CN102659883B - DiHT-type universal hapten and artificial antigen of food radiation marker, and preparation method and application thereof - Google Patents

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

Food irradiation mark DiHT class universal hapten, artificial antigen and preparation method thereof and application

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 through the research of decades, as a kind of efficient, ecological, safe new and high technology, to guarantee food hygienic safety, reduce post-harvest loss, giving a impetus to trade plays irreplaceable effect with consumption.Its application approve by increasing country, according to the preliminary statistics, existing 38 countries and regions, the whole world have approved 548 kinds of food and seasonings can use radiation treatment.But in view of the singularity of irradiated food, the universal standard of CAC irradiated food and the regulation of a lot of country all specify will identify irradiated food, specify in the general rule of CAC irradiated food, the maximum absorbed dose of irradiated food should more than 10kGy, even if the irradiation composition of the finished product is formed be less than 1%, also must force to indicate this product postdose on label.So far, CAC has promulgated the authentication method of ten kinds of irradiated foods, as thermoluminescence (TL) method, spectrum (ESR) method and light release light (PSI) method, DNA comet method etc., 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 professional to operate.Along with normally carrying out of irradiated food international trade, various countries are further urgent to the requirement of identification of irradiated foodstuffs technology, in international trade, and especially customs and commodity inspection department, need the sample of qualification to get more and more, be 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 greatly, the advantage such as safely and fast, be used widely in hazardous material such as detection drug residue of food and mycotoxins etc. etc.There are reports for the explorative research of enzyme immunoassay in identification of irradiated foodstuffs, as (the Christopher T.Elliott such as study on determination method of Food irradiation specific product 2-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 synthesis of artificial antigen is that in food immunodetection, antibody is prepared and sets up the step of immune analysis method most critical, and haptenic synthesis is the basis of artificial antigen synthesis.Most of haptens is the micromolecular compound that molecular weight is less than 1000, and itself does not possess immunogenicity, therefore, first small molecules and the sub-coupling of carrier proteins must prepare complete antigen.If small haptens can not directly by bi-functional cross-linking agent and carrier proteins covalent coupling, then need first to produce active function group by derivatization process on small molecules, in this, as haptens, then with carrier protein coupling.DiHT is a kind of small molecules, relative molecular weight is 244.24, for haptens, recognition site cannot be provided for immune t-cell, not possess immunogenicity, so before preparation artificial antigen, to transform its end group, because DiHT does not originally have with it with the active group of protein molecule, must modify haptens, connecting arm suitable in modification.The key of hapten design is the feature structure retaining former test substance as much as possible, and introduces suitable connecting arm and the active group with carrier protein couplet in position.The introducing position of the connecting arm of different structure, different coupling methods or connecting arm, the sensitivity that the ELISA affecting follow-up foundation detects and specificity.At present, be that haptens is prepared synthetic antibody and artificial antigen and carried out enzyme immunoassay (ELISA) and rarely have report with dihydro deoxythymidine (DiHT) derivative.

Summary of the invention

The present invention, according to the demand in above-mentioned field and blank, provides a kind of Food irradiation mark DiHT universal hapten, artificial antigen and preparation method thereof and application.The present invention for initial reactant, applies the derivative of succinyl oxide method synthesizing dihydro deoxythymidine, i.e. haptens DiHT-COOH with dihydro deoxythymidine (DiHT); Mixed anhydride method and active ester method is adopted respectively haptens and carrier proteins BSA, OVA to be carried out coupling, prepare artificial antigen and the coating antigen of dihydro deoxythymidine, this artificial antigen immunity BALB/c mouse prepares polyvalent antibody, and indirect non-competing ELISA method detects to tire and reaches 1: 2.56 × 10 ⁴, polyvalent antibody is to the IC of DiHT ₅₀for 191.8669ng/mL, show that obtained DiHT antiserum(antisera) has higher affinity to DiHT class material, and productive rate height of the present invention is effective, simple and efficient, for the Food irradiation immunological identification technology set up based on the special auxiliary hydrolysis products DiHT of food provides basis.

A kind of dihydro deoxythymidine class universal hapten, has following molecular structure:

A kind of dihydro deoxythymidine class artificial antigen, is characterized in that: the molecule using following structure, the molecule obtained with carrier protein couplet,

Described carrier proteins refers to bovine serum albumin BSA or Protalbinic acid OVA, and the antigen prepared is respectively immunogen and coating antigen.

