CN105388282B - A kind of method for coating of ELISA kit antigen indirect coated elisa plate - Google Patents
A kind of method for coating of ELISA kit antigen indirect coated elisa plate Download PDFInfo
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Abstract
A kind of method for coating of ELISA kit antigen indirect coated elisa plate, comprises the following steps：(1) it is coated with；(2) close：Closed using the 0.01mol/L PBSs of 10g/L isinglasses；(3) target antigen is captured；(4) 0.01mol/L containing 5% sucrose and 1~5% bovine serum albumin(BSA) is added, the solid phase antigen stabilizer of the phosphate buffers of pH 7.2~7.6 is drained, produces antigen indirect coated elisa plate.The ELISA Plate storage life that present invention coating is obtained significantly improved stability and storage life that antigen indirect is coated with ELISA kit up to more than 1 year.
Description
Technical field
The present invention relates to a kind of method for coating of ELISA Plate, and in particular to a kind of ELISA kit antigen indirect is coated with enzyme
The method for coating of target.
Background technology
Antigen indirect coating refers to specific antibody (monoclonal antibody is how anti-) coated elisa plate, Ran Houzai first with certain antigen
By the specific binding of antigen-antibody, capture target antigen is on ELISA Plate, for assembling ELISA kit.Antigen indirect
Coating prepares ELISA Plate and is advantageous in that：One contributes to promote the target antigen albumen of gene engineering method expression to form it certainly
Right conformation, is reacted by the specific affinity of antigen-antibody, envelope antigen has been further purified, has made albumen knot by washing process
Structure is closer to natural conformation, so as to improve the sensitivity and specificity of diagnostic method；Two be the target through more than half purifying and renaturation
Antigen protein can just meet requirement.
At present, domestic ELISA kit quality is unstable, main reason is that direct or indirect antigen coat ELISA Plate
Stability is poor, causes ELISA kit storage life short；And the antigen stabilizer of in the market, it is not only expensive, and source
It is unstable, have a strong impact on the development of China ELISA class diagnostic kits.Therefore, the coating technique of antigen coat ELISA Plate is
Ensure the key of ELISA kit quality.
The content of the invention
The technical problems to be solved by the invention are to provide that a kind of stability is good, the low ELISA examinations of long shelf-life, cost
The method for coating of agent box antigen indirect coated elisa plate.
The technical scheme that the present invention solves the use of its technical problem is that a kind of ELISA kit antigen indirect is coated with enzyme mark
The method for coating of plate, comprises the following steps：
(1) it is coated with：Monoclonal antibody or polyclonal antibody are diluted to work with the carbonate buffer solutions of 0.05mol/L pH 9.6
Make the μ g/mL of concentration 1~2, add ELISA Plate, add 95 per hole~105 μ L, (18~25 DEG C) coatings are stayed overnight at room temperature, then use PBS
Wash 3 times；
(2) close：The μ L of 0.01mol/L PBSs 95~105 per Kong Jiahan 10g/L isinglasses, 35~38 DEG C of envelopes
1~2h is closed, is then washed with PBS 3 times；
(3) target antigen is captured：Target antigen is diluted to the μ of working concentration 0.5~1.5 with 0.01mol/L PBSs
G/mL, ELISA Plate is added per the μ L of hole 95~105, and then 18~25 DEG C of 1~1.5h of capture reaction are washed 3 times with PBS；
(4) solid phase antigen stabilizer is added：Under the situation that ELISA Plate hole is moistened, 120~150 μ L solid phase antigens are added per hole
Stabilizer, room temperature acts on 20~40min, abandons raffinate；ELISA Plate is placed in the clean room of room epidemic disaster 20%~30%, dehumidifier
Machine is drained 10~12 hours, produces antigen indirect coated elisa plate；Wherein, the solid phase antigen stabilizer is containing 5% sucrose and 1
The phosphate buffer of the 0.01mol/L of~5% bovine serum albumin(BSA), pH 7.0~7.6.
Further, in step (1), the monoclonal antibody is foot and mouth disease virus 3A monoclonal antibodies, and working concentration is 2 μ
g/mL；The polyclonal antibody is foot and mouth disease virus 2C polyclonal antibodies, and working concentration is 1 μ g/mL.
Further, in step (4), the solid phase antigen stabilizer is containing 5% sucrose and 1% bovine serum albumin(BSA)
0.01mol/L, pH 7.5 phosphate buffer, adds 0.1% thiomersal preservative, and 37 DEG C of placements are made for 4 weeks.
