CN104914249B - Quickly detect colloidal gold immune chromatography test and the preparation method of Soybean Kunitz Trypsin enzyme inhibition factor - Google Patents
Quickly detect colloidal gold immune chromatography test and the preparation method of Soybean Kunitz Trypsin enzyme inhibition factor Download PDFInfo
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Abstract
The present invention relates to colloidal gold immune chromatography test and the preparation method of a kind of quick detection Soybean Kunitz Trypsin enzyme inhibition factor; this reagent paper principle based on double-antibody sandwich detection KTI; reagent paper contains supporting layer, adsorption layer, protective layer, and adsorption layer is followed successively by sample pad, pad, cellulose membrane and adsorptive pads from test lead.Pad is adsorbed with the specific antibody of KTI, cellulose membrane is provided with the detection line with KTI rabbit source Anti-TNF-α liquid solution specking and with goat anti-mouse igg antibody or the nature controlling line of rabbit anti-mouse IgG antibody solution specking, and the specific antibody of KTI uses the KTI Mus resource monoclonal antibody of Au colloidal nanoparticles labelling.The immune chromatography test paper high specificity of the present invention, highly sensitive, detection is easy, directly perceived, accurately, low cost, applied widely, it is easy to popularization and application.
Description
Technical field
The present invention relates to colloidal gold immune chromatography test and the preparation method of a kind of quick detection Soybean Kunitz Trypsin enzyme inhibition factor.
Background technology
Rich in protein in Semen sojae atricolor, generally 35% ~ 38%, aminoacid Compositional balance, and rich in unsaturated fatty acid, is human body and the important vegetable protein of animal body and vegetable oil source.Owing to its cultivated area is wide, yield is big, is widely used in processing raw material of food and feedstuff the most always, has high use value for human health and Animal nutrition.But Semen sojae atricolor also contains multiple antinutritional factor, these antinutritional factor have impact on the development and utilization of Semen sojae atricolor to a great extent.
Soybean trypsin inhibitor (Kunitz trypsin inhibitor, KTI) being one of Main Soybean Antinutritional Factors, belong to many peptides or protein, molecular weight is between 7975 ~ 21500D, about it is made up of 72 ~ 197 amino acid residues, accounts for the 6% of soybean protein.KTI is separated in 1945 first by Kunitz from Semen sojae atricolor, and its molecular weight is 20 ~ 25kD, 181 aminoacid and a small amount of cysteine 2 disulfide bond formed form.KTI can suppress the tryptic activity of pancreatic secretion in humans and animals intestinal, reduces digesting and assimilating of protein, causes pancreatic regeneration and hypertrophy, causes the problems such as growth retardation, induction anaphylaxis.Therefore, people explore various method in production practices and bean product are carried out suitable processed, and so that soybean trypsin inhibitor is reduced or eliminated, but these methods all can not be completely eliminated its impact.So setting up effectively, the content of KTI is particularly important during detection method detects food and feedstuff accurately.
Immune chromatography test paper detecting method has the advantages such as low cost, quick, portable and easy operation, may be used for the qualitative and quantitative detection of batch samples, accuracy and sensitivity also are able to meet requirement, it is simple that this method analyzes process, detection as KTI has unique advantage, is the detection technique needing to first develop.But at present still without the report about KTI immune chromatography test paper detecting method, will become a developing direction of soybean trypsin inhibitor detection method in this way.
Summary of the invention
The technical problem to be solved in the present invention: the deficiency existed for prior art, it is provided that a kind of immune chromatography test paper special, sensitive, easy, safe, quickly detection KTI and preparation method thereof.
Technical scheme:
The colloidal gold immune chromatography test of a kind of quick detection KTI, bottom is supporting layer, and intermediate layer is adsorption layer, and protective layer is fixed on adsorption layer, and adsorption layer is followed successively by sample pad, pad, cellulose membrane and adsorptive pads from test lead.It is provided with the detection line with KTI rabbit source Anti-TNF-α liquid solution specking and with goat anti-mouse igg antibody or the nature controlling line of rabbit anti-mouse IgG antibody solution specking at cellulose membrane；Described pad is adsorbed with the KTI Mus resource monoclonal antibody of Au colloidal nanoparticles labelling.
