CN104086648A - Preparation method and application of progesterone complete antigen - Google Patents
Preparation method and application of progesterone complete antigen Download PDFInfo
- Publication number
- CN104086648A CN104086648A CN201410229282.2A CN201410229282A CN104086648A CN 104086648 A CN104086648 A CN 104086648A CN 201410229282 A CN201410229282 A CN 201410229282A CN 104086648 A CN104086648 A CN 104086648A
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- CN
- China
- Prior art keywords
- ova
- progesterone
- solution
- gained
- complete antigen
- 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.)
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- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 title claims abstract description 63
- 239000000427 antigen Substances 0.000 title claims abstract description 41
- 102000036639 antigens Human genes 0.000 title claims abstract description 41
- 108091007433 antigens Proteins 0.000 title claims abstract description 41
- 229960003387 progesterone Drugs 0.000 title claims abstract description 32
- 239000000186 progesterone Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 239000007788 liquid Substances 0.000 claims description 10
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 150000007942 carboxylates Chemical class 0.000 claims description 3
- 239000013078 crystal Substances 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 10
- 230000003053 immunization Effects 0.000 abstract description 6
- 150000001718 carbodiimides Chemical class 0.000 abstract description 3
- 108010058846 Ovalbumin Proteins 0.000 abstract 2
- 229940092253 ovalbumin Drugs 0.000 abstract 2
- 239000011248 coating agent Substances 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000036039 immunity Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- BFZHCUBIASXHPK-QJSKAATBSA-N 11alpha-hydroxyprogesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)C[C@H]2O BFZHCUBIASXHPK-QJSKAATBSA-N 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- VKHAHZOOUSRJNA-GCNJZUOMSA-N mifepristone Chemical compound C1([C@@H]2C3=C4CCC(=O)C=C4CC[C@H]3[C@@H]3CC[C@@]([C@]3(C2)C)(O)C#CC)=CC=C(N(C)C)C=C1 VKHAHZOOUSRJNA-GCNJZUOMSA-N 0.000 description 1
- 229960003248 mifepristone Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005201 scrubbing Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- RINCXYDBBGOEEQ-UHFFFAOYSA-N succinic anhydride Chemical compound O=C1CCC(=O)O1 RINCXYDBBGOEEQ-UHFFFAOYSA-N 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- C07K14/77—Ovalbumin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
Abstract
The invention discloses a preparation method and application of a progesterone complete antigen. The immunizing antigen is prepared by using a carbodiimide (COD) method to selectively couple activated 11alpha-hydroxyl progesterone (11alpha-OH-P4) with ovalbumin (OVA). Finally the prepared immunizing antigen is used to prepare antiserum through an immunized mouse, and the optimum coating concentration for detecting the antigen is determined.
Description
Technical field
The present invention relates to biological field, be specifically related to a kind of preparation method and application thereof of progesterone complete antigen.
Background technology
Because progesterone is a kind of small-molecule substance, do not possess immunogenicity, cannot make it inject after body and produce antibody.Conventional method is to utilize coupling agent as medium, prepares complete antigen by the coupling of haptens and carrier proteins.The difference of coupling method and the selection of carrier proteins have a certain impact to the immunocompetence tool of complete antigen.
Summary of the invention
For addressing the above problem, the invention provides a kind of preparation method and application thereof of progesterone complete antigen.
For achieving the above object, the technical scheme that the present invention takes is:
The preparation method of progesterone complete antigen, comprises the steps:
S1, by 100mg11 α-OH-P
4be dissolved in 5mL anhydrous pyridine with 30mg hemisuccinic acid carboxylate, in 95 DEG C of water-baths, after water-bath 90min, 95 DEG C of oven dry, are converted into yellow crystal 109.3mg.
S2, in 0.5mL DMF, dissolve the 11 α-OH-P of 30mg S1 gained
4-HS (11 Alpha-hydroxy progesterone hemisuccinic acid ester);
S3, again 8.94mg NHS and 16.02mg DCC are dissolved in respectively in 0.01mL DMF;
S4, above-mentioned S2 gained solution is added in S3 gained solution after, 90min vibrates under room temperature;
S5, get A, two parts of 7mL PBS solution of B, in A solution, add 95.2mg BSA, in B solution, add after 95.2mg OVA, add respectively in S4 gained solution, stir at 4 DEG C and spend the night; ;
S6, S5 gained A, B liquid are respectively charged in dialysis tubing, at 0.1mol/L NaHCO
3in 2.2L, dialyse 4.5 hours, in 2.2L deionized water, dialyse 5 hours, three times, finally put into the PBS solution of pH=7.0, middle dialysed overnight;
Liquid in S7, taking-up dialysis tubing, 4 DEG C of refrigerated centrifuges, the centrifugal 30min of 10000rpm, discards precipitation, gets supernatant liquor;
S8, by S7 gained supernatant liquor vacuum lyophilization, prepare P
4-BSA conjugate and P
4-OVA conjugate.