The method for coating of the ELISA kit antigen indirect coated elisa plate of the present invention can make indirect antigen coat ELISA Plate
Storage life reach more than 1 year, significantly improve the stability and storage life of ELISA kit；And antigen stabilizer used
Formula it is simple, antigen-antibody reaction is not interfered with, material is easily obtained, it is unrestricted to originate, applied widely.
Embodiment
The present invention is further illustrated with reference to embodiment.
1st, materials and methods
(1) material：Carbonate buffer solution (Na2CO3/NaHCO3) and gelatin be purchased from SIGMA companies；The non-knot of foot and mouth disease virus
Structure albumen 3A is that the preparation preservation of this laboratory is prepared using conventional method with 3B monoclonal antibodies；Horseradish peroxidase (HRP)
3B monoclonal antibodies commission Nanjing Jin Sirui corporate logos are marked, titration working concentration is 1: 5000；Foot and mouth disease virus non-structural
Albumen 2C epitopes area's polyclonal antibody is prepared by this laboratory using conventional method, using the 2C epitopes area protein immunization man of expression
It is prepared by rabbit；Foot and mouth disease virus nonstructural protein 3A BC is expressed with reference to documents below：Cao Yimei, Liu Xin, Lu Zengjun, wait mouthfuls of hoof
Expression [J] the journal of animal science and veterinary medicine of epidemic disease poison nonstructural protein gene 3ABC in Escherichia coli, 2004,35 (1):115-
118；Foot and mouth disease virus Nonstructural protein 2C 3AB expression is carried out with reference to documents below：Zhang little Li, Tian Meina, Lu Zengjun, etc.
.FMDV the combinational expression of Nonstructural protein 2C Primary epitope area and 3AB genes and activity analysis bioengineering journals, 2009,25
(1):10-15[J]。
(2) nonstructural protein 3A BC and 2C3AB fusion proteins expression, purifying and renaturation are carried out with reference to documents below：
Cao Yimei, Liu Xin, Lu Zengjun, wait tables of the foot and mouth disease virus nonstructural protein genes 3ABC in Escherichia coli
Up to [J] journal of animal science and veterinary medicine, 2004,35 (1):115-118.
Cao Yimei, Lu Zengjun, Liu Xin, wait ETECs to express the purification renaturation of FMDV 3ABC non-structural proteins
With the Chinese animal doctor's science and technology of Activity determination [J], 2003,33 (8):22-25.
(3) preparation of non-structural protein 3B monoclonal antibodies is carried out with identification with reference to documents below：
Li Yongliang, Tian Meina, Lu Zengjun, wait the preparation and identification of foot and mouth disease virus non-structural protein 3B monoclonal antibodies
[J] Jiangsu's agriculture journals, 2009,25 (2):296-300.
Embodiment 1：The method for coating of foot and mouth disease virus 3A monoclonal antibodies coating capture 3ABC ELISA Plates
(1) it is coated with：It is dense to working with coating buffer solution (carbonate buffer solutions of 0.05mol/L pH 9.6) dilution 3A monoclonal antibodies
Spend for 1：10000, ELISA Plate is added, 100 μ L are added per hole, 4 DEG C overnight, are washed 5 times with PBS.
(2) close：Add confining liquid (PBS solution for containing 1% isinglass) 100 μ L, 37 DEG C of closing 1h per hole, washed with PBST
3 times.
(3) 3ABC albumen is captured：It is 1 μ g/mL to dilute 3ABC albumen to working concentration with 0.01mol/L PBSs,
ELISA Plate is added per the μ L of hole 100,37 DEG C are reacted 1h, and PBS is washed 3 times.
(4) solid phase antigen stabilizer is added：Prepare sucrose containing various concentrations (50g/L and 100g/L) and various concentrations cow's serum
The 0.01mol/L phosphate buffers (pH 7.5) of albumin (10,30,50 and 100g/L), detecting its protection, coating is anti-indirectly
The effect of former stability.120 μ L, room temperature effect 30min are added per hole, raffinate is abandoned；ELISA Plate puts room epidemic disaster 20%~30%
Clean room, dehumidifier drains about 12 hours.Thoroughly dry ELISA Plate is put in transparent plastic packaging bag, plus drier is vacuumized
Sealing preserve.
(5) test serum is added：With PBST dilution NSP antibody strong positive, weakly positive and negative control sera containing 1%BSA
(1: 5 dilution), ELISA Plate is added per the μ L of hole 100, is reacted at room temperature 16~20 hours, PBST is washed 5 times, is patted dry.