The described supporting layer toughness material not absorbed water is made；Sample pad glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made；Pad glass fibre cotton, polyester film, acetate fiber or nylon membrane are made；Cellulose membrane nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made；Adsorptive pads absorbent filter or filter paper for oil are made.Stealthy detection line on cellulose membrane and stealthy nature controlling line be arranged in parallel "??" orthoscopic, or be other similar markings.
The quickly preparation method of the colloidal gold immune chromatography test of detection KTI: include that the KTI Mus resource monoclonal antibody with Au colloidal nanoparticles labelling (gold mark monoclonal antibody) prepares pad, line is detected with the Anti-TNF-α liquid solution specking stealth of KTI rabbit source, with goat anti-mouse igg antibody or rabbit anti-mouse IgG antibody solution specking stealth nature controlling line, on supporting layer, then paste sample pad, pad, cellulose membrane and adsorptive pads.Being coated with protecting film on described sample pad, pad and adsorptive pads, on the protecting film that sample pad is corresponding with pad intersection, specking has sample mark line, finally adds up-protective layer.
This reagent paper make use of the principle of double-antibody sandwich detection antigen, after reagent paper test lead inserts testing sample solution, solution to be measured drives the gold mark monoclonal antibody in KTI to be measured and pad to spread to cellulose membrane together by siphonage, and eventually penetrates the absorbent material layer of handle end.In diffusion process, KTI to be measured can first combine with gold mark monoclonal antibody, and then the rabbit source polyclonal antibody detection line of specking is combined on cellulose membrane, gold labeling antibody and rabbit source polyclonal antibody combine from the different antigenic determinants on KTI molecule detected in sample respectively, KTI is sandwiched between gold mark monoclonal antibody and rabbit source polyclonal antibody, formed gold mark monoclonal antibody-antigen-rabbit source multi-resistance complex, i.e. double-antibody sandwich pattern, display redness detection the marking "?”；On cellulose membrane, sheep or the rabbit anti-mouse IgG antibody nature controlling line of specking also can be combined with gold labeling antibody, the red Quality Control marking of formation "?", i.e. two Red Sigils "??" it is expressed as the positive；Otherwise time in sample solution without KTI, then can not be combined with gold labeling antibody, can not be combined by the rabbit source polyclonal antibody detection line of specking on cellulose membrane equally, do not show the red detection marking, and sheep or the rabbit anti-mouse IgG antibody nature controlling line of specking can be combined with gold labeling antibody on cellulose membrane, the red nature controlling line marking of display "?", formed a Red Sigil "?" it is expressed as feminine gender.No matter result is positive or negative, all can show the red Quality Control marking, if not showing, represents that reagent paper lost efficacy.
The positive beneficial effect of the present invention:
(1) high specificity, highly sensitive.Reagent paper of the present invention is prepared from based on double-antibody sandwich principle, and two kinds of antibody are respectively Mus resource monoclonal antibody and the rabbit source polyclonal antibody of Au colloidal nanoparticles labelling.Being formed without covalent bond between gold grain and antibody molecule in gold labeling antibody, the two is combined by the Van der Waals force between the charges of different polarity, and the specificity of colloid gold label antagonist and affinity impact are the least, and have higher mark rate.Test shows, this reagent paper has higher sensitivity, can detect the trace KTI of 100ng/mL；Glycinin, β-conglycinin of high concentration, Semen sojae atricolor Bowman-Brik trypsin ihhibitor and soybean agglutinin is detected respectively with this reagent paper, concentration is 20mg/mL, result detection line does not develops the color, illustrate that this reagent paper and these several soybean proteins all do not have cross reaction, there is good specificity.
