CN108896770A - A kind of method for building up of the kit of pGEX -4T-V recombinant protein as antigen coat elisa plate - Google Patents
A kind of method for building up of the kit of pGEX -4T-V recombinant protein as antigen coat elisa plate Download PDFInfo
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- CN108896770A CN108896770A CN201811046537.6A CN201811046537A CN108896770A CN 108896770 A CN108896770 A CN 108896770A CN 201811046537 A CN201811046537 A CN 201811046537A CN 108896770 A CN108896770 A CN 108896770A
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
A kind of method for building up the invention discloses pGEX -4T-V as the kit of antigen coat elisa plate, includes the following steps：Antigen coat concentration 1:1500,37 DEG C are coated with 2 hours, discard liquid PBST and wash 3 times, 3min/ times；37 DEG C of skimmed milk power 300ul of 5% are closed 1 hour, are discarded confining liquid PBST and are washed 3 times, 3min/ times；Add the sheep blood serum 1 of detection:100,37 DEG C of effect 45min discard blood-serum P BST and wash 3 times, 3min/ times；Anti- sheep secondary antibody diluted concentration 1:15000,100ul/ holes, 25 DEG C of effect 45min discard liquid PBST and wash 3 times, 3min/ times；Add the hole TMB developing solution 100ul/, 25 DEG C of effect 3-5min；The hole 2M sulfuric acid terminate liquid 100ul/, 450nm measure ELISA OD value.
Description
Technical field
The invention belongs to field of biotechnology, it is related to a kind of pGEX -4T-V as the kit of antigen coat elisa plate
Method for building up.
Background technique
Existing home and overseas technology is to be coated with elisa plate using peste des petits ruminants N protein, and the competitive ELISA of foundation is examined
Disconnected method.This method sensibility and specificity is poor.
Summary of the invention
It is an object of the present invention to overcome the shortcomings of the prior art and provide a kind of pGEX -4T-V to be used as antigen packet
By the method for building up of the kit of elisa plate.
Its specific technical solution is：
A kind of method for building up of the kit of pGEX -4T-V as antigen coat elisa plate, includes the following steps：
(1) antigen coat concentration 1:1500,37 DEG C are coated with 2 hours, discard liquid PBST and wash 3 times, 3min/ times.
(2) close 1 hour for 37 DEG C of 5% skimmed milk power 300ul, discard confining liquid PBST and wash 3 times, 3min/ times.
Plus detection sheep blood serum (Nigera75/1 live vaccine was immunized) 1:100,37 DEG C of effect 45min, discard serum
PBST is washed 3 times, 3min/ times.
(4) anti-sheep secondary antibody diluted concentration 1:15000,100ul/ holes, 25 DEG C of effect 45min discard liquid PBST and wash 3 times,
3min/ times.
(5) plus the hole TMB developing solution 100ul/, 25 DEG C of effect 3-5min.
(6) the hole 2M sulfuric acid terminate liquid 100ul/, 450nm measure ELISAOD value.
Compared with prior art, beneficial effects of the present invention are：
The peste des petits ruminants immune antiboidy potency for the sheep blood serum sample detection that the present invention is immunized is high.The present invention test to it is related
The crucial difference of report is exactly the V protein for the peste des petits ruminants Nigero75/1 vaccine virus applied.The present invention is using indirect
ELISA method, secondary antibody can reinforce signal, and different measurement analyses can be done there are many selection.Serum to be detected is then protected
The immunoreactivity that it is most is stayed.It is easy to operate.
With the comparative situation of commercial kit：(V- indirect ELISA) of the pGEX -4T-V as antigen coat elisa plate
The competitive ELISA kit of detection method and ID.vet company detects comparing result, and 172 parts of sheep blood serum sample detection results meet
Rate reaches 97.6%.
Detailed description of the invention
Fig. 1 is flow chart of the pGEX -4T-V as the method for building up of the kit of antigen coat elisa plate；
Fig. 2 is result of the pGEX -4T-V as antigen coat elisa plate detection sheep blood serum.
Fig. 3 is pGEX -4T-V as antigen coat elisa plate (V- indirect ELISA) detection method and ID.vet company
Competitive ELISA detects the result of 80, sheep blood serum sample comparisons.
Fig. 4 is pGEX -4T-V as antigen coat elisa plate (V- indirect ELISA) detection method and ID.vet company
Competitive ELISA detects the result of 92, sheep blood serum sample comparisons.
Specific embodiment
Technical solution of the present invention is described in more detail with reference to the accompanying drawings and examples.
As shown in Figure 1, pGEX -4T-V protein concentration is determined using BCA method determination of protein concentration kit, according to
Square matrix method determines the best antigen coat concentration of pGEX -4T-V protein, determines the optium concentration of secondary antibody, establishes indirect ELISA and examine
Disconnected method, we determined that：
(1) antigen coat concentration 1:1500,37 DEG C are coated with 2 hours, discard liquid PBST and wash 3 times, 3min/ times.
(2) close 1 hour for 37 DEG C of 5% skimmed milk power 300ul, discard confining liquid PBST and wash 3 times, 3min/ times.