(6) enzyme-added mark 3B monoclonal antibodies：Enzyme mark 3B monoclonal antibodies are diluted to most suitable working concentration with serum dilution, per the μ L of hole 100
ELISA Plate is added, 1h is reacted at room temperature, PBST is washed 5 times, patted dry.
(7) substrate colour developing is added：Tmb substrate solution is added per the μ L of hole 100,37 DEG C of lucifuges react 10~15min.
(8) terminate：0.3mol/L H are added per hole2SO4Make reaction terminating.
Determine OD values：450nm wavelength OD values are determined with ELIASA.
Calculating blocking rate PI, PI=of the positive control with weakly positive control sample to 3B monoclonal antibody combination antigens, (the 1- positives are right
According to OD450/ negative control OD450) × 100%.
Embodiment 2：The method for coating of foot and mouth disease virus 2C polyclonal antibodies coating capture 2C3AB ELISA Plates
The how anti-coated elisa plate in foot and mouth disease virus 2C epitopes area (1: 20000) is utilized with differing only in for embodiment 1,
Capture Nonstructural protein 2C 3AB (1 μ g/mL), other reaction conditions and reagent embodiment 1.
Embodiment 3：The stability of antigen indirect coated elisa plate and the measure of storage life
1st, antigen indirect coated elisa plate storage life is determined
ELISA Plate is respectively placed in 4 DEG C and 37 DEG C, 1 times a week contrasting detection NSP antibody positives, weakly positive and negative control
Serum, continuous detection 6 weeks.If 37 DEG C are placed 4 weeks, ELISA Plate detected value does not have significant change, then the storage life of 4 DEG C of ELISA Plate
Up to 1 year.
2nd, antigen indirect coated elisa plate estimation of stability
Negative control sera does not have NSP antibody, will not add HRP mark monoclonal antibodies or many with indirect coated antigen binding
After anti-, OD450 values reach highest；Weakly positive is compareed contains different amounts of NSP antibody with positive control serum, with envelope antigen knot
Close, block the combination of 3B monoclonal antibodies, the reduction of OD450 values；Therefore, by calculating the positive blocking rate with weakly positive control serum, with
And the OD450 values of negative control, the stability and detection sensitivity of envelope antigen can be reflected, if blocking rate and feminine gender are right
According to the no significant changes of OD450 values, then it is considered that indirect envelope antigen is activity stabilized, detection performance does not change.
3rd, not Detection results of synantigen stabilizer treatment antigen indirect coated elisa plate
5% sucrose, 1%BSA+5% sucrose, 3%BSA+5% sucrose, 5%BSA+5% sucrose and 10%BSA+5% will be contained
The PBS of sucrose, adds the thiomersal preservative of 0.1/100 concentration, and 37 DEG C are placed 4 weeks, obtains not synantigen stable
Liquid, handles right with blocking ELISA detection NSP antibody positives, weakly positive and feminine gender after 3ABC antigen indirect coated elisa plates respectively
According to the blocking rate value of serum, it the results are shown in Table shown in 1.
Synantigen stabilizing solution processing ELISA Plate does not detect positive, weakly positive control serum PI values to table 1-
As shown in Table 1, when only adding the PBS containing 5% sucrose as antigen stabilizing solution, process is dried up in ELISA Plate
Middle antigen loss amount is big, so as to cause signal intensity to decline；And addition contains sucrose and BSA PBS is steady as antigen
When determining liquid, antigen active is improved, and detects signal enhancing；But with the rise of BSA concentration, the sensitivity detected to weak positive serum
Property decline, illustrate the inhibition that is combined with to antigen and antibody when BSA amounts are too high, influence detection sensitivity, while BSA consumptions
Increase, production cost consequently also increases.Therefore, the optimum formula of solid phase antigen stabilizing solution be containing 5% sucrose, 1%BSA with it is appropriate
The PBS of preservative.
The Detection of Stability result of antigen stabilizer treatment ELISA Plates of the table 2- containing 5% sucrose and 1%BSA
As shown in Table 2, with the antigen stabilizer treatment 3ABC antigen indirect coated elisa plates containing 5% sucrose and 1%BSA,
37 DEG C preserve 49 days after, although negative control OD450 values decline 24.6%, detection weakly positive compare sensitiveness not under
Drop, illustrates that amount of antigen is declined slightly, the sensitiveness on detection does not influence significantly, remains able to ensure wanting for detection sensitivity
Ask.Preserved 4 weeks according to 37 DEG C, the term of validity that 1 year is preserved equivalent to 4 DEG C is calculated, using the method for coating of the present invention, Neng Goubao
Demonstrate,prove the antigen indirect coated elisa plate storage life of at least 1 year.
Claims (2)
1. a kind of method for coating of ELISA kit antigen indirect coated elisa plate, it is characterised in that
(1) it is coated with：Monoclonal antibody is diluted with the carbonate buffer solutions of 0.05mol/L pH 9.6 or polyclonal antibody is dense to working
1~2 μ g/mL are spent, ELISA Plate is added, add 95 per hole~105 μ L, coating is stayed overnight at room temperature, is then washed with PBS 3 times；
(2) close：0.01mol/L PBSs 95~105 μ L per Kong Jiahan 10g/L isinglasses, 35~38 DEG C of closings 1~
2h, is then washed 3 times with PBS；
(3) target antigen is captured：Target antigen is diluted to the μ g/mL of working concentration 0.5~1.5 with 0.01mol/L PBSs,
ELISA Plate is added per the μ L of hole 95~105, then 18~25 DEG C of 1~1.5h of capture reaction are washed 3 times with PBS；
(4) solid phase antigen stabilizer is added：Under the situation that ELISA Plate hole is moistened, 120~150 μ L solid phase antigens are added per hole stable
Agent, room temperature acts on 20~40min, abandons raffinate；ELISA Plate is placed in the clean room of room epidemic disaster 20%~30%, and dehumidifier is taken out
It is dry 10~12 hours, produce antigen indirect coated elisa plate；Wherein, the solid phase antigen stabilizer is by mass fraction 5%
The phosphate buffer of sucrose and the bovine serum albumin(BSA) 0.01mol/L, pH 7.5 of mass fraction 1%, adds mass fraction 0.1%
Thiomersal preservative, 37 DEG C of placements are made for 4 weeks.
2. the method for coating of ELISA kit antigen indirect coated elisa plate according to claim 1, it is characterised in that
In step (1), the monoclonal antibody is foot and mouth disease virus nonstructural protein 3A monoclonal antibody, and working concentration is 2 μ g/mL；
The polyclonal antibody is foot and mouth disease virus Nonstructural protein 2C polyclonal antibody, and working concentration is 1 μ g/mL.
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| CN106053785A (en) * | 2016-05-18 | 2016-10-26 | 北京北方生物技术研究所有限公司 | Solid antibody pre-coating method for competitive chemiluminescent immunoreactions |
| CN106405093A (en) * | 2016-08-31 | 2017-02-15 | 北京市农林科学院 | Detection kit for detecting infectious bovine rhinotracheitis virus antibody |
| CN108181467B (en) * | 2018-02-06 | 2019-03-12 | 长春祈健生物制品有限公司 | A kind of preparation method of the stable antigen slide for the detection of FAMA method |
| CN108802378A (en) * | 2018-05-31 | 2018-11-13 | 重庆中元汇吉生物技术有限公司 | A kind of test strips that the nature controlling line of test strips is coated with solution and is prepared with this solution |
| CN109293748B (en) * | 2018-09-26 | 2021-04-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Foot-and-mouth disease virus non-structural protein 3A epitope peptide and application thereof |
| CN109557307A (en) * | 2018-12-04 | 2019-04-02 | 中国农业科学院兰州兽医研究所 | A kind of visualization quick detection kit of O-shaped foot and mouth disease virus and preparation method thereof |
| CN109851675B (en) * | 2018-12-24 | 2020-09-01 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Foot-and-mouth disease diagnostic kit and foot-and-mouth disease diagnostic antigen used by same |
| CN109851662B (en) * | 2018-12-24 | 2020-09-01 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Foot-and-mouth disease virus recombinant protein and related biological material and application thereof |
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| US6982087B2 (en) * | 2000-09-07 | 2006-01-03 | The University Of North Carolina At Chapel Hill | Vectors derived from South African Arbovirus No. 86 |
| CN102175852A (en) * | 2011-01-06 | 2011-09-07 | 云南省畜牧兽医科学院 | Detection method for solid phase competition ELISA (Enzyme Linked Immuno Sorbent Assay) of foot and mouth disease |
| CN102662062B (en) * | 2012-04-17 | 2015-05-27 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and method for detecting nonstructural protein (NSP) antibody of foot-and-mouth disease virus (FMDV) |
| CN103063830B (en) * | 2012-12-21 | 2016-04-13 | 杭州茂天赛科技有限公司 | A kind of preparation method of ELISA pre-coated elisa plate |
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