(2) easy, quickly.Use reagent paper of the present invention, it is not necessary to other any reagent and instruments, can execute-in-place.Only reagent paper need to be inserted in test sample liquid, after 10min, i.e. can determine that testing result.
(3) result display is vivid, directly perceived, accurately.Reagent paper of the present invention with display Red Sigil "??" and "?" (or similar character) as the positive of detection and negative marker, i.e. show on cellulose membrane two Red Sigils "??" (detection line and nature controlling line), represent in test sample containing KTI；Show a Red Sigil "?" (nature controlling line), represent in test sample without KTI.This result can the most directly be observed, it is determined that vivid, directly perceived, accurate, simple and clear, is difficult to the artificially erroneous judgement such as false positive and false negative occur.
(6) expense is saved.It is applicable to carry out Site Detection, low cost, saves a large amount of expensive instrument and cost of equipment.
(7) applied widely, it is simple to popularization and application.Present invention achieves the principle based on double-antibody sandwich application in quickly detection KTI colloidal gold immune chromatography test, this reagent paper is easy and simple to handle, the needs of different levels personnel can be met, including customs quarantine control, health quarantine, quality-monitoring, agricultural and animal products manufacturing enterprise and feed manufacturing emterprise etc..The present invention ensuring food safety, protect the consumer health for soybean allergy and ensure that the animal and fowl fodder aspect such as safely is extremely important, there is obvious economic benefit and social benefit.
(8) affinity costant of monoclonal antibody prepared by the present invention reaches 109L/mol, and the specificity of monoclonal antibody is preferable, the cross reacting rate using indirect competitive ELISA to measure with other soybean proteins is identified, these several soybean protein competitor include glycinin, beta-conglycinin, Semen sojae atricolor Bowman-Brik trypsin ihhibitor and soybean agglutinin, with the half-inhibition concentration (IC of KTI50) and the IC of each competitor50Percentage ratio as cross reacting rate, substantially there is no cross reaction between monoclonal antibody and this several soybean protein competitor, result shows that it has good specificity for KTI.
Accompanying drawing explanation
Fig. 1 is the structural representation of the immune chromatography test paper of the present invention.
Fig. 2 is the plan structure schematic diagram of the immune chromatography test paper of the present invention.
In figure, 1 is supporting layer, and 2 is sample pad, and 3 is pad, and 4 is cellulose membrane, and 5 is adsorptive pads, and 6 is detection line, and 7 is nature controlling line, and 8-1 is test lead protecting film, and 8-2 is handle end protecting film, and 9 is sample mark line.
Detailed description of the invention
Following example are to preferably explain the present invention, it is not intended that be particularly limited to the present invention, if not otherwise specified, percentage composition therein all can be regarded as percentage by weight.
The preparation process of reagent paper of the present invention includes: the steps such as the assembling of the preparation of KTI rabbit source polyclonal antibody, the preparation of KTI Mus resource monoclonal antibody, the preparation of pad, the preparation of sample pad, the preparation of cellulose membrane and reagent paper.
