A kind of Western blotting film and preparation method thereofTechnical field
The invention belongs to protein analysis field, more particularly, to a kind of Western blotting film and preparation method thereof withUsing.
Background technology
Western blotting (Western Blot) technology is, by Protein transfer to film, then to be detected using antibodyTechnology.Western blotting is usually used in identifying certain albumen, and can carry out quantitative and semi-quantitative analysis to albumen.With reference to chemiluminescence inspectionSurvey, can simultaneously compare the expression difference of multiple sample same proteins.There is the high-resolution of SDS-PAGE due to Western blottingThe high specific and sensitiveness of power and solid-phase immunoassay, a kind of routine techniques as analysis of protein.
Film used by immunoblot assay is nitrocellulose filter, nylon membrane or polyvinylidene fluoride film, is shaped as strip(《Antibody technique experiment guide》, Science Press, 2002.9).The film is only capable of carrying out the single detection of single sample to be tested,If the Parallel testing of multiple samples to be tested need to be carried out, after needing to be transferred to the plurality of albumen sample on different films, forDifferent films are individually incubated, while being vibrated to accelerate reaction speed.Therefore had the disadvantages that using the film：First, when Parallel testing is carried out, reaction efficiency is low；2nd, because Western blotting film needs to be immersed in sample to be tested solution,Required sample size is big, expends reagent, and be difficult to trace detection；3rd, film needs to be developed the color after being dried and takes pictures, then entersRow analysis, so as in situ detection cannot be realized；4th, generally it is immersed in different windows or reactive tank due to film, different filmsReaction condition slightly have difference, so as to influence the accuracy of Western blotting.Disadvantage mentioned above also result in enter using the film indirectlyThe structure of the automatic detection instrument of row Western blotting is complex, and production cost is higher.
The content of the invention
For the disadvantages described above or Improvement requirement of prior art, the invention provides a kind of Western blotting film and its preparationMethod, its object is to film surface is divided into multiple hydrophilic regions, so as to realize the Parallel testing of multiple albumen samples.
To achieve the above object, according to one aspect of the present invention, there is provided a kind of Western blotting film, the Diagnosis of SghistosomiasisMark film surface has the multiple parallel strip hydrophilic region being spaced with hydrophobic region, and the strip hydrophilic region has oneOr multiple lands, there is target antigen, the target antibody for combining Western blotting on the land.
Preferably, the strip hydrophilic region has multiple lands, and the multiple land includes test block and rightAccording to area.
As it is further preferred that the check plot includes positive control area, negative control area or conjugate check plot.
Preferably, the material of the strip hydrophilic region is nitrocellulose, nylon or Kynoar.
Preferably, the hydrophobic region has silylation.
As it is further preferred that the material of the hydrophobic region is dimethyl silicone polymer or derivatives thereof.
Preferably, the width of the strip hydrophilic region and hydrophobic region is 1mm~5mm.
Preferably, the Western blotting film along the direction of strip hydrophilic region around being made as spool structure.
As it is further preferred that a diameter of 2cm~6cm of the spool structure.
As it is further preferred that the spool structure is wound in support shaft.
As it is further preferred that the support shaft is cylindric or cylindrical shape.
It is another aspect of this invention to provide that a kind of preparation method of above-mentioned Western blotting film is additionally provided, including：
The combining target antigen on substrate film, to form the quantity identical junction belt with target antigen, the substrate filmMaterial be nitrocellulose, nylon or Kynoar；
Hydrophobic region is formed on substrate film with hydrophobic coating, substrate film multiple strip hydrophilic regions is divided into, togetherWhen junction belt is divided into land so that each strip hydrophilic region have combined with the quantity identical of target antigenArea.
It is another aspect of this invention to provide that a kind of preparation method of above-mentioned Western blotting film is additionally provided, including：
The parallel patch of multiple strip hydrophilic films is formed on hydrophobic film so that have and multiple bars on the hydrophobic filmShape hydrophilic film formed multiple parallel strip hydrophilic region, the strip hydrophilic region with hydrophobic region separately.
Preferably, the hydrophobic film surface has the groove being engaged with strip hydrophilic film, and the groove is used for solidDetermine strip hydrophilic film.
In general, by the contemplated above technical scheme of the present invention compared with prior art, due to by hydrophobic regionDomain, multiple parallel strip hydrophilic regions are divided into by Western blotting film, can obtain following beneficial effect：
1st, on same Western blotting film, the sample of multiple target antibodies can be detected, so as to improve parallel inspectionThe reaction efficiency of survey；
2nd, Western blotting film has the multiple parallel strip hydrophilic region being spaced with hydrophobic region, by hydrophilic regionAbsorption affinity combined with target antibody, so as to more save reagent, while being applied to trace detection；
3rd, Western blotting film is combined due to the absorption affinity by hydrophilic region with target antibody, so that required is moltenLiquid measure is less, simplifies detecting step, is more suitable in situ detection；
4th, the sample of multiple target antibodies is incorporated on same Western blotting film, so as to be more suitable for identicalUnder the conditions of reacted, it is easier to carry out parallel control, improve the accuracy rate of detection.