As preferably, in described step S5, the PH of PBS solution is 7.
The invention also discloses the application of the prepared progesterone complete antigen of aforesaid method.
The present invention passes through carbodlimide method (EDC) by 11 α-hydroxyprogesterone (11 α-OH-P of activation
4), with oralbumin (OVA), select and carry out coupling, prepare immunizing antigen.Finally the immunizing antigen of preparation is prepared to antiserum(antisera) by immune mouse, and the coated concentration of the best of definite detectable antigens.
Embodiment
In order to make objects and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
This concrete preparation method who implements to provide a kind of progesterone complete antigen, comprises the steps:
S1, by 100mg11 α-OH-P
4be dissolved in 5mL anhydrous pyridine with 30mg hemisuccinic acid carboxylate, in 95 DEG C of water-baths, after water-bath 90min, 95 DEG C of oven dry, are converted into yellow crystal 109.3mg.
S2, in 0.5mL DMF, dissolve the 11 α-OH-P of 30mg S1 gained
4-HS (11 Alpha-hydroxy progesterone hemisuccinic acid ester);
S3, again 8.94mg NHS and 16.02mg DCC are dissolved in respectively in 0.01mL DMF;
S4, above-mentioned S2 gained solution is added in S3 gained solution after, 90min vibrates under room temperature;
S5, get A, two parts of 7mL PBS solution of B (PH=7), in A solution, add 95.2mg BSA, in B solution, add after 95.2mg OVA, add respectively in S4 gained solution, stir at 4 DEG C and spend the night; ;
S6, S5 gained A, B liquid are respectively charged in dialysis tubing, at 0.1mol/L NaHCO
3in 2.2L, dialyse 4.5 hours, in 2.2L deionized water, dialyse 5 hours, three times, finally put into the PBS solution of pH=7.0, middle dialysed overnight;
Liquid in S7, taking-up dialysis tubing, 4 DEG C of refrigerated centrifuges, the centrifugal 30min of 10000rpm, discards precipitation, gets supernatant liquor;
S8, by S7 gained supernatant liquor vacuum lyophilization, prepare P
4-BSA conjugate and P
4-OVA conjugate.
The invention also discloses the application of the prepared progesterone complete antigen of aforesaid method.
Embodiment
Prepare antiserum(antisera)
Choose 10 of male BALB/c mouse in age in 6-8 week, with self-control P
4-BSA immunizing antigen carries out repeatedly immunity.First immunisation: get 1.0mg/mL50 μ LP
4after-BSA and the complete emulsification of equivalent FCA, to every mouse peritoneal injection, immunity time 14d; Second immunisation: get 1.0mg/mL50 μ LP
4-BSA and the complete emulsification of equivalent FIA, to every mouse peritoneal injection, immunity time 14d; Booster immunization: get 1.0mg/mL50 μ LP
4-BSA, to every mouse peritoneal injection, immunity time 10d.Finally mouse exoculation is gathered to blood sample, through centrifugation serum, the qualification of antiserum(antisera) character.
Indirect ELISA test
1. by pre-configured antigen coated liquid dilution detectable antigens, in each hole, add 100 μ L, under 37 DEG C of conditions, leave standstill after 30min, be placed in 4 DEG C of refrigerator 12h;
2. every hole adds pre-configured confining liquid 200 μ L, 37 DEG C of thermostat container incubation 2h;
3. every hole adds pre-configured washings 100-200 μ L, so repetitive scrubbing 3 times; Each washing about 3 minutes;
4. will add with the serum of PBS dilution, every hole adds 100 μ L, wet box leaves standstill 1h at 37 DEG C;
5. washing methods is the same;
6. will in the every hole of (1: 2000) HRP-ELIAS secondary antibody with PBS dilution, add 100 μ L, wet box standing and reacting 1h at 37 DEG C;
7. washing methods is the same;
8. will in every hole, add 100 μ L substrates, at 37 DEG C, lucifuge leaves standstill 1h;
9. 50 μ L stop buffers will be added in every hole;
10. enzyme plate is read to every hole OD490nm value (replication is averaged for 3 times) by microplate reader.
