CN110128529B - Synthetic method of nortestosterone artificial antigen - Google Patents
Synthetic method of nortestosterone artificial antigen Download PDFInfo
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- CN110128529B CN110128529B CN201910288482.8A CN201910288482A CN110128529B CN 110128529 B CN110128529 B CN 110128529B CN 201910288482 A CN201910288482 A CN 201910288482A CN 110128529 B CN110128529 B CN 110128529B
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- NPAGDVCDWIYMMC-IZPLOLCNSA-N nandrolone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 NPAGDVCDWIYMMC-IZPLOLCNSA-N 0.000 title claims abstract description 74
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- 229960000892 attapulgite Drugs 0.000 claims abstract description 31
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
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- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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Images
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Steroid Compounds (AREA)
Abstract
The invention discloses a synthesis method of nortestosterone artificial antigen, which is characterized by comprising the following steps: (1) The preparation of the attapulgite-nano ferric oxide powder is prepared by purifying the attapulgite, activating the attapulgite and mixing the attapulgite and the nano ferric oxide powder; (2) Stirring and dissolving the 19-nor-testosterone hapten with N, N-dimethylformamide, and then adding the attapulgite-nano ferric oxide powder obtained in the step (1) to obtain a solution 1; (3) Dissolving bovine serum albumin in PBS, regulating the pH value, adding N, N-dimethylformamide, and stirring to dissolve to obtain a solution 2; (4) And (3) dropwise adding the solution 1 obtained in the step (2) into the solution 2 obtained in the step (3), carrying out light-shielding oscillation reaction, dialyzing with distilled water after the reaction is finished, dialyzing with PBS, sub-packaging when ultraviolet scanning dialyzate does not have a small molecular absorption peak, and refrigerating to obtain the ultraviolet scanning dialyzate. The method prepares the nor-testosterone artificial antigen by using the attapulgite-nano ferric oxide powder to prepare the nor-testosterone artificial antigen with BSA.
Description
Technical Field
The invention belongs to the technical field of biology for animals, and particularly relates to a method for synthesizing a nortestosterone artificial antigen.
Background
Amide bonds are one of the most basic chemical structures found in nature, and constitute the backbone of important biological peptides and proteins, found in many natural products and pharmaceutical compounds. The compounds developed by the three international pharmaceutical companies were sampled and counted, 55% of the compounds having amide bonds. The preparation of amine compounds by reduction of amides is also a common transformation in organic synthesis. Currently, the main method of forming amide bonds is still a coupling reagent method using atoms with poor economy. The direct amidation reaction is catalyzed, i.e. no activating reagent is added, and the carboxylic acid and the amine are condensed to form amide under the action of the catalyst. The method can avoid a plurality of defects caused by using an activating reagent, and is an ideal green path for synthesizing the amide.
Nano ferric oxide is a multifunctional material. When the size of the ferric oxide particles is as small as nano-scale (1-100 nm), the surface atomic number, specific surface area, surface energy and the like are sharply increased along with the reduction of the particle size, so that the characteristics of small size effect, quantum size effect, surface effect, macroscopic quantum tunneling effect and the like are shown, and the ferric oxide particles have good optical properties, magnetism, catalytic performance and the like. The attapulgite clay is a clay mineral with attapulgite as a main component. The attapulgite is a crystalline hydrated magnesium aluminum silicate mineral, has unique layer chain structure characteristics, has lattice substitution in the structure, and helps the crystal to contain a certain amount of Na+, ca2+, fe3+, al3+, and the crystal is needle-shaped, fibrous or fiber-gathered. The attapulgite has unique good colloid properties of dispersion, high temperature resistance, salt and alkali resistance and the like and higher adsorption capacity. Because of the unique crystal structure of the attapulgite, the attapulgite has a plurality of special physical and chemical properties and technological properties.
Some scientific research units at home and abroad develop steroid antigens to prepare various antigens, and all the antigens are chemically synthesized, so that the main problem at present is that the molecular binding ratio of BSA and nortestosterone is about 1:20, and the immune effect is affected due to low binding rate.