1, the preparation of KTI rabbit source polyclonal antibody:
Take cleaning grade 3 monthly age new zealand white rabbit, carry out immunity with KTI solution, subcutaneous point 4 ~ 6 injections of cervical region.First immunisation dosage is 200 g/, mixes with equivalent Freund's complete adjuvant (FCA) with aseptic PBS dilution KTI, the fully emulsified 10min of 25000r/min；Booster immunization dosage increases, and is 300 g/, immunity 4 times, mixes with equivalent incomplete Freund's adjuvant (FIA) with aseptic PBS dilution KTI, the fully emulsified 10min of 25000r/min.Immunity 4 times altogether, each immunization interval 3 weeks, after last immune 30 days, measure titer with ELISA method, and identify its Sensitivity and Specificity, Culling heart blood also separates and collects serum.With saturated ammonium sulfate salting out method purified rabbit source polyclonal antibody, i.e. take 1 part of serum and add 2 parts of PBS(pH7.2) mixing, add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, 10000rpm is centrifuged 30min, abandon supernatant, again with appropriate PBS(pH7.2) dissolution precipitation, add saturated ammonium sulfate solution to final concentration 33%, put 4 DEG C of refrigerator 12h, 4 DEG C, 10000rpm is centrifuged 30min, abandon supernatant, with appropriate PBS(pH7.2) dissolution precipitation, put 4 DEG C of interior PBS(pH7.2 of refrigerator) dialyse 48 ~ 72h, liquid is changed 6 ~ 8 times in centre, 4 DEG C, 12000rpm is centrifuged 15min, collect supernatant, obtain the KTI rabbit source polyclonal antibody of preliminary purification.Then Protein is used
G affinity column is further purified, first with 10 times of column volume PBS start buffer balance chromatographic columns, after the KTI rabbit source polyclonal antibody of preliminary purification is diluted with start buffer 1:1, it is loaded after being filtered by the 0.22 disposable sterilized filter of μm, wash with the start buffer stream of 10 times of column volumes subsequently, remove uncombined antibody, finally with elution buffer (the 0.1mol/L glycine-HCI of 2 times of column volumes, pH 2.5) eluting, with filling neutralizer (0.1mol/L Tris-HCl, pH 9.0) centrifuge tube collect, more fully dialyse with PBS, standby.
2, the preparation of KTI Mus resource monoclonal antibody:
Take SPF level 6 ~ 8 week old BALB/c mouse, carry out immunity with KTI solution.Dosage is 50 g/, and dorsal sc divides 4 ~ 6 injections.Immunity 4 times, first immunisation aseptic PBS dilution KTI mixes with equivalent FCA, and booster immunization aseptic PBS dilution KTI mixes with equivalent FIA, the fully emulsified 10min of 25000r/min, immunization interval 3 weeks.Determine that antibody titer is not added with adjuvant with the dosage of 50 g/ after meeting the requirements and carries out superpower immunity, hole under immune mouse socket of the eye was taken a blood sample in 3 ~ 4 days afterwards, separate positive serum；De-neck is lethal, and with alcohol-pickled mice 5 ~ 10min sterilization body surface of volume ratio 75%, sterile working takes its spleen, shredded by spleen and grind, and filters through 120 mesh nylon gauzes, and 1000rpm is centrifuged 10min, collects splenocyte.By 1 × 108Splenocyte mix in the ratio of 10:1 with NS0 myeloma cell, 1000rpm is centrifuged 10min, abandons supernatant, and cell precipitate is slowly added to the PEG1500 effect 1min of 0.7 ~ 1.0mL in 37 DEG C of water-baths, it is then slowly added into serum-free 1640 culture medium 15mL, to terminate the effect of PEG；37 DEG C of water-baths 5 ~ 10 min, 1000rpm are centrifuged 10min and abandon supernatant, are resuspended in HAT Selective agar medium by cell precipitate, and add 96 porocytes cultivation plate hole (100 L/ hole, L ~ 200), are placed in 37 DEG C, the CO of 5%2Incubator is cultivated.Cultivate 10 ~ 14 days, carry out positive hole sizer choosing with indirect ELISA and indirect competitive ELISA method, select strong positive, suppression ratio height, the eugonic hole of cell to carry out 2 ~ 3 limited dilution clonings, after the amplification culture of positive hole, set up hybridoma cell strain.Inducing ascites method in using Mice Body and obtain ascites, the more purified Mus resource monoclonal antibody that obtains, purification process uses the saturated ammonium sulfate salting out method in the polyclonal antibody preparation of above-mentioned rabbit source and affinity chromatography.
