ℃ of refrigerators more than the airtight 2h of leaving standstill, the centrifuging and taking supernatant is called reaction solution 2.Take by weighing OVA 40mg, be dissolved in respectively among the PB of 4mL, 0.05mol/L, pH=8.0.Reaction solution 2 stirrings are added in the protein solution, rise to stirring at room reaction 1h then gradually, slow stirring reaction spends the night under 4 ℃.Be respectively charged into reaction solution in the dialysis tubing next day, under the PBS of 0.01mol/L, pH=7.4 and 4 ℃ of conditions, dialyses; Every 6h changes dialyzate one time; Change altogether 6 times, in dialyzate, can't check till the haptin uv-absorbing, get DiHT-COOH-OVA (Fig. 4) solution; Be sub-packed in the frozen pipe ,-20 ℃ frozen.
The evaluation of embodiment 3. artificial antigens
The evaluation of artificial antigen is mainly identified through UV UV scanning and two kinds of methods of SDS-PAGE gel electrophoresis.
(1) UV scanning is identified
Artificial antigen and BSA are diluted to proper concn, in wavelength 190～400nm scope, scan, obtain haptenic UV spectrum.
(2) the SDS-PAGE gel electrophoresis is identified
Selecting to concentrate gum concentration is 5%, and resolving gel concentration is 12%, and concentrating glue voltage is 120v, and separation gel voltage is 60v, and applied sample amount is respectively 20 μ L and 40 μ L, adopts coomassie brilliant blue staining, takes a picture with the ultraviolet gel imaging system and observe in the decolouring back.
The mensuration of embodiment 4. artificial antigen coupling ratios
According to lambert's one Beer's law, wavelength one timing, the uv-absorbing of mixture has additive properties.Adopt the total length ultraviolet spectrophotometer that haptin, carrier proteins and conjugate are scanned, confirm according to absorption peak whether coupling is successful
[17]The binding ratio calculation formula is following:
Wherein: OD
Conjugate--the conjugate light absorption value; OD
Protein--the protein light absorption value; OD
Hapten--the haptin light absorption value; C
Hapten--haptin concentration; C
Protein--protein concn; M
Protein--protein molecular weight; M
Hapten--antigen molecular
The preparation of embodiment 5. polyvalent antibodies
DiHT-COOH-BSA is diluted with saline water; Add the abundant mixing emulsification of equivalent Freund's complete adjuvant; The subcutaneous multi-point injection in the BALB/c mouse back; Initial immunity dosage is 100 μ g/, and later 2 weeks of every interval change Freund's complete adjuvant into Freund's incomplete adjuvant and carry out booster immunization with method, and immunizing dose progressively increases on a small quantity.From the 4th immunity, 1 week docking end in each immunity back is got blood, and serum spends the night in 4 ℃ of placements, and the centrifugal 15min of 5000r/min abandons deposition, and upper serum places 4 ℃ of refrigerators subsequent use
[18], press 1: 100 times of dilution during use.
With DiHT-COOH-OVA as coating antigen. with DiHT-COOH-BSA antiserum(antisera) doubling dilution; Adopt indirect non-competing ELISA method antagonistic Serum to detect; The reaction back is measured the OD value down in the 450nm wavelength; Every hole is surveyed 4 times, averages, and polyclonal antibody is measured through indirect competitive ELISA (CI-ELISA) haptenic inhibiting rate.Carry out according to best antigen-antibody working concentration and optimal conditions; With haptin with PBST (0.01mol/LPBS, pH=7.4 contain 0.05%Tween-20) compound concentration be respectively 156.25,312.5,625,1250,2500,5000, the standardized solution of 10000ng/mL; With the antiserum(antisera) extension rate is X-coordinate; The OD value is an ordinate zou, sets up competition and suppresses curve, calculates concentration (IC in the inhibition
50).
To mark article DiHT and after the reaction of Fig. 2 synthetic route, obtain white powder; With DiHT with obtain white powder solution and carry out TLC and detect; Developping agent is a methyl alcohol: methylene dichloride: acetate=1: 9: 0.1 (v/v/v); Result such as Fig. 5, the phosphor dot that obtains white solid obviously is different from the raw material point, calculates white solid R
f=0.48, tentatively obtaining white solid is title product Hapten, but also need synthesize further checking.