Brief description of the drawings
Fig. 1 is the top view of the embodiment of the present invention 1；
Fig. 2 is the manufacturing process schematic diagram of the embodiment of the present invention 1；
Fig. 3 is the manufacturing process schematic diagram of the embodiment of the present invention 2；
Fig. 4 is the manufacturing process schematic diagram of the embodiment of the present invention 3；
Fig. 5 is the structural representation of the embodiment of the present invention 4；
In all of the figs, identical reference be used for represent identical element or structure, wherein：1- Western blottings are thinFilm, 11- hydrophobic regions, 12- hydrophilic regions, 13- lands, 2- support shafts.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and ExamplesThe present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, andIt is not used in the restriction present invention.As long as additionally, technical characteristic involved in invention described below each implementation methodNot constituting conflict each other can just be mutually combined.
The invention provides a kind of Western blotting film 1, the surface of Western blotting film 1 has between hydrophobic region 11Every multiple strip hydrophilic regions 12, the material of the strip hydrophilic region 12 is nitrocellulose, nylon or Kynoar,So as to combining target antigen, the hydrophobic region 11 is usually the organic hydrophobic polymer with silylation, while being also possible to takeWith siloxy or silicone hydroxyl, such as dimethyl silicone polymer or derivatives thereof；
The strip hydrophilic region 12 has one or more lands 13 along its length, has on the land 13There is target antigen, the target antigen is used to be combined with the target protein of Western blotting；When the strip hydrophilic region 12 includesDuring multiple lands 13, land 13 can be divided into test block and check plot, and test block is used to be combined with target protein, and comparesArea can be divided into positive control area, negative control area or conjugate check plot according to its function；Wherein, positive control area generally containsThere is the antigen that can be combined with other compositions in solution to be measured in addition to target antibody, for example, when with blood as solution to be measured,The target antigen of positive control area can then use serum antibody, and positive control area colour developing proves that Western blotting film 1 does not go bad, andSolution type to be measured is errorless；And negative control area can be coated with unlabelled secondary antibody so that negative control area at the standard conditionsCannot develop the color, if it develops the color, it was demonstrated that the abnormal conditions such as there may be Western blotting film 1 rotten, it should change Western blotting film 1And redeterminate；And the target antigen of conjugate check plot be IgG conjugates, IgM conjugates or IgA conjugates, due to IgG,IgM and IgA are common immunoglobulin, and the presence of conjugate check plot may indicate that whether target antibody successfully combines.
The width of the strip hydrophilic region 12 and hydrophobic region 11 is usually 1mm~5mm, is being easy to do Western blottingWhile test, it is ensured that each strip hydrophilic region 12 is spaced completely, while effectively using the space of Western blotting film 1.
For the ease of storing and using, the Western blotting film 1 can be along the direction of strip hydrophilic region around cylindricOr the support shaft 2 of cylindrical shape is around the spool structure for being made as a diameter of 2cm~6cm, as shown in Figure 5.
The preparation method of the Western blotting film 1 is roughly divided into two kinds：
The first：The combining target antigen on substrate film, it is described to form the quantity identical junction belt with target antigenThe material of substrate film is nitrocellulose, nylon or Kynoar；Hydrophobic region 11 is formed on substrate film with hydrophobic coating,Multiple strip hydrophilic regions 12 are divided into by substrate film, while by junction belt land 13 corresponding with junction belt so that everyIndividual strip hydrophilic region 12 all has the quantity identical land 13 with target antigen；Hydrophobic coating needs are nontoxic, andLess than 37 degree can dry, in order to avoid the activity of influence target antigen, the hydrophobic coating in such as patent document CN201380057250.
Second：The parallel patch of multiple strip hydrophilic films is formed on hydrophobic film so that have on the hydrophobic filmWith multiple strip hydrophilic films formed strip hydrophilic region 12, the strip hydrophilic region 12 with hydrophobic region 11 separately；The hydrophobic film surface can be provided with the groove being engaged with strip hydrophilic film, be combined simultaneously with strip hydrophilic filmIt is fixed so that surface of the surface less than hydrophobic place where strip hydrophilic region 12, can so be further ensured that strip is hydrophilicReaction solution between region 12 will not interact mixing.