The affinity costant being measured Mus resource monoclonal antibody by ELISA saturation can reach 109L/mol.Utilizing Sigma Mus resource monoclonal antibody hypotype indentifying substance, the hypotype identifying this monoclonal antibody is IgG1 type.The cross reacting rate that the specificity of monoclonal antibody measures with other soybean proteins initially with indirect competitive ELISA is identified, these several soybean protein competitor include glycinin (glycinin), beta-conglycinin (β-conglycinin), Semen sojae atricolor Bowman-Brik trypsin ihhibitor and soybean agglutinin, with the half-inhibition concentration (IC of KTI50) and the IC of each competitor50Percentage ratio as cross reacting rate, result is as shown in table 1, does not substantially have cross reaction between monoclonal antibody and this several soybean protein competitor, and result shows that it has good specificity for KTI.
Table 1 KTI Mus resource monoclonal antibody and the cross reaction of competitor
| Competitor | Half-inhibition concentration IC50(ng/mL) | Cross reacting rate CR(%) |
| Glycinin | >1.0×105 | <0.5 |
| β-conglycinin | >1.0×105 | <0.5 |
| Bowman-Brik trypsin ihhibitor | >1.0×105 | <0.5 |
| Soybean agglutinin | >1.0×105 | <0.5 |
3, the preparation of sample pad
Prepared by sample pad glass fibre cotton, nylon membrane, polyvinylidene fluoride pvdf membrane or polyester film, fibrous material is cut into the band of wide 1.5cm specification, puts it into immersion 30min in sample pad confining liquid, in 37 DEG C of drying, standby.
4, the preparation of pad
Use reduction of sodium citrate method prepare colloidal gold solution, i.e. boiling 0.01 ~ 0.02% chlorauric acid solution in add 1% newly prepare sodium citrate solution, it is thus achieved that the colloidal gold solution of diameter 15 ~ 20nm, with the K of 0.1mol/L2CO3Regulation pH value, to 8.5 ~ 9.5, is put 2 ~ 8 DEG C and is saved backup.With the labelling of 1:2000 ratio KTI Mus resource monoclonal antibody to be marked added in the colloidal gold solution of pH 8.5 ~ 9.5, after labelling 30min, add the BSA of 10%, stand 30min, 4 DEG C, 13000rpm is centrifuged 30min and abandons supernatant.Precipitate and redissolve with the 0.02mol/L sodium tetraborate solution containing 2% BSA, 4 DEG C, 13000rpm is centrifuged 30min and abandons supernatant.By resuspended for the TB re-suspension liquid (composition of re-suspension liquid: contain the sodium azide of the BSA and 0.03% of 1% in 0.02mol/L sodium tetraborate solution) of Au colloidal nanoparticles precipitation 25mL, it is thus achieved that the KTI Mus resource monoclonal antibody of colloid gold label.Prepared by the colloidal gold labeled monoclonal antibody specking that 2 ~ 5 times dilute pad on glass fibre silk floss, and 56 DEG C of dried sealings are kept in Dark Place.
5, the preparation of cellulose membrane
Cellulose membrane nitrocellulose filter, pure cellulose film or carboxylated cellulose film, cut into the band of wide 1.5cm specification, with point sample instrument diverse location specking KTI rabbit source polyclonal antibody and goat anti-mouse igg antibody (or rabbit anti-mouse IgG antibody) respectively on cellulose membrane, make stealthy detection line and nature controlling line, with 2% after drying in 40 DEG C
BSA solution adequate closure, after drying at room temperature, sealing is kept in Dark Place standby.
Wherein the preparation method of goat anti-mouse igg (or rabbit anti-mouse IgG) antibody is as follows:
KTI negative mice serum IgG is extracted first with saturated ammonium sulfate salting out method, then with 200 g ~ 500 g/ dosage only through subcutaneous or intramuscular injection cleaning grade goat or new zealand white rabbit 3 ~ 4 times, after final immunization 30 days, with ELISA measure its serum titer reach more than 1:10000 time, heart or arterial blood drawing, separate and collect hyper-immune serum, extract sheep anti-Mouse or rabbit anti-mouse IgG antibody, for the specking of KTI Test paper nature controlling line with saturated ammonium sulfate salting out method and affinity chromatography.