The haptenic NMR spectrum of embodiment 8.DiHT-COOH is identified
Can know from detected result, (1HNMR) (DMSO δ ppm): 11.056 (s, H ,-COOH), 9.860 (s, H, 3-NH), 5.803 (s, H; N-C-H), 4.737 (s, H ,-OH), 4.207 (s, 2H ,-CH2), 3.566 (s, H; H-3 '), 3.229 (s, H, H-4 '), 3.409 (t, 2H ,-CH2), 2.706～2.780 (t; 2H ,-CH2), 2.069 (s, H, 5-H), 2.228 (s, 2H ,-CH2); 1.242 (5-CH3), but assorted peak has appearred in 7.298-7.396 for m, 3H, and this possibly be because to cross the pillar purifying incomplete, but the peak of target substance occurs, and prove that product is exactly a haptin, and the coupling that can carry out artificial antigen prepares.
The evaluation of embodiment 9. artificial antigens
(1) UV identifies
BSA, OVA, DiHT-COOH, DiHT-COOH-BSA and five samples of DiHT-COOH-OVA scan through ultraviolet spectrophotometer; The result shows; BSA and OVA have maximum absorption band at 278nm and 279nm wavelength respectively, and the maximum absorption band of DiHT-COOH-BSA and DiHT-COOH-OVA is respectively in 260nm and 287nm wavelength.Close additive theory and can know according to what UV absorbed; Characteristic peak after the integration does not overlap with haptin and carrier; Skew has appearred; And be respectively 9.8: 1 and 12: 1 with the coupling ratio of BSA and OVA according to lambert's one Beer's law calculating DiHT-COOH, it is generally acknowledged that coupling ratio can produce specific antibody preferably at 5: 1～30: 1 in the scope, the coupling ratio explanation haptin that this institute obtains is successful with carrier B SA and OVA coupling respectively.
(2) SDS-PAGE gel electrophoresis
The DiHT-COOH-BSA conjugate is carried out SDS-PAGE identify, Fig. 9 shows that the mobility of its BSA of mobility ratio of synthetic artificial antigen DiHT-COOH-BSA band is smaller, shows the molecular mass increase of the molecular mass of artificial antigen than its respective carrier.Think that this is because carrier connects the haptenic result gone up certain number, electrophoresis result show DiHT-COOH with BSA success coupling.Traction phenomenon has in various degree all appearred in the synthetic antigen of 4 and 5 bands among Fig. 9 in the corresponding position, maybe be because carrier protein and inconsistent the causing of small-molecule substance bonded molecule number.Randomness when the just micromolecular average number of each carrier protein bonded that the UV scanning method calculates, the result of SDS-PAGE then link to carrier and haptin and disparity have more really reacts.
The mensuration that embodiment 10. polyclonal antibodies are tired
The anti-DiHT-COOH-BSA serum titer of table 1
Immunity back mice serum is tired as shown in table 1 through indirect non-competing ELISA detected result, and mouse all can produce higher positive antiserum(antisera) of tiring, and wherein 1, No. 3 mice serum antibody titers of mouse all reach 1: 10
4, it is low slightly that No. 2 mouse antibodies are tired, but also can reach 1: 6.4 * 10
3This result shows that synthetic artificial antigen Hapten-BSA can stimulate animal to produce antibody well, but because the difference of animal individual, tiring that each mouse produces is slightly different.
The affinity qualification result of DiHT is seen table 2, and analytical table 2 can know that in the 156.25-10000ng/mL scope, 3 mouse have all produced inhibition, select No. 3 mouse serum, with its OD
450Value is converted into B/B
0Lg [DiHT/100] is being carried out regression analysis, and the sero-fast competition of DiHT suppresses curve such as Figure 10, and the equation of linear regression that mouse polyvalent antibody standard suppresses curve is v=-0.2300x+0.5651, coefficient R
2=0.9905, be 191.8669ng/mL according to the regression equation calculation polyvalent antibody to the IC50 of DiHT, show that the DiHT antiserum(antisera) that obtains has higher affinity to DiHT.
Table 2 mouse resisting anteserum is tired to the inhibition of DiHT
The detection test of embodiment 12. artificial antigens of the present invention.
With embodiment 11 determined antigen-antibody best effort concentration; Detect antibody that antigen prepd of the present invention obtains to dihydro deoxythymidine, deoxythymidine, 1 through indirect Elisa method; The specificity of analog molecules such as 3-dimethyl-thymus pyrimidine and thymus pyrimidine; The result is as shown in table 1, and antiserum(antisera) is to the analogue of antigenic structure with the present invention preparation, like dihydro deoxythymidine, deoxythymidine, 1; 3-dimethyl-thymus pyrimidine and thymus pyrimidine etc. have tangible cross reaction, and trimethoprim and the not fork reaction of 2-chloropyrimide to having textural difference.