Plus detection sheep blood serum (Nigera75/1 live vaccine was immunized) 1:100,37 DEG C of effect 45min, discard serum
PBST is washed 3 times, 3min/ times.
(4) anti-sheep secondary antibody diluted concentration 1:15000,100ul/ holes, 25 DEG C of effect 45min discard liquid PBST and wash 3 times,
3min/ times.
(5) plus the hole TMB developing solution 100ul/, 25 DEG C of effect 3-5min.
(6) the hole 2M sulfuric acid terminate liquid 100ul/, 450nm measure ELISAOD value.As a result see Fig. 2, immune sheep blood serum sample
The peste des petits ruminants immune antiboidy potency of detection is high.
The prokaryotic expression of the present invention V protein of peste des petits ruminants Nigero75/1 vaccine virus, is named as pGEX -4T-V, wraps
By elisa plate, peste des petits ruminants antibody is detected.We have detected 276 parts of immune sheep blood using the coated elisa plate of V protein
Clearly, and compared with competitive ELISA agent box is commercialized, coincidence rate reaches 98.5%.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Claims (2)
1. a kind of method for building up of pGEX -4T-V as the kit of antigen coat elisa plate, which is characterized in that including following
Step：
(1) antigen coat concentration 1:1500,37 DEG C are coated with 2 hours, discard liquid PBST and wash 3 times, 3min/ times；
(2) close 1 hour for 37 DEG C of 5% skimmed milk power 300ul, discard confining liquid PBST and wash 3 times, 3min/ times；
Plus detection sheep blood serum 1:100,37 DEG C of effect 45min discard blood-serum P BST and wash 3 times, 3min/ times；
(4) anti-sheep secondary antibody diluted concentration 1:15000,100ul/ holes, 25 DEG C of effect 45min discard liquid PBST and wash 3 times,
3min/ times；
(5) plus the hole TMB developing solution 100ul/, 25 DEG C of effect 3-5min；
(6) the hole 2M sulfuric acid terminate liquid 100ul/, 450nm measure ELISAOD value.
2. method for building up of the pGEX -4T-V according to claim 1 as the kit of antigen coat elisa plate, special
Sign is that sheep blood serum described in step (3) is that Nigera75/1 live vaccine was immunized.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811046537.6A CN108896770A (en) | 2018-09-08 | 2018-09-08 | A kind of method for building up of the kit of pGEX -4T-V recombinant protein as antigen coat elisa plate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811046537.6A CN108896770A (en) | 2018-09-08 | 2018-09-08 | A kind of method for building up of the kit of pGEX -4T-V recombinant protein as antigen coat elisa plate |
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| CN108896770A true CN108896770A (en) | 2018-11-27 |
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| CN201811046537.6A Pending CN108896770A (en) | 2018-09-08 | 2018-09-08 | A kind of method for building up of the kit of pGEX -4T-V recombinant protein as antigen coat elisa plate |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7303913B2 (en) * | 2004-11-05 | 2007-12-04 | Republic Of Korea (Management: Ministry Of Agriculture And Forestry, National Veternary Research) | Rapid diagnostic methods of Peste des Petits Ruminants using recombinant nucleocapsid protein expressed in insect cells and monoclonal antibody |
| CN105158480A (en) * | 2015-08-05 | 2015-12-16 | 中国农业科学院兰州兽医研究所 | Kit for detecting peste des petits ruminants virus hemagglutinin protein antibody and application method of kit |
| CN107238702A (en) * | 2017-06-22 | 2017-10-10 | 中国动物疫病预防控制中心 | Detect the enzyme linked immunological kit of PPR virus antibody |
-
2018
- 2018-09-08 CN CN201811046537.6A patent/CN108896770A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7303913B2 (en) * | 2004-11-05 | 2007-12-04 | Republic Of Korea (Management: Ministry Of Agriculture And Forestry, National Veternary Research) | Rapid diagnostic methods of Peste des Petits Ruminants using recombinant nucleocapsid protein expressed in insect cells and monoclonal antibody |
| CN105158480A (en) * | 2015-08-05 | 2015-12-16 | 中国农业科学院兰州兽医研究所 | Kit for detecting peste des petits ruminants virus hemagglutinin protein antibody and application method of kit |
| CN107238702A (en) * | 2017-06-22 | 2017-10-10 | 中国动物疫病预防控制中心 | Detect the enzyme linked immunological kit of PPR virus antibody |
Non-Patent Citations (4)
| Title |
|---|
| S.BHADOURIYA ET AL.: "Prokaryotic Expression and Confirmation of Non-structural Protein C of Peste-Des-Petits-Ruminants Virus", 《INTERNATIONAL JOURNAL OF CURRENT MICROBIOLOGY AND APPLIED SCIENCES》 * |
| 严欢 等: "小反刍兽疫病毒P蛋白及非结构蛋白V表达特异性研究", 《中国兽疫杂志》 * |
| 王东方: "小反刍兽疫间接ELISA抗体检测方法建立及初步应用", 《中国优秀硕士学位论文全文数据库》 * |
| 黄华欣: "小反刍兽疫病毒Nigeria75/1株M蛋白的原核表达及单克隆抗体的制备和初步应用", 《中国优秀硕士学位论文全文数据库》 * |
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Application publication date: 20181127 |