6, the assembling of immune chromatography test paper:
Sample pad, pad, cellulose membrane, adsorptive pads are pasted the most successively on the supporting layer with binding agent, each other intersection overlap 2 ~ 3mm, be then cut into the wide reagent paper of 3 ~ 4mm.
7, the Cleaning Principle of immune chromatography test paper:
After reagent paper test lead inserts testing sample solution, solution to be measured drives the golden labeling antibody in KTI to be measured and pad to spread to cellulose membrane together by siphonage, and eventually penetrates the absorbent material layer of handle end.In diffusion process, KTI to be measured can first combine with golden mark monoclonal antibody, and then the KTI rabbit source polyclonal antibody detection marking of specking is combined on cellulose membrane, display detection line, formation redness detection tape "?”；On cellulose membrane, sheep or the rabbit anti-mouse IgG antibody Quality Control marking of specking also can be combined with gold labeling antibody, the red Quality Control tape of formation "?", i.e. two red tapes "??" it is expressed as the positive；Otherwise time in sample solution without KTI, then can not be combined with gold labeling antibody, can not be combined by the KTI rabbit source polyclonal antibody detection marking of specking on cellulose membrane equally, do not show red detection tape, and sheep or the rabbit anti-mouse IgG antibody Quality Control marking of specking can be combined with gold labeling antibody on cellulose membrane, the red Quality Control tape of display "?", formed a red zone "?" it is expressed as feminine gender.If not having any red tape to show on cellulose membrane, then show that reagent paper lost efficacy.
The structure of reagent paper of the present invention, detection method and characteristic is illustrated below with example.
Embodiment one: see Fig. 1 and Fig. 2.In figure, 1 is supporting layer, makes with plastic slice bar；2 is sample pad, makes with glass fibre cotton；3 is pad, is adsorbed with the glass fibre cotton of the KTI Mus resource monoclonal antibody of Au colloidal nanoparticles labelling；4 is cellulose membrane, uses nitrocellulose filter；The adsorptive pads 5 of handle end is made with absorbent filter.Sample pad 2, pad 3, cellulose membrane 4, adsorptive pads 5 are pasted the most successively and be fixed on supporting layer 1, and intersection fiber overlaps each other each other.Cellulose membrane 4 is provided with stealthy detection line 6, makes with KTI rabbit source Anti-TNF-α liquid solution；Stealthy nature controlling line 7 goat anti-mouse igg antibody solution specking on cellulose membrane is made, and two lines band is arranged in parallel, formation combination trace band "??”。
8-1 is to cover the sample end white protecting film on sample pad 2 and pad 3; 8-2 is to cover other color protecting film (such as yellow) on adsorptive pads 5; 9 is sample mark line; this mark line is positioned on the white protecting film that sample pad 2 is corresponding with pad 3 intersection at deflection sample pad 2 side about 0.5cm, is printed on arrow and Max printed words on the left of mark line on protecting film.
The preparation of testing sample and detecting step:
Detection defatted milk powder: by sample with containing 0.01mol/L NaHSO3Aqueous solution dissolve with the solid-liquid ratio of 1:15 after, the NaOH of 1mol/L adjusts pH to 9.0, stirs 1h, mixed solution 10000rpm at normal temperatures is centrifuged 30min under 45 DEG C of water bath condition, separates supernatant and is used for detecting.
Operational approach: KTI reagent paper sample end inserted in testing sample, insertion depth is less than mark line, within about 10 ~ 20 seconds, takes out reagent paper, horizontal positioned, observes and judge testing result after 10min.
Result judge: if (a) positive show on cellulose membrane two red trace bands "??", represent that testing result is positive, illustrate in testing sample containing KTI；If (b) feminine gender show on cellulose membrane a red trace band "?", represent that testing result is negative, illustrate in testing sample without KTI；Do not had red zone to show on cellulose membrane if c () is lost efficacy, then showed that reagent paper lost efficacy.