Table 1
Claims (9)
1. dihydro deoxythymidine class universal hapten has following molecular structure:
2. the artificial general antigen of a dihydro deoxythymidine class; It is characterized in that: use the molecule of following structure, the molecule that coupling carrier albumen obtains on the carboxylic group of
.
3. the general antigen of manual work according to claim 2, said carrier proteins refers to bovine serum albumin BSA or Protalbinic acid OVA, the antigen for preparing is respectively immunogen and coating antigen.
4. claim 2 or the general antigenic preparation method of 3 said manual works comprise being prepared as follows step:
(1) synthetic universal hapten;
(2) said universal hapten and carrier protein couplet;
Said haptin DiHT-COOH and carrier protein BSA link coupled synthetic route are:
Said haptin DiHT-COOH and carrier protein OVA link coupled synthetic route are:
5. the general antigenic preparation method of the said manual work of claim 4, the preparation process of said universal hapten is:
(1) take by weighing DiHT 5mg and succinyl oxide 16.4mg, be dissolved in the 5mL anhydrous pyridine, backflow 10h is stirred in 60 ℃ of heating;
(2) after reaction finishes, in solution, add the 10mL methylene dichloride, collect lower floor's mixture, use 10mL 5% (v/v) salt pickling 3 times then, discard dichloromethane layer, residue is used the 10mL ether dissolution, and with 10mL de-ionized washing 3 times, purifying gets haptin.
6. claim 4 or 5 described preparing methods adopt mixed anhydride method that said universal hapten and carrier proteins BSA coupling are prepared immunogen or coating antigen, and step is following:
A, HT-COOH powder 6.68mg are dissolved in the 3mL dry DMF, and vibration is stirred, and adds 40 μ L tri-n-butylamines, and ice bath adds isobutyl chlorocarbonate 40 μ L down, 4 ℃ of stirring reaction 1h, after rise to room temperature 1h, solution 1,
B, title 120mg BSA are dissolved in the borate buffer solution (pH=8.7) of 12mL 0.2mol/L, get solution 2,
C, solution 1 dropwise joined in the solution 2 under stirring at room go, in 15 ℃ of following stirred overnight, 4 ℃ of zero(ppm) water of mixed solution stir dialysis; 4h changes water once, changes altogether 6 times, in dialyzate, can not survey till the haptin uv-absorbing; Lyophilize gets DiHT-COOH-BSA;
Or adopt the active ester method preparation that said universal hapten and carrier proteins OVA coupling are prepared immunogen or coating antigen, step is following:
A, take by weighing DiHT-COOH powder 6.68mg and be dissolved in the 1mL dry DMF, then with 80mg DCC heating for dissolving in the 3mL dry DMF, the DCC dissolving is cooled to room temperature is added in the MBC solution; Add 28mg NHS; Stirring reaction 5h under the room temperature puts in 4 ℃ of refrigerators more than the airtight 2h of leaving standstill the centrifuging and taking supernatant then; Get solution 3
B, take by weighing OVA 40mg, be dissolved in respectively among the PB of 4mL, 0.05mol/L, pH=8.0, solution 4,
C, solution 3 mixed be added to solution 4, rise to stirring at room reaction 1h then gradually, in 4 ℃ down slow stirring reactions spend the night; Be respectively charged into reaction solution in the dialysis tubing next day, under the PBS of 0.01mol/L, pH=7.4 and 4 ℃ of conditions, dialyses; Every 6h changes dialyzate one time; Change altogether 6 times, in dialyzate, can't check till the haptin uv-absorbing, get DiHT-COOH-OVA.
7. claim 2 or the general antigen of 3 described manual works are used for detecting the antibody of dihydro deoxythymidine class or the application of antiserum(antisera) as immunogen in preparation.
8. claim 2 or the general antigen of 3 described manual works are adopting indirect Elisa method to detect in the dihydro deoxythymidine class application as coating antigen.