Antigen P
4the suitableeest coated concentration of-OVA
Coated antigen P
4-OVA is best, and weaker concn is utilized square formation titration measuring, i.e. the suitableeest coated concentration.Laterally with antiserum(antisera) coated (respectively with A-F name), according to 1: 4.0 × 10
3, 1: 8.0 × 10
3, 1: 1.6 × 10
4, 1: 3.2 × 10
4, 1: 6.4 × 10
4with 1: 1.28 × 10
5weaker concn coated with enzyme plate in.Longitudinally (name with a-g respectively) and use detectable antigens P
4-OVA is by 1: 1.0 × 10
1, 1: 2.0 × 10
1, 1: 4.0 × 10
1, 1: 8.0 × 10
1, 1: 1.6 × 10
2, 1: 3.2 × 10
2, 1: 1.28 × 10
3weaker concn coated with enzyme plate in, in ELISA test, establish horizontal h and i is respectively negative control and blank.Working method is tested with indirect ELISA, and result is with OD
490nm value is close to 1.0 the P that is judged to be
4the suitableeest coated concentration of-OVA.
The specificity of progesterone residue in antiserum(antisera) and complete antigen
By the antigen P of suitable coated concentration
4-OVA and OVA are coated in respectively in enzyme plate, with after confining liquid sealing, by 50 μ LP
4-BSA adds in 12 enzyme plate holes.25 μ g/ml progesterone standard substance are put into methyl alcohol dilution (as starting multiple proportions, doing 12 gradients according to 1: 10), and vibration mixes 20min, and every hole adds 50 μ L in above-mentioned 12 enzyme plate holes afterwards.Be contrast by PBS damping fluid with the methanol mixed without progesterone.Operate according to indirect ELISA test method, measure every hole OD490
nmvalue.
The specificity of antiserum(antisera) and free progesterone
By the antigen P of suitable coated concentration
4-OVA is coated in enzyme plate, with after confining liquid sealing, multiple proportions antiserum(antisera) and blank (PBS damping fluid) is operated according to indirect ELISA test method, measures every hole OD
490after nm value, calculate respectively ratio and the P in OVA hole and blank hole
4the ratio in-OVA hole and OVA hole.
The suitableeest coated concentration determination result of antigen P4-OVA
Carry out antigen P by square formation method
4-OVA is the qualification of suitable coated concentration.Result shows (table 1): P
4the coated concentration of-OVA is higher, OD
450nmbe worth higher.OD makes discovery from observation
450nmvalue is that 1.0 holes are antigen P
4the coated concentration of-OVA is 1: 160, and sero-fast weaker concn is 1: 32000, therefore set it as the suitableeest coated concentration.
Table 1 antigen P
4the suitableeest coated concentration of-OVA
| ? | A | B | C | D | E | F |
| a | 1.701 | 1.688 | 1614 | 1.567 | 1.485 | 1.402 |
| b | 1.655 | 1.614 | 1.576 | 1.504 | 1.472 | 1.386 |
| c | 1.596 | 1.484 | 1.421 | 1.387 | 1.312 | 1.299 |
| d | 1.573 | 1.405 | 1.376 | 1.259 | 1.168 | 1.097 |
| e | 1.487 | 1.375 | 1.294 | 1.101 | 1.059 | 0.976 |
| f | 1.409 | 1.291 | 1.124 | 1.000 | 0.904 | 0.645 |
| g | 1.392 | 1.215 | 1.075 | 0.912 | 0.786 | 0.618 |
| h | 0.038 | 0.038 | 0.039 | 0.039 | 0.040 | 0.040 |
| i | 0.038 | 0.039 | 0.038 | 0.039 | 0.038 | 0.038 |
The specific reaction result of progesterone residue in antiserum(antisera) and complete antigen
The specific reaction of the antiserum(antisera) (1: 32000) of measuring optimal concentration by ELISA to progesterone residue in complete antigen molecule, the results are shown in Table 2, and laterally label is 1-10 (1-7 is that extent of dilution is the antiserum(antisera) of 1: 32000).By OVA and P
4after-OVA is coated with respectively, obtain OD490 by detecting antiserum(antisera) specific reaction
nmvalue shows: in enzyme plate, the ratio in the anti-hole of OVA and blank hole (being N/B) is 1.0, and P
4the ratio (being P/N) in-OVA hole and OVA hole is 10.03.Therefore, the antiserum(antisera) of this experiment preparation and OVA self are without any specificity, and P
athe antibody activity that-OVA detects in hole is the specific reaction for progesterone residue in molecule.