Above-mentioned Western blotting film 1 can be applied to immune-blotting method, so that indirect method carries out chemiluminescence detection as an example, toolBody is comprised the following steps：
S1. add corresponding with the quantity of strip hydrophilic region 12, is being set at the top 0.1mm of strip hydrophilic region 12~2mmLiquid pipe, by various solution to be measured by liquid-feeding tube making an addition to the different surfaces of strip hydrophilic region 12, in making solution to be measuredTarget antibody is completely combined with the target antigen in junction belt；Simultaneously so that the strip hydrophilic region 12 of liquid-feeding tube with 1cm/s~The speed of 15cm/s does relatively reciprocating motion, and while motion, solution to be measured can be still slowly added in strip by liquid-feeding tubeThe surface of hydrophilic region 12, in case liquid evaporation, is 0.1mm~1mm to keep the liquid level on strip hydrophilic region 12；HereinIn the case of, it is computed, even if the solution to be measured that will be remained in liquid-feeding tube is counted, the volume of required solution to be measured is also only neededSeveral tens of microliters to milliliter level；
S2. collector tube can be set in one end of each strip hydrophilic region 12, one end of collector tube is away from strip hydrophilic region 12Top 0.1mm~2mm, other end connection vavuum pump, after completion of the reaction, vavuum pump aspirates strip hydrophilic region by collector tubeUnnecessary solution to be measured on 12；
S3. in mode same in S1-S2., the solution to be measured in former liquid-feeding tube is substituted with buffer solution, Western blotting is thinThe solution to be measured on the surface of film 1 is rinsed well；Because buffer solution is usually the lower-cost solution such as PBS, relative to solution to be measured,More volume can be added；
S4. in mode same in S1-S2., the buffer solution in former liquid-feeding tube is substituted with two corresponding anti-solution so that secondary antibody is completeCombined with target antibody；
S5. in mode same in S3., the two corresponding anti-solution in former liquid-feeding tube is substituted with buffer solution, by Western blotting film 1The two corresponding anti-solution on surface is rinsed well；
S6. in mode same in S1-S2., the buffer solution in former liquid-feeding tube is substituted with chemiluminescence agent solution so that markThe secondary antibody recorded a demerit lights；
S7. in mode same in S3., the chemiluminescence agent solution in former liquid-feeding tube is substituted with buffer solution, by Diagnosis of SghistosomiasisThe chemiluminescence agent solution on the surface of mark film 1 is rinsed well；
S8. adaptive immune trace testing result.
Embodiment 1
Fig. 1 is the Western blotting film 1 of the embodiment of the present invention 1, and its material is nylon membrane；The surface of Western blotting film 1With the multiple strip hydrophilic regions 12 being spaced with hydrophobic region 11, the width of the strip hydrophilic region 12 and hydrophobic region 11Degree is 1mm, and the strip hydrophilic region 12 has one or more lands 13, anti-with target on the land 13It is former；
The preparation method of the Western blotting film 1 resists as shown in Fig. 2 combining four kinds of targets first on the substrate film of nylonOriginal, to form four junction belts, as shown in Fig. 2 a-b；Then hydrophobic region 11 is formed on substrate film with hydrophobic coating, willSubstrate film is divided into multiple strip hydrophilic regions 12, at the same by junction belt be divided into junction belt quantity identical land 13,So that each strip hydrophilic region 12 has the quantity identical land 13 with target antigen, as shown in Figure 2 c, that is, obtainWestern blotting film 1 shown in Fig. 1.The composition of hydrophobic coating is the tetraethyl orthosilicate of 1.448wt%, the ten of 0.1588wt%Dialkyl group trimethoxy silane, the ammonium hydroxide of 0.0698wt%, the HCl of 0.1938wt%, the ethanol of 208wt% andThe softened water of 78.1198wt%.
Embodiment 2
Fig. 3 is the manufacturing process schematic diagram of the Western blotting film 1 of the embodiment of the present invention 2, first places dimethyl silicone polymer(PDMS) substrate, then will have multiple lands 13, and width is viscous with the interval of 3mm for the cellulose nitrate film of 3mmIt is formed on substrate.
Embodiment 3
Fig. 4 is the manufacturing process schematic diagram of the Western blotting film 1 of the embodiment of the present invention 3, and the difference with embodiment 2 is,On the PDMS substrates, at interval of 5mm, there are 5mm grooves wide, width can be just for the polyvinylidene difluoride film of 5mmIt is sticked with the groove.
Embodiment 4
Fig. 5 is the structural representation of 4 Western blotting film of the embodiment of the present invention 1, and the difference with embodiment 1 is that length isThe Western blotting film 1 of 10cm is wound in the support shaft 2 of diameter about 3.2cm.
As it will be easily appreciated by one skilled in the art that the foregoing is only presently preferred embodiments of the present invention, it is not used toThe limitation present invention, all any modification, equivalent and improvement made within the spirit and principles in the present invention etc., all should includeWithin protection scope of the present invention.