Embodiment two: test paper structure and embodiment one are essentially identical, and difference is: sample pad 2 is made with nylon membrane, and cellulose membrane 4 uses pure cellulose film.
Detection feedstuff: first pulverized by sample, crosses 60 mesh sieves, then with petroleum ether, the sample after pulverizing is carried out ungrease treatment.Sample preparation methods subsequently is with embodiment one.
Operational approach and result judge with embodiment one.
Embodiment three: test paper structure and embodiment one are essentially identical, and difference is: sample pad 2 polyvinylidene fluoride pvdf membrane is made, stealthy Quality Control trace 7 rabbit anti-mouse IgG antibody solution makes on cellulose membrane, and cellulose membrane 4 uses carboxylated cellulose film.
Detection feedstuff: sample preparation methods is with embodiment two.
Operational approach and result judge with embodiment one.
Embodiment four: test paper structure and embodiment one are essentially identical, and difference is: sample pad 2 is made with polyester film, and cellulose membrane 4 uses carboxylated cellulose film.Detection sample, operational approach and result judge with embodiment one.
Embodiment five: test paper structure and embodiment one are essentially identical, and difference is: sample pad 2 is made with nylon membrane, stealthy Quality Control trace 7 rabbit anti-mouse IgG antibody solution is made on cellulose membrane.Detection sample, operational approach and result judge with embodiment one.
Embodiment six: the Sensitivity and Specificity test of reagent paper of the present invention.
Sensitivity: take the KTI standard solution of ten gradients, concentration is respectively 0ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 800ng/mL, 1600ng/mL, 3200ng/mL, 6400ng/mL, 12800ng/mL.Respectively taking the standard solution 100mL of above-mentioned concentration, detect with the reagent paper in embodiment one, observed result after 10min, to detect the sensitivity of line colour developing evaluation of result reagent paper.Result shows, the sensitivity of reagent paper can reach 100ng/mL.
Specificity: detect glycinin, β-conglycinin of high concentration, Semen sojae atricolor Bowman-Brik trypsin ihhibitor and soybean agglutinin respectively by this detection paper, concentration is 20mg/mL, result detection line does not develops the color, illustrate that this reagent paper and these several soybean proteins all do not have cross reaction, there is good specificity.
Claims (9)
1. the colloidal gold immune chromatography test of a quick detection KTI; bottom is supporting layer; intermediate layer is adsorption layer; protective layer is fixed on adsorption layer; adsorption layer is followed successively by sample pad, pad, cellulose membrane and adsorptive pads from test lead, is characterised by: be provided with the stealthy detection line with KTI rabbit source Anti-TNF-α liquid solution specking and with goat anti-mouse igg antibody or the stealthy nature controlling line of rabbit anti-mouse IgG antibody solution specking at cellulose membrane；Described pad is adsorbed with the KTI Mus resource monoclonal antibody of Au colloidal nanoparticles labelling；
Wherein the preparation method of KTI rabbit source polyclonal antibody is as follows:
Taking cleaning grade 3 monthly age new zealand white rabbit, carry out immunity with KTI solution, subcutaneous point 4 ~ 6 injections of cervical region, first immunisation dosage is 200 g/, mixes with equivalent Freund's complete adjuvant with aseptic PBS dilution KTI, the fully emulsified 10min of 25000r/min；Booster immunization dosage increases, it it is 300 g/, immunity 4 times, mix with equivalent incomplete Freund's adjuvant with aseptic PBS dilution KTI, the fully emulsified 10min of 25000r/min, altogether immunity 4 times, immunization interval 3 weeks every time, last immunity measured titer with ELISA method after 30 days, and identified its Sensitivity and Specificity, and Culling heart blood also separates and collects serum；With saturated ammonium sulfate salting out method purified rabbit source polyclonal antibody, i.e. take the PBS that 1 part of serum adds 2 parts of pH7.2, mixing, add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, 10000rpm is centrifuged 30min, abandon supernatant, again with the PBS dissolution precipitation of appropriate pH7.2, add saturated ammonium sulfate solution to final concentration of 33%, put 4 DEG C of refrigerator 12h, 4 DEG C, 10000rpm is centrifuged 30min, abandon supernatant, PBS dissolution precipitation with appropriate pH7.2, put the PBS 48 ~ 72h of 4 DEG C of interior pH7.2 of refrigerator, liquid is changed 6 ~ 8 times in centre, 4 DEG C, 12000rpm is centrifuged 15min, collect supernatant, obtain the KTI rabbit source polyclonal antibody of preliminary purification；
Then Protein G affinity column is used to be further purified, first balance chromatographic column with the PBS start buffer of 10 times of column volumes, after the KTI rabbit source polyclonal antibody of preliminary purification is diluted with start buffer 1:1, it is loaded after being filtered by the disposable sterilized filter of 0.22 μm, washes with the start buffer stream of 10 times of column volumes subsequently, remove unconjugated antibody, finally with the elution buffer eluting of 2 times of column volumes, collect with the centrifuge tube filling neutralizer, more fully dialyse with PBS, standby；Described elution buffer is the glycine-HCI of 0.1mol/L, and pH value is 2.5；Neutralizer be Tris-HCl, pH value of 0.1mol/L be 9.0.
Immune chromatography test paper the most according to claim 1, is characterized in that: the described supporting layer toughness material not absorbed water is made；Sample pad glass fibre cotton, nylon membrane, PVDF membrane or polyester film are made；Pad glass fibre cotton, polyester film, acetate fiber or nylon membrane are made；Cellulose membrane nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made；Adsorptive pads absorbent filter or filter paper for oil are made.
Immune chromatography test paper the most according to claim 1, is characterized in that: described stealthy detection line and stealthy nature controlling line be arranged in parallel "??" orthoscopic trace；Being coated with protective layer on described sample pad, pad and adsorptive pads, on the protective layer that sample pad is corresponding with pad intersection, specking has sample mark line.
Immune chromatography test paper the most according to claim 1, is characterized in that: wherein the preparation method of KTI Mus resource monoclonal antibody is as follows:
Taking SPF level 6 ~ 8 week old BALB/c mouse, carry out immunity with KTI solution, dosage is 50 g/, dorsal sc divides 4 ~ 6 injections, immunity 4 times, and first immunisation dilutes KTI with aseptic PBS, mix with equivalent FCA, booster immunization aseptic PBS dilution KTI mixes with equivalent FIA, the fully emulsified 10min of 25000r/min, immunization interval 3 weeks, after determining that antibody titer meets the requirements, it is not added with adjuvant with 50 g/ dosage only and carries out superpower immunity, hole under immune mouse socket of the eye was taken a blood sample in 3 ~ 4 days afterwards, separate positive serum；De-neck is lethal, and with alcohol-pickled mice 5 ~ 10min sterilization body surface that volume ratio is 75%, sterile working takes its spleen, shredded by spleen and grind, and filters through 120 mesh nylon gauzes, and 1000rpm is centrifuged 10min, collects splenocyte, by 1 × 108Splenocyte mix in the ratio of 10:1 with NS0 myeloma cell, 1000rpm is centrifuged 10min, abandons supernatant, and cell precipitate is slowly added to the PEG1500 effect 1min of 0.7 ~ 1.0mL in 37 DEG C of water-baths, it is then slowly added into serum-free 1640 culture medium 15mL, to terminate the effect of PEG；37 DEG C of water-baths 5 ~ 10 min, 1000rpm are centrifuged 10min and abandon supernatant, are resuspended in HAT Selective agar medium by cell precipitate, and add 96 porocyte culture plates