The specificity that table 2 antiserum(antisera) reacts with progesterone in complete antigen
The specific reaction of antiserum(antisera) and free progesterone
The free progesterone specific reaction of antiserum(antisera) and different concns the results are shown in Table 3.The extent of dilution of free progesterone is higher, OD490
nmbe worth highlyer, the free progesterone molecule that proves different concns progesterone residue in coated complete antigen can be combined with antiserum(antisera) competitively.
The specificity of the antiserum(antisera) (1: 32000) of mensuration optimal concentration to progesterone residue reaction in complete antigen molecule, the results are shown in Table 2, and laterally label is 1-10 (1-7 is 1: 32000 antiserum(antisera) of extent of dilution).By OVA and P
4after-OVA is coated with respectively, by detecting sero-fast OD490
nmvalue shows: in enzyme plate, the ratio in OVA hole and blank hole (being N/B) is 1.0, and P
4the ratio (being P/N) in-OVA hole and OVA hole is 10.03.Therefore, the antiserum(antisera) of this experiment preparation and OVA self are without any specificity, P
4the antibody activity that-OVA detects in hole is the specific reaction occurring because of progesterone residue in its molecule.
The specificity that table 3 antiserum(antisera) reacts with free progesterone
This concrete enforcement by succinyl oxide method 11 α-OH-P
4activation, can with carrier proteins generation specific binding; Use carbodiimide method successfully to prepare progesterone complete antigen P
4-OVA, and the information of having preserved molecular structure in free progesterone; The complete antigen P of preparation
4-OVA can stimulate BALB/c mouse to produce the immunne response of Mifepristone, determined simultaneously antiserum(antisera) and detectable antigens the suitableeest weaker concn.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. the preparation method of progesterone complete antigen, is characterized in that, comprises the steps:
S1, by 100mg11 α-OH-P
4be dissolved in 5mL anhydrous pyridine with 30mg hemisuccinic acid carboxylate, in 95 DEG C of water-baths, after water-bath 90min, 95 DEG C of oven dry, are converted into yellow crystal 109.3mg.
S2, in 0.5mL DMF, dissolve the 11 α-OH-P of 30mg S1 gained
4-HS;
S3, again 8.94mg NHS and 16.02mg DCC are dissolved in respectively in 0.01mL DMF;
S4, above-mentioned S2 gained solution is added in S3 gained solution after, 90min vibrates under room temperature;
S5, get A, two parts of 7mL PBS solution of B, in A solution, add 95.2mg BSA, in B solution, add after 95.2mg OVA, add respectively in S4 gained solution, stir at 4 DEG C and spend the night; ;
S6, S5 gained A, B liquid are respectively charged in dialysis tubing, at 0.1mol/L NaHCO
3in 2.2L, dialyse 4.5 hours, in 2.2L deionized water, dialyse 5 hours, three times, finally put into the PBS solution of pH=7.0, middle dialysed overnight;
Liquid in S7, taking-up dialysis tubing, 4 DEG C of refrigerated centrifuges, the centrifugal 30min of 10000rpm, discards precipitation, gets supernatant liquor;
S8, by S7 gained supernatant liquor vacuum lyophilization, prepare P
4-BSA conjugate and P
4-OVA conjugate.
2. the preparation of progesterone complete antigen according to claim 1, is characterized in that, in described step S5, the PH of PBS solution is 7.
3. the application of progesterone complete antigen as prepared in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410229282.2A CN104086648A (en) | 2014-05-22 | 2014-05-22 | Preparation method and application of progesterone complete antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410229282.2A CN104086648A (en) | 2014-05-22 | 2014-05-22 | Preparation method and application of progesterone complete antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104086648A true CN104086648A (en) | 2014-10-08 |
Family
ID=51634431
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410229282.2A Pending CN104086648A (en) | 2014-05-22 | 2014-05-22 | Preparation method and application of progesterone complete antigen |
Country Status (1)
| Country | Link |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949940A (en) * | 2010-08-11 | 2011-01-19 | 吉林大学 | Detection kit of cow milk progesterone content |
| CN103163291A (en) * | 2013-03-09 | 2013-06-19 | 黑龙江八一农垦大学 | Kit and operation method thereof |
-
2014
- 2014-05-22 CN CN201410229282.2A patent/CN104086648A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949940A (en) * | 2010-08-11 | 2011-01-19 | 吉林大学 | Detection kit of cow milk progesterone content |
| CN103163291A (en) * | 2013-03-09 | 2013-06-19 | 黑龙江八一农垦大学 | Kit and operation method thereof |
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Application publication date: 20141008 |