The preparation method of above-mentioned artificial antigen, comprises following preparation process:

(1) universal hapten is synthesized;

(2) described universal hapten and carrier protein couplet;

The synthetic route of described synthesis universal hapten is:

The synthetic route of described haptens DiHT-COOH and carrier protein BSA coupling is:

The synthetic route of described haptens DiHT-COOH and carrier protein OVA coupling is:

The preparation process of described universal hapten is:

(1) take DiHT 5mg and succinyl oxide 16.4mg, be dissolved in 5mL anhydrous pyridine, 60 DEG C of heating, stir, backflow 10h;

(2) after reaction terminates, in solution, add 10mL methylene dichloride, collect lower floor's mixture, then use 10mL 5% (v/v) salt pickling 3 times, discard dichloromethane layer, residue 10mL ether dissolution, wash 3 times with 10mL deionization, purifying obtains haptens

Immunogen is prepared in described universal hapten and carrier proteins BSA and OVA coupling and coating antigen adopts mixed anhydride method and active ester method to prepare respectively, and concrete steps are as follows:

(1) mixed acid anhydride prepares artificial antigen DiHT-COOH-BSA

A, HT-COOH powder 6.68mg is dissolved in 3mL dry DMF, vibration stir, add 40 μ L tri-n-butylamines, add isobutyl chlorocarbonate 40 μ L under ice bath, 4 DEG C of stirring reaction 1h, after rise to room temperature 1h, obtain solution 1;

B, title 120mg BSA are dissolved in the borate buffer solution (pH=8.7) of 12mL 0.2mol/L, obtain solution 2;

C, solution 1 dropwise joined in solution 2 under stirring at room temperature and goes, stir at 15 DEG C and spend the night.Mixed solution 4 DEG C of distilled water stir dialysis, and 4h changes water once, changes 6 times altogether, until do not measure haptens uv-absorbing in dialyzate, lyophilize, obtains DiHT-COOH-BSA,

(2) active ester method prepares coating antigen DiHT-COOH-OVA

A, take DiHT-COOH powder 6.68mg and be dissolved in 1mL dry DMF, then by 80mg DCC heating for dissolving in 3mL dry DMF.DCC dissolving is cooled to room temperature be added in MBC solution, adds 28mg NHS, stirred at ambient temperature reaction 5h.Then put airtight standing more than 2h in 4 DEG C of refrigerators, centrifuging and taking supernatant liquor, obtains solution 3;

B, take OVA 40mg, be dissolved in the PB of 4mL, 0.05mol/L, pH=8.0 respectively, obtain solution 4;

C, to be mixed by solution 3 and be added to solution 4, then rise to stirring at room temperature reaction 1h gradually, at 4 DEG C, slow stirring reaction spends the night.Next day, be respectively charged into by reaction solution in dialysis tubing, dialyse under PBS and the 4 DEG C condition of 0.01mol/L, pH=7.4, every 6h changes a dialyzate, changes 6 times altogether, till can't check haptens uv-absorbing, obtains DiHT-COOH-OVA solution in dialyzate.

Above-mentioned artificial antigen as immunogen for the preparation of the application detected in dihydro deoxythymidine antibody-like or antiserum(antisera).

Above-mentioned artificial antigen is adopting the application as coating antigen in indirect Elisa method detection dihydro deoxythymidine class.

The polyvalent antibody that described artificial antigen prepares as immunogen immune animal, polyclonal antibody, antibody or hybridoma.

The key point of enzyme-linked immunoassay method is that preparation has immunogenic artificial antigen, and the preparation of high-titer antigen is the important prerequisite obtaining specific antibody and ensure immunodetection susceptibility, and wherein haptenic preparation is again key prepared by antigen.The auxiliary hydrolysis products of Food irradiation specificity-dihydro deoxythymidine DiHT is a kind of small molecules, relative molecular weight is 244.24, for haptens, recognition site cannot be provided for immune t-cell, do not possess immunogenicity, so before preparation artificial antigen, to transform its end group, because DiHT does not originally have the active group with protein molecule with it, must modify haptens, analyze in this test, research, explore multiple method and modify connecting arm, and adopt TLC and NMR to characterize during the course, find to adopt succinyl oxide method to be modified by the terminal hydroxyl of DiHT, effect is better, requirement of experiment can be reached.Haptens prepared by the present invention has the common trait of the auxiliary hydrolysis products of Food irradiation specificity-dihydro deoxythymidine DiHT quasi-molecule structure, the antiserum(antisera) prepared as immunogen immune animal can to the material specific binding with this molecular characterization, i.e. the auxiliary hydrolysis products of specific recognition Food irradiation specificity-dihydro deoxythymidine DiHT quasi-molecule.In order to can with carrier protein molecule coupling, need to introduce connecting arm and carboxyl, the synthetic route that antigen of the present invention adopts is based on take into account the selection of connecting arm and carboxyl introducing position to the impact of the three-D space structure of synthetic product, avoid the space conformation of the molecular structure characteristic that have impact on synthetic product due to the introducing of connecting arm as far as possible, unaffected to ensure the structure as epitope point.

The present invention has prepared immunogen DiHT-COOH-BSA and coating antigen DiHT-COOH-OVA by making haptens and protein molecule with mixed anhydride method, active ester method, coupling ratio is between 5: 1 ~ 30: 1, meet small molecule immune requirement, the inventive method is easy and simple to handle, and side reaction is few.The qualification of UV UV spectrum and SDS-PAGE method is mainly adopted to carry out to the authentication method of immunogen and detectable antigens.Because carrier proteins and haptens all have distinctive ultra-violet absorption spectrum, so conjugate and carrier proteins are analyzed by UV scanning, the change of the uv-spectrogram of generation can be observed very intuitively, the maximum absorption band of DiHT-COOH-BSA and DiHT-COOH-OVA is respectively at 260nm and 287nm wavelength place, the artificial antigen characteristic peak of synthesis does not overlap with haptens and carrier, thus can identify coupling success by uv scan.SDS-PAGE is also conventional authentication method, and the molecular weight due to conjugate is greater than the molecular weight of carrier, and its rate of migration when electrophoresis is different, and after dyed and decolouring, two bands will be in different positions, therefore also can prove out coupling whether success.

The preparation of efficient antigen is the important prerequisite obtaining high-affinity antibody, select at the carrier proteins of hapten conjugation artificial immunization antigen, have rabbit serum proteins (RSA), human serum protein (HSA), thyroglobulin (TG) etc.The selection of protein carrier is usual and had heterology by the animal of immunity, and the immunne response that such animal body produces is comparatively strong, thus easily obtains high-titer antibody.But also someone thinks that oneself protein also can be used as carrier, and immune effect is better than heterologous protein.Select BSA as the carrier proteins of immunizing antigen in the present invention, OVA is as the carrier proteins of envelope antigen.These two kinds of albumen physicochemical property are stablized, and contain comparatively high lysine content and free amino group group, and economical and practical, draw materials conveniently.Animal immune experiment result shows, and dosage, the number of times of immunity, the selection of adjuvant all can have an impact to the result of immunity.Immunizing dose is 150 μ g/ for the first time, the immunity of employing tail vein injection, serious side reaction is there is after result mouse immune, rear scheme is adjusted to dorsal sc multi-point injection, immunizing dose is only reduced to 100 μ g/, result shows that immune effect is better, successfully obtains high-titer, the good polyvalent antibody of specificity, lays a good foundation for developing ELISA detection irradiated food test kit further later.

DiHT and DiHT-COOH haptens white powder solution is carried out TLC detection, developping agent is methyl alcohol: methylene dichloride: acetic acid=1: 9: 0.1 (v/v/v), the phosphor dot obtaining white solid is obviously different from raw material point, Rf is 0.48, and tentatively obtaining white solid is target product DiHT-COOH.

Compose detected result from haptens DiHT nucleus magnetic resonance (1H-NMR), the peak of target substance occurs, proves that product is exactly haptens, can carry out the coupling preparation of artificial antigen.

The qualification of artificial antigen is identified mainly through UV UV scanning and SDS-PAGE gel electrophoresis two kinds of methods.

According to the conjunction additive theory that ultra-violet absorption spectrum and UV absorb, characteristic peak after integration does not overlap with haptens and carrier, there is skew, and be respectively 9.8: 1 and 12: 1 according to the coupling ratio of lambert one Beer's law calculating DiHT-COOH and BSA and OVA, it is generally acknowledged that coupling ratio can produce good specific antibody in 5: 1 ~ 30: 1 scope, coupling ratio that the present invention obtains illustrate haptens respectively with carrier B SA and OVA coupling success.

SDS-PAGE gel electrophoresis qualification is carried out to DiHT-COOH-BSA conjugate, smaller according to the mobility of its BSA of mobility ratio of the artificial antigen DiHT-COOH-BSA band of the known synthesis of SDS-PAGE gel electrophoresis qualification result, show that the molecular mass of artificial antigen increases than the molecular mass of its respective carrier.This is because carrier connects the haptenic result having gone up certain number, and electrophoresis result shows the DiHT-COOH successful coupling with BSA.In SDS-PAGE gel electrophoresis qualification result figure all there is traction phenomenon in various degree in the synthetic antigen of 4 and 5 bands in corresponding position, being that the molecule amount that combines due to carrier protein and small-molecule substance is inconsistent causes.The micromolecular average number that just each carrier protein that UV scanning method calculates combines, the randomness when result of SDS-PAGE is then connected to carrier and haptens and disparity have reacts more really.

Immunized mice serum titer is known through indirect non-competing ELISA detected result, mouse all can produce the positive antiserum of more efficient valency, show that the artificial antigen Hapten-BSA synthesized can produce antibody by stimulating animal well, but due to the difference of animal individual, what each mouse produced tires slightly different.

From the affinity qualification result of DiHT, within the scope of 156.25-10000ng/mL, 3 mouse all create suppression.Be that research object sets up regression equation with haptens, show that obtained DiHT antiserum(antisera) has higher affinity to DiHT.

Accompanying drawing explanation

Fig. 1 is dihydro deoxythymidine molecular structure

Fig. 2 is DiHT hapten synthesis route map

Fig. 3 is the synthetic route chart of haptens DiHT-COOH and carrier protein BSA coupling

Fig. 4 is the synthetic route chart of haptens DiHT-COOH and carrier protein OVA coupling

Fig. 5 is Hapten TLC detected result figure

Wherein A:DiHT; B: haptens

Fig. 6 be haptens DiHT nucleus magnetic resonance ( ¹h-NMR) spectrogram

Fig. 7 is the uv absorption spectra of haptens, BSA and immunizing antigen conjugate

Fig. 8 is the uv absorption spectra of haptens, OVA and coating antigen conjugate

Fig. 9 is the SDS-PAGE electroresis appraisal 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 the sero-fast Competitive assays graphic representation of DiHT

Embodiment

The invention provides following embodiment is to understand the present invention further better; be not limited to described preferred forms; content of the present invention and protection domain are not construed as limiting; anyone the identical or akin product with the present invention drawn under enlightenment of the present invention and with the combination of any prior art, all drops within protection scope of the present invention.

The preparation of embodiment 1. haptens and qualification

Take DiHT 5mg and succinyl oxide 16.4mg, be dissolved in 5mL anhydrous pyridine.60 DEG C of heating, stir, backflow 10h.After reaction terminates, 10mL methylene dichloride is added in solution, collect lower floor's mixture, then use 10mL 5% (v/v) salt pickling 3 times, discard dichloromethane layer, residue 10mL ether dissolution, 3 times are washed, ether layer anhydrous magnesium sulfate drying, vacuum filtration with 10mL deionization, after purification by silica gel column chromatography, after ether volatilization, obtain oily matter.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 shown in Fig. 2.

The synthesis of embodiment 2.DiHT artificial antigen

Mixed anhydride method and active ester method is adopted respectively haptens DiHT and carrier proteins BSA, OVA to be carried out coupling.

(1) mixed acid anhydride prepares artificial antigen DiHT-COOH-BSA method

Taking DiHT-COOH powder 6.68mg is dissolved in 3mL dry DMF, vibration stir, add 40 μ L tri-n-butylamines, add isobutyl chlorocarbonate 40 μ L under ice bath, 4 DEG C of stirring reaction 1h, after rise to room temperature 1h, this be called reaction solution 1.Claim 120mg BSA to be dissolved in the borate buffer solution (pH=8.7) of 12mL 0.2mol/L, this is solution 2.Reaction solution 1 is dropwise joined in solution 2 under stirring at room temperature and goes, stir at 15 DEG C and spend the night.Mixed solution 4 DEG C of distilled water stir dialysis, and 4h changes water once, changes 6 times altogether, until do not measure haptens uv-absorbing in dialyzate, lyophilize, obtain DiHT-COOH-BSA (Fig. 3), packing is for subsequent use.

(2) active ester method prepares coating antigen DiHT-COOH-OVA

Taking DiHT-COOH powder 6.68mg is dissolved in 1mL dry DMF, then by 80mg DCC heating for dissolving in 3mL dry DMF.DCC dissolving is cooled to room temperature be added in MBC solution, adds 28mg NHS, stirred at ambient temperature reaction 5h.Then put airtight standing more than 2h in 4 DEG C of refrigerators, centrifuging and taking supernatant liquor, is called reaction solution 2.Take OVA 40mg, be dissolved in the PB of 4mL, 0.05mol/L, pH=8.0 respectively.Being stirred by reaction solution 2 is added in protein solution, and then rise to stirring at room temperature reaction 1h gradually, at 4 DEG C, slow stirring reaction spends the night.Next day, reaction solution is respectively charged in dialysis tubing, dialyse under PBS and the 4 DEG C condition of 0.01mol/L, pH=7.4, every 6h changes a dialyzate, change 6 times altogether, can't check haptens uv-absorbing in dialyzate till, obtain DiHT-COOH-OVA (Fig. 4) solution, be sub-packed in cryopreservation tube ,-20 DEG C frozen.

The qualification of embodiment 3. artificial antigen

The qualification of artificial antigen is identified mainly through UV UV scanning and SDS-PAGE gel electrophoresis two kinds of methods.

(1) UV scanning qualification

Artificial antigen and BSA are diluted to proper concn, scan within the scope of wavelength 190 ~ 400nm, obtain haptenic UV spectrum.

(2) SDS-PAGE gel electrophoresis qualification

Select concentrated gum concentration to be 5%, resolving gel concentration is 12%, and concentrated 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 observe after decolouring with ultraviolet gel imaging system.

The mensuration of embodiment 4. artificial antigen coupling ratio

According to lambert one Beer's law, wavelength one timing, the uv-absorbing of mixture has additive properties.Total length ultraviolet spectrophotometer is adopted to scan haptens, carrier proteins and conjugate, whether successful according to absorption peak determination coupling ^[17].In conjunction with as follows than calculation formula:

Wherein: OD _conjugate--conjugate light absorption value; OD _protein--protein light absorption value; OD _hapten--haptens light absorption value; C _hapten--haptens concentration; C _protein--protein concn; M _protein--protein molecular weight; M _hapten--antigen molecular

The preparation of embodiment 5. polyvalent antibody

By DiHT-COOH-BSA normal saline dilution, add equivalent Freund's complete adjuvant and fully mix emulsification, in BALB/c mouse dorsal sc multi-point injection, initial immunity dosage is 100 μ g/, changing Freund's complete adjuvant into Freund's incomplete adjuvant at interval of 2 weeks carries out booster immunization with method later, and immunizing dose progressively increases on a small quantity.From the 4th immunity, after each immunity, 1 week docking end gets blood, and serum spends the night in 4 DEG C of placements, and the centrifugal 15min of 5000r/min, abandons precipitation, and it is for subsequent use that upper serum is placed in 4 DEG C of refrigerators ^[18], by 1: 100 times of dilution during use.

The indirect non-competing ELISA method of embodiment 6. measures antiserum titre

Using DiHT-COOH-OVA as coating antigen. by DiHT-COOH-BSA antiserum(antisera) doubling dilution, indirect non-competing ELISA method antagonistic Serum is adopted to detect, under 450nm wavelength, OD value is measured after reaction, every hole surveys 4 times, average, polyclonal antibody is measured by indirect competitive ELISA (CI-ELISA) haptenic inhibiting rate.Carry out according to best antigen-antibody working concentration and optimal conditions, by haptens PBST (0.01mol/LPBS, pH=7.4, containing 0.05%Tween-20) compound concentration is respectively 156.25,312.5,625,1250,2500,5000, the standardized solution of 10000ng/mL, with antiserum(antisera) extension rate for X-coordinate, OD value is ordinate zou, sets up Competitive assays curve, calculates concentration (IC in suppressing ₅₀).

The haptenic preparation of embodiment 7. and qualification

Mark product DiHT is obtained white powder after the reaction of Fig. 2 synthetic route, by DiHT with obtain white powder solution and carry out TLC detection, developping agent is methyl alcohol: methylene dichloride: acetic acid=1: 9: 0.1 (v/v/v), result is as Fig. 5, the phosphor dot obtaining white solid is obviously different from raw material point, calculates white solid R _f=0.48, tentatively obtaining white solid is target product Hapten, but also needs to synthesize further checking.

The haptenic NMR (Nuclear Magnetic Resonance) spectrum qualification of embodiment 8.DiHT-COOH

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 (m, 3H, 5-CH3), but there is assorted peak in 7.298-7.396, this may be incomplete owing to crossing pillar purifying, but the peak of target substance occurs, prove that product is exactly haptens, the coupling preparation of artificial antigen can be carried out.

The qualification of embodiment 9. artificial antigen

(1) UV qualification

BSA, OVA, DiHT-COOH, DiHT-COOH-BSA and DiHT-COOH-OVA five samples scan through ultraviolet spectrophotometer, result shows, BSA and OVA has maximum absorption band at 278nm and 279nm wavelength place respectively, and the maximum absorption band of DiHT-COOH-BSA and DiHT-COOH-OVA is respectively at 260nm and 287nm wavelength place.According to the conjunction additive theory that UV absorbs, characteristic peak after integration does not overlap with haptens and carrier, there is skew, and be respectively 9.8: 1 and 12: 1 according to the coupling ratio of lambert one Beer's law calculating DiHT-COOH and BSA and OVA, it is generally acknowledged that coupling ratio can produce good specific antibody in 5: 1 ~ 30: 1 scope, this institute obtain coupling ratio illustrate haptens respectively with carrier B SA and OVA coupling success.

(2) SDS-PAGE gel electrophoresis

Carry out SDS-PAGE qualification to DiHT-COOH-BSA conjugate, the mobility that Fig. 9 shows its BSA of mobility ratio of the artificial antigen DiHT-COOH-BSA band of synthesis is smaller, shows that the molecular mass of artificial antigen increases than the molecular mass of its respective carrier.Think, this is because carrier connects the haptenic result having gone up certain number, and electrophoresis result shows the DiHT-COOH successful coupling with BSA.In Fig. 9 all there is traction phenomenon in various degree in the synthetic antigen of 4 and 5 bands in corresponding position, may be that the molecule amount that combines due to carrier protein and small-molecule substance is inconsistent causes.The micromolecular average number that just each carrier protein that UV scanning method calculates combines, the randomness when result of SDS-PAGE is then connected to carrier and haptens and disparity have reacts more really.

The mensuration that embodiment 10. polyclonal antibody is tired

The anti-DiHT-COOH-BSA serum titer of table 1

Immunized mice serum titer is as shown in table 1 through indirect non-competing ELISA detected result, and mouse all can produce the positive antiserum of more efficient valency, and its small mouse 1, No. 3 mice serum antibody titers all reach 1: 10 ⁴, No. 2 mouse antibodies are tired slightly low, but also can reach 1: 6.4 × 10 ³.This result shows that the artificial antigen Hapten-BSA synthesized can produce antibody by stimulating animal well, but due to the difference of animal individual, what each mouse produced tires slightly different.

Embodiment 11. affinity of antibody measures

The affinity qualification result of DiHT is in table 2, and analytical table 2 is known, and within the scope of 156.25-10000ng/mL, 3 mouse all create suppression, selects No. 3 mouse serum, by its OD ₄₅₀value is converted into B/B ₀carrying out regression analysis to Lg [DiHT/100], the sero-fast Competitive assays curve of DiHT is as Figure 10, and mouse polyvalent antibody standard suppresses the equation of linear regression of curve to be v=-0.2300x+0.5651, coefficient R ²=0.9905, be 191.8669ng/mL according to regression equation calculation polyvalent antibody to the IC50 of DiHT, show that obtained DiHT antiserum(antisera) has higher affinity to DiHT.

The suppression of table 2 mouse resisting anteserum to DiHT is tired

The detection experiment of embodiment 12. artificial antigen of the present invention.

By embodiment 11 determined antigen-antibody best effort concentration, antibody that antigen of the present invention prepares is detected to dihydro deoxythymidine by indirect Elisa method, deoxythymidine, 1, the specificity of the analog molecules such as 3-dimethyl thymus pyrimidine and thymus pyrimidine, result is as shown in table 1, antiserum(antisera) is to the analogue of structure with antigen prepared by the present invention, as dihydro deoxythymidine, deoxythymidine, 1, 3-dimethyl thymus pyrimidine and thymus pyrimidine etc. have obvious cross reaction, and comparatively reaction is not pitched to the trimethoprim and 2-chloropyrimide with textural difference.

Table 1

Claims (10)

1. a dihydro deoxythymidine class universal hapten, has following molecular structure:

2. the artificial general antigen of dihydro deoxythymidine class, is characterized in that: the molecule using following structure:

carboxylic group on the molecule that obtains of coupling carrier albumen.

3. artificial general antigen according to claim 2, described carrier proteins refers to bovine serum albumin BSA or Protalbinic acid OVA, and the antigen prepared is respectively immunogen and coating antigen.

4. the preparation method of artificial general antigen described in claim 2, comprises following preparation process:

(1) synthesize universal hapten according to claim 1, the route of described synthesis is:

(2) described universal hapten and carrier protein couplet.

5. preparation method according to claim 4, described carrier proteins refers to bovine serum albumin BSA or Protalbinic acid OVA;

The synthetic route of described universal hapten and carrier protein BSA coupling is:

The synthetic route of described universal hapten and carrier protein OVA coupling is:

6. the preparation method of artificial general antigen described in claim 5, the preparation process of described universal hapten is:

(1) take dihydro deoxythymidine 5mg and succinyl oxide 16.4mg, be dissolved in 5mL anhydrous pyridine, 60 DEG C of heating, stir, backflow 10h;

(2) after reaction terminates, in solution, add 10mL methylene dichloride, collect lower floor's mixture, then use 10mL5% (v/v) salt pickling 3 times, discard dichloromethane layer, residue 10mL ether dissolution, wash 3 times with 10mL deionization, purifying obtains haptens.

7. the preparation method described in claim 5 or 6, adopt mixed anhydride method that described universal hapten and carrier proteins BSA coupling are prepared immunogen or coating antigen, step is as follows:

A, universal hapten powder 6.68mg are dissolved in 3mL dry DMF, vibration stir, add 40 μ L tri-n-butylamines, add isobutyl chlorocarbonate 40 μ L under ice bath, 4 DEG C of stirring reaction 1h, after rise to room temperature 1h, obtain solution 1,

B, title 120mg BSA are dissolved in the borate buffer solution (pH=8.7) of 12mL0.2mol/L, obtain solution 2,

C, dropwise to be joined in solution 2 by solution 1 and to go under stirring at room temperature, stir at 15 DEG C and spend the night, mixed solution 4 DEG C of distilled water stir dialysis, 4h changes water once, changes 6 times altogether, until do not measure haptens uv-absorbing in dialyzate, lyophilize, obtains universal hapten-BSA;

Or adopt active ester method preparation that described universal hapten and carrier proteins OVA coupling are prepared immunogen or coating antigen, step is as follows:

A, take universal hapten powder 6.68mg and be dissolved in l mL dry DMF, then by 80mg DCC heating for dissolving in 3mL dry DMF, DCC dissolving is cooled to room temperature to be added in MBC solution, add 28mg NHS, stirred at ambient temperature reaction 5h, then puts airtight standing more than 2h in 4 DEG C of refrigerators, centrifuging and taking supernatant liquor, obtain solution 3

B, take OVA40mg, be dissolved in the PB of 4mL, 0.05mol/L, pH=8.0 respectively, obtain solution 4,

C, solution 3 mixed be added to solution 4, then stirring at room temperature reaction lh is risen to gradually, at 4 DEG C, slow stirring reaction spends the night, next day, reaction solution is respectively charged in dialysis tubing, dialyses under PBS and the 4 DEG C condition of 0.01mol/L, pH=7.4, every 6h changes a dialyzate, change 6 times altogether, can't check haptens uv-absorbing in dialyzate till, obtain universal hapten-OVA.

8. the artificial general antigen described in Claims 2 or 3 as immunogen for the preparation of the application detected in the antibody of dihydro deoxythymidine class or antiserum(antisera).

9. the artificial general antigen described in Claims 2 or 3 is adopting the application as coating antigen in indirect Elisa method detection dihydro deoxythymidine class.

10. the antiserum(antisera) that the artificial general antigen described in Claims 2 or 3 prepares as immunogen immune animal.