Disclosure of Invention
The first object of the present invention is to overcome the above-mentioned drawbacks by providing a method for synthesizing nor-testosterone artificial antigen.
A second object of the present invention is to provide a nortestosterone artificial antigen obtained according to the above method.
The aim of the invention is realized by the following technical scheme:
a method for synthesizing nortestosterone artificial antigen, comprising the steps of:
(1) Preparation of attapulgite-nano ferric oxide powder
The preparation method comprises the steps of purifying attapulgite, activating the attapulgite and mixing the attapulgite with nano ferric oxide powder;
(2) Preparation of solution 1
Stirring and dissolving the 19-nor-testosterone hapten with N, N-dimethylformamide, and then adding the attapulgite-nano ferric oxide powder obtained in the step (1) to obtain a solution 1;
(3) Preparation of solution 2
Dissolving bovine serum albumin in PBS, regulating the pH value, adding N, N-dimethylformamide, and stirring to dissolve to obtain a solution 2;
(4) Reaction
And (3) dropwise adding the solution 1 obtained in the step (2) into the solution 2 obtained in the step (3), carrying out light-shielding oscillation reaction, dialyzing with distilled water after the reaction is finished, dialyzing with PBS, sub-packaging when ultraviolet scanning dialyzate does not have a small molecular absorption peak, and refrigerating to obtain the ultraviolet scanning dialyzate.
In the step (1), the nano iron oxide powder and the activated attapulgite are mixed according to a weight ratio of 1:1, and the mixture is carried out on a solid mixer for 0.5h.
Further, in the step (2), the mass volume ratio of the 19-nor-testosterone hapten to the N, N-dimethylformamide is 1:40g/mL, and the weight ratio of the 19-nor-testosterone hapten to the attapulgite-nano ferric oxide powder is 1:10-80, preferably 1:60.
Further, in the step (3), the mass-volume ratio of the bovine serum albumin to the BSA is 33:1mg/mL, the pH value is adjusted to 8.5, the mass-volume ratio of the bovine serum albumin to the N, N-dimethylformamide is 33:1mg/mL, and the mixture is stirred for 1h at 4 ℃.
Further, in the step (4), the shaking reaction is carried out at 1-5 ℃, preferably 4 ℃, for 1-5 hours, preferably 4 hours, for 2d of dialysis time with distilled water, 3d of dialysis time with PBS, and the refrigerating temperature is-20 ℃.
Further, the nortestosterone artificial antigen has the following structural formula:
a nortestosterone artificial antigen obtainable by any one of the methods described above.
The inventor finds that nano ferric oxide can catalyze the amidation reaction of long-chain carboxylic acid and long-chain amine to obtain better catalytic effect. According to the invention, the catalyst is adsorbed in the concave-convex rod cavity, so that the slow release is realized, the contact time of the catalyst and reactants is prolonged, and the catalytic effect is improved.
The invention has the following beneficial effects:
according to the synthesis method of the nortestosterone artificial antigen, provided by the invention, the coupling of the nortestosterone and the BSA can be successfully realized through a novel composite catalyst, namely the attapulgite-nano ferric oxide powder, the nortestosterone artificial antigen is prepared, the yield of catalyzing the reaction is high, the molecular combination ratio of the BSA and the nortestosterone is 1:38, the operation is simple, and a novel method is provided for the development of biological medicines; the obtained nortestosterone has ideal effect, can improve lambing rate after immunization, and has better immunization effect after continuous use for many years.
Drawings
FIG. 1 is a 17 beta-19-NT succinate mass spectrum of NT hapten.
FIG. 2 is a 1H-NMR spectrum of NT hapten.
FIG. 3 is an infrared spectrum of BSA, NT and NT-BSA.
FIG. 4 is an ultraviolet scan of NT artificial antigen.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and explanation only and is not intended to limit the present invention.
Example 1
Synthetic method of nortestosterone artificial antigen
The technology for artificially controlling the ewe to produce the double lambs by using the method of reproduction immunology and reproduction endocrinology is an emerging biological reproduction technology with low cost, good effect, simplicity, convenience and practicability and no side effect, and a steroid antigen capable of improving the ovulation rate and the lambing rate of the ewe is constructed. The invention can successfully realize the coupling of the nortestosterone and the BSA by a novel composite catalyst, and prepares the nortestosterone artificial antigen.
1. Experimental materials:
19-nor-testosterone (NT): purchased from Shanghai Guanyu bioengineering Co., ltd;
bovine Serum Albumin (BSA): (content 96%) salt city Saibao biotechnology Co., ltd;
the remaining reagents were all analytically pure reagents.
Dialysis bag (8000-14000 Da) Solarbio product;
phosphate buffer (PBS, pH 7.4);
attapulgite: jingfu county, gansu province, industry and trade Limited liability company;
n, N-Dimethylformamide (DMF);
nano iron oxide powder: and Shanghai Ling, metal materials Co., ltd.
2. Experimental method
2.1 NT hapten synthesis
Accurately weighing 100mg of NT, dissolving in 10mL of anhydrous pyridine, then adding 180mg of succinic anhydride, and stirring and reacting for 24 hours at 50 ℃ in a dark place. The reaction product was concentrated with a nitrogen blower to give a pale yellow paste, which was purified with 5wt% NaHCO 3 After dissolution, diethyl ether is used for extraction, H 2 SO 4 And (5) acidifying. The supernatant is discarded after centrifugation, the residue is dried by anhydrous sodium sulfate, and light yellow solid is obtained after recrystallization, namely hapten 17 beta-19-NT succinate. And (3) carrying out mass spectrum and 1H nuclear magnetic resonance spectrum identification on the reaction product.
2.2 immune antigen (NT-BSA) Synthesis
Preparing attapulgite-nano ferric oxide powder:
activating attapulgite:
1) Purification of attapulgite: grinding and crushing attapulgite, screening to be less than 2mm, mixing the attapulgite with acid water with the pH value of 2 in a cement pond at a ratio of 1:2 (W/W), mechanically stirring for 1h, standing for 12h, centrifugally drying and filtering on a filter cloth, paving the dried attapulgite on a clean cement land, naturally drying, mixing the attapulgite with deionized water in the cement pond at a ratio of 1:2 (W/W), mechanically stirring for 0.5h, standing for 5h, centrifugally drying and filtering on the filter cloth, paving the dried attapulgite on the clean cement land, and naturally drying; 2) Activation of attapulgite: and (3) placing the prepared attapulgite into an oven, heating to 250 ℃ for 0.5h, further heating to 300 ℃, maintaining for 1h, starting to cool, and taking out the attapulgite after the temperature is cooled to room temperature.
The nano iron oxide powder and activated attapulgite are purchased to obtain a mixture of 1:1 (W/W) on a solid mixer for 0.5h. Thus obtaining the attapulgite-nano ferric oxide powder.
50mg of NT hapten was dissolved in 2mL of N, N-Dimethylformamide (DMF) under stirring, and 3g of attapulgite-nanosized iron oxide powder was added.
66mg of BSA was weighed out and dissolved in 2mL of PBS, the pH was adjusted to about 8.5, 2mL of N, N-Dimethylformamide (DMF) was added thereto, and the mixture was dissolved by stirring at 4℃for 1 hour.
The reaction principle is as follows:
3. results and analysis
3.1 (+) ESI-MS identification of NT hapten: as in fig. 1. The molecular ion peak of fragment ion with m/z of 375.59 as hapten can be attributed to 17 beta-19-NT succinate. The fragment ion peak with m/z of 275.85 is the hapten molecule desquamation-CO (CH) 2 ) 2 COOH formed fragment ions; the fragment ion having m/z of 257.65 was hapten despeck-OCO (CH 2 ) 2 COOH forms an ionic peak. The mass spectrometry analysis result shows that the reaction product is the target compound, namely 17 beta-19-NT succinate.
3.2 hapten Nuclear magnetic resonance analysis
From FIG. 2 hapten 1H NMR (400 MHz, C 3 D 6 And O) identification result: δ9.8 to 10.8 (d, 1H, cooh), 5.85 (s, 1H, -ch=c), 3.6 to 3.96 (t, 1H, CH-O-C (=o)), 2.69 to 2.89 (q, 2H, CH) 2 -C(＝O)C＝C)，2.42～2.55(t，2H，CH 2 -C(＝O)O＝C)，2.58～2.62(d，2H，CH 2 (＝O)O)，1.17(q，3H，CH 3 -C)。
3.3 Infrared Spectroscopy identification of Artificial antigens
The identification result is shown in FIG. 3.BSA and NT-BSA have similar absorptions in the 2500-3200 cm-1 and 1660-1500 cm-1 regions 18, which are characteristic peaks of amino acids in BSA, indicating that BSA is contained in the synthetic artificial antigen. The NT standard has a strong absorption peak at 3300cm-1, which is a characteristic peak of hydroxyl on the 17 # carbon atom, and the artificial antigen has no strong absorption peak, thus proving that hapten reconstruction and coupling are successful. The NT standard has strong absorption peaks of carbonyl and methyl between 1700cm < -1 > and 1380-1460 cm < -1 >, and after BSA is coupled with hapten, the absorption peak is obviously enhanced, which indicates that the characteristic functional group is unchanged. From this, it was confirmed that the synthesis of NT-BSA artificial antigen was successful.
3.4 determination of Artificial antigen coupling Rate
The ultraviolet scan results are shown in fig. 4. The maximum absorption peak wavelengths of NT standard and BSA were 247nm and 279nm, respectively. The absorption peaks of the artificial antigen at these two wavelengths were significantly changed, indicating that the coupling of NT with BSA was successful. The coupling ratio of NT to BSA was calculated to be 38:1 based on the artificial antigen coupling ratio.
4 optimization of the Synthesis Process
4.1 Effect of raw Material ratio
Fixing the reaction temperature and the reaction time. The effect of the NT hapten/attapulgite/nano iron oxide powder weight ratio on yield was examined and the results are shown in Table 1.
TABLE 1 influence of catalyst on yield
| Numbering device | NT hapten/attapulgite-nano ferric oxide powder weight | Yield% | |
| 1 | 1:10 | 52.5 | |
| 2 | 1:20 | 55.9 | |
| 3 | 1:40 | 63.2 | |
| 4 | 1:60 | 66.7 | |
| 5 | 1:80 | 59.8 |
As can be seen from table 1, the product yield increased significantly with increasing weight ratio of catalyst attapulgite to nano iron oxide powder, but when the weight ratio of attapulgite to nano iron oxide powder was increased to 1:60, the yield decreased instead. Therefore, the weight ratio of the NT hapten to the attapulgite to the nano ferric oxide powder can be controlled to be 1:10-80, and the preferable weight ratio is 1:60.
4.2 influence of reaction time on yield
The reaction temperature and the catalyst amount are fixed. The effect of reaction time on yield was examined and the results are shown in Table 2.
TABLE 2 influence of reaction time on yield
| Numbering device | Reaction time | Yield% | |
| 1 | 1 | 60.4 | |
| 2 | 2 | 62.5 | |
| 3 | 3 | 64.2 | |
| 4 | 4 | 66.5 | |
| 5 | 5 | 64.5 |
As is clear from Table 2, the reaction time had a certain effect on the yield, and the product yield increased with the increase of the reaction time from 1 to 5 hours, but after 4 hours, the increase was not significant. The reaction time can be controlled to be 1-5h, and the reaction time is determined to be 4h advantageously from the viewpoints of cost and yield.
4.3 influence of reaction temperature on yield
The reaction time and the catalyst amount are fixed. The effect of reaction time on product yield was examined and the results are shown in Table 3.
TABLE 3 influence of reaction temperature on yield
| Numbering device | Reaction temperature/. Degree. | Yield% | |
| 1 | 1 | 60.3 | |
| 2 | 2 | 62.9 | |
| 3 | 3 | 64.4 | |
| 4 | 4 | 67.8 | |
| 5 | 5 | 65.2 |
As is clear from Table 3, the reaction temperature had a certain influence on the yield, and the product yield increased with an increase in the reaction temperature from 1 to 5 ℃. The reaction temperature may be controlled to 1-5℃and is advantageously determined to 4℃from the standpoint of yield.
5. Conclusion and hope
According to the invention, firstly, nano ferric oxide is adsorbed in an attapulgite cavity to catalyze nortestosterone and BSA to form amide bond connection, and the result shows that: the catalyst has high yield of catalyzing the reaction, the molecular combination ratio of BSA and nortestosterone is 1:38, the experimental operation is simple, clinical data shows that the double-embryo rate is highest, a more ideal result is obtained, a synthetic method of the nortestosterone artificial antigen is innovated, and a new method is provided for the development of biological medicines.
Application of nortestosterone antigen in double-lamb production
The invention aims to improve the breeding rate of mutton sheep and further bring the economic benefit of the fine hair sheep industry into play. In 2017, the inventor performs nor-testosterone antigen comparison experiments in Gansu Kaiyue sheep farm, scientifically evaluates the effect of nor-testosterone antigen on sheep reproductive force, and provides basis for large-area popularization.
Test materials and methods
1. Test variety
Hybrid mutton sheep of small-tailed han sheep ewe and pure-bred non-horn Taosite ram.
2. Test antigen
The nortestosterone antigen is prepared by diluting the dialysis bag with 50mL of water (so that 2mg of antigen is added to 100mL of distilled water), and storing in a refrigerator at 4deg.C for use.
3. Number and grouping of experimental sheep
A total of 280 adult ewes with healthy Gansu Kaiyue sheep farm are selected. The kit is divided into two groups at random according to age, fetal time, weight and the like, wherein one group is a treatment group, nortestosterone antigen is injected, and the other group is a control group without nortestosterone antigen. The experimental sheep and the control sheep are the same group, and the feeding supervision conditions are identical.
4. Nortestosterone antigen injection
Treatment group ewes were each injected with 2mL of nortestosterone antigen (cervical subcutaneous) 7 and 4 weeks prior to dosing.
5. Seed-mix
Artificial insemination was performed conventionally using fresh semen.
6. Feeding management
6.1, the treatment or control group of the ewes at two test points are respectively the same group, namely, the conditions of grazing, supplementary feeding, mating, lambing and the like are completely the same, and all the tested ewes have earmarks and numbers and accurately perform the works of recording, registering and the like.
6.2 weighing. All the tested ewes were weighed on a empty stomach on the day of the first nortestosterone antigen injection.
And 6.3, feeding materials. All pregnant ewes were tested and fed concentrate (mixture of corn, oil residue and skin) at 6 weeks before parturition. Each feed was fed 200 grams daily, either for control or treatment group, until the lambing was completed.
Experimental results
1. Lambing situation
The tested ewes at the two test points were then normally lambed until the lambing was substantially completed. The result shows that the nortestosterone antigen has obvious effect on improving the reproduction rate of the mutton sheep. A total of 200 nortestosterone antigen treated ewes were born with 197 fetuses, 281 lambs were born with a reproduction rate of 140.5%, and the treated group had a double lamb rate of 42.64% (84/197). Details are shown in Table 4.
Table 4 results of nortestosterone antigen control experimental lambing (only, foetus,%)
As seen in table 4, the lambing rate of the treated group of ewes was significantly higher than that of the control group. Namely, the double lambing rate of the nortestosterone antigen treated group ewe is improved by 32.92 percent compared with the control group. Proved to have obvious effect on improving the fertility of the meat-producing hybrid ewe.
2. Lamb condition
2.1 survival of lambs from nortestosterone antigen treatment the survival rate was substantially the same as that of the control group and all survived.
2.2 lamb birth weight, treatment and control group, no significant difference exists between the birth weight of single lamb, double lamb and the birth weight of male and female lambs, the birth weight of the lamb at two test points is higher than that of double lamb, and the birth weight of male lamb is higher than that of female lamb, and the common law is met. It was thus demonstrated that nor-testosterone antigen had no adverse effect on embryo development in the lambs.
The nortestosterone antigen can effectively improve the fertility of meat type hybrid sheep. If under the same condition, the double-birth rate is greatly improved. The method is simple and easy, and has stable and reliable effect and no side effect.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for synthesizing nortestosterone artificial antigen, which is characterized by comprising the following steps:
(1) Preparation of attapulgite-nano ferric oxide powder
The preparation method comprises the steps of purifying attapulgite, activating the attapulgite and mixing the attapulgite with nano ferric oxide powder;
(2) Preparation of solution 1
Stirring and dissolving the 19-nor-testosterone hapten with N, N-dimethylformamide, and then adding the attapulgite-nano ferric oxide powder obtained in the step (1) to obtain a solution 1;
(3) Preparation of solution 2
Dissolving bovine serum albumin in PBS, regulating the pH value, adding N, N-dimethylformamide, and stirring to dissolve to obtain a solution 2;
(4) Reaction
And (3) dropwise adding the solution 1 obtained in the step (2) into the solution 2 obtained in the step (3), carrying out light-shielding oscillation reaction, dialyzing with distilled water after the reaction is finished, dialyzing with PBS, sub-packaging when ultraviolet scanning dialyzate does not have a small molecular absorption peak, and refrigerating to obtain the ultraviolet scanning dialyzate.
2. A method for synthesizing nortestosterone artificial antigen according to claim 1, wherein in step (1), said nano iron oxide powder is mixed with activated attapulgite in a weight ratio of 1:1, and is carried out on a solid mixer for 0.5h.
3. A method for synthesizing a nortestosterone artificial antigen according to claim 1, wherein in step (2), the mass to volume ratio of the 19-nortestosterone hapten to the N, N-dimethylformamide is 1:40g/mL, and the weight ratio of the 19-nortestosterone hapten to the attapulgite-nano iron oxide powder is 1:10-80.
4. A method for synthesizing a nortestosterone artificial antigen according to claim 3, wherein the weight ratio of said 19-nortestosterone hapten to said attapulgite-nano iron oxide powder is 1:60.
5. A method of synthesizing nortestosterone artificial antigen according to claim 1, wherein in step (3), the mass-to-volume ratio of bovine serum albumin to BSA is 33:1mg/mL, the pH is adjusted to 8.5, the mass-to-volume ratio of bovine serum albumin to N, N-dimethylformamide is 33:1mg/mL, and stirring is performed for 1 hour at 4 ℃.
6. A method for the synthesis of nortestosterone artificial antigen according to claim 1, wherein in step (4), the shaking reaction is carried out at 1-5 ℃ for a period of time of 1-5 hours, preferably 4 hours, the dialysis time against distilled water is 2d, the dialysis time against pbs is 3d, and the refrigeration temperature is-20 ℃.
7. A method for the synthesis of nortestosterone artificial antigen according to claim 6, wherein in said step (4), the temperature of the shaking reaction is 4 ℃.
8. A method of synthesizing a nortestosterone artificial antigen according to claim 1, wherein said nortestosterone artificial antigen has the following structural formula:
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| RO122695B1 (en) * | 2007-01-18 | 2009-11-30 | Institutul Naţional De Cercetare-Dezvoltare Pentru Fizică Şi Inginerie Nucleară "Horia Hulubei" | Process for obtaining nandrolone-3-carboxymethyloxime- bovine serum albumin-alkaline phosphatase (nand-3cmo-asb-fa) enzymatic marker |
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| CN102336830A (en) * | 2010-07-27 | 2012-02-01 | 天津科技大学 | Anti-nandrolone specific antibody |
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| EP1846434B1 (en) * | 2005-02-04 | 2009-03-25 | Government of the United States of America, Represented by the Secretary, Department of Health and Human Services | NANDROLONE 17ß-CARBONATES |
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| CN101769921A (en) * | 2008-12-30 | 2010-07-07 | 王武康 | Direct competitive enzyme-linked immunosorbent assay kit for assaying nortestosterone (nandrolone) |
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