9. the antiserum(antisera), polyclonal antibody, antibody or the hybridoma that prepare as the immunogen immune animal of claim 2 or the general antigen of 3 described manual works.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210047848.0A CN102659883B (en) | 2012-01-16 | 2012-02-27 | DiHT-type universal hapten and artificial antigen of food radiation marker, and preparation method and application thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210012971.9 | 2012-01-16 | ||
| CN201210012971 | 2012-01-16 | ||
| CN201210047848.0A CN102659883B (en) | 2012-01-16 | 2012-02-27 | DiHT-type universal hapten and artificial antigen of food radiation marker, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102659883A true CN102659883A (en) | 2012-09-12 |
| CN102659883B CN102659883B (en) | 2015-01-07 |
Family
ID=46769491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210047848.0A Expired - Fee Related CN102659883B (en) | 2012-01-16 | 2012-02-27 | DiHT-type universal hapten and artificial antigen of food radiation marker, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102659883B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101863981A (en) * | 2010-04-21 | 2010-10-20 | 广东工业大学 | Preparation method of anti-bisphenol A monoclonal antibody |
| CN101962368A (en) * | 2010-08-17 | 2011-02-02 | 华南农业大学 | Cyanurate hapten, artificial antigen and antibody, and preparation method and applications thereof |
| CN101161679B (en) * | 2007-11-01 | 2011-07-20 | 江南大学 | Method for preparing dexamethasone artificial antigen |
| CN102241764A (en) * | 2011-05-05 | 2011-11-16 | 南京工业大学 | Preparation method and application of pyrene artificial immunogen PY-BSA |
-
2012
- 2012-02-27 CN CN201210047848.0A patent/CN102659883B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161679B (en) * | 2007-11-01 | 2011-07-20 | 江南大学 | Method for preparing dexamethasone artificial antigen |
| CN101863981A (en) * | 2010-04-21 | 2010-10-20 | 广东工业大学 | Preparation method of anti-bisphenol A monoclonal antibody |
| CN101962368A (en) * | 2010-08-17 | 2011-02-02 | 华南农业大学 | Cyanurate hapten, artificial antigen and antibody, and preparation method and applications thereof |
| CN102241764A (en) * | 2011-05-05 | 2011-11-16 | 南京工业大学 | Preparation method and application of pyrene artificial immunogen PY-BSA |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102659883B (en) | 2015-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | An immunochromatographic strip sensor for sildenafil and its analogues | |
| CN102516148B (en) | Half antigen and antigen of tenuazonic acid and preparation method and application thereof | |
| CN101245032A (en) | Leuco malachite green hapten, produced antibody and application of the antibody | |
| CN103739703A (en) | Glycocholic acid immunogen, anti-glycocholic acid specific antibody and detection reagent | |
| CN104327183B (en) | A kind of paclobutrazol artificial antigen and preparation method and the application of polyclonal antibody | |
| CN101962358A (en) | Ciprofloxacin hapten, artificial antigen and antibody and preparation method and application thereof | |
| CN105301252A (en) | Immunomagnetic bead for enrichment and purification of ochratoxin A, and preparation method and application of immunomagnetic bead | |
| CN105712970A (en) | Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof | |
| CN106366021A (en) | Urethane hapten composition and artificial antigen composition, and preparation methods and application thereof | |
| CN103018440B (en) | A kind of clopidol colloidal gold chromatographic test strip or card | |
| CN102875671A (en) | Clothianidin antigen, antibody and application thereof | |
| CN104387431A (en) | Preparation method of high-specificity ribavirin artificial antigens | |
| Wang et al. | Detection of triclabendazole and three metabolites in bovine muscle samples with a gold nanoparticle-based lateral flow immunoassay | |
| CN105277423A (en) | Immunomagnetic bead used for vomitoxin enrichment purifying and preparation method and application thereof | |
| CN102250238A (en) | Method for synthesizing swainsonine antigen | |
| CN105624119B (en) | The general monoclonal antibody of anti-capsaicine, Dihydrocapsaicin, the synthetic capsaicin of hybridoma cell strain YQQD8 and its generation | |
| CN104383889B (en) | A kind of Dihydrocapsaicin polyclonal antibody adsorbent, immune affinity column and its preparation method and application | |
| CN104710488A (en) | Synthesis method of half-antigen and complete antigen of tulathromycin | |
| CN103589688B (en) | The monoclonal antibody of anti-three kinds of organophosphorus pesticides and application thereof | |
| CN105319356A (en) | Immunomagnetic bead for aflatoxin B1 enrichment purification and preparation method and application thereof | |
| CN102659883B (en) | DiHT-type universal hapten and artificial antigen of food radiation marker, and preparation method and application thereof | |
| CN103145633B (en) | Novel melamine antigen and antibody and application | |
| CN109438424A (en) | Ribavirin haptens and artificial antigen and the preparation method and application thereof | |
| CN113046325B (en) | Vitamin K 3 Monoclonal antibody hybridoma cell strain and application thereof | |
| CN111499637B (en) | Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150107 Termination date: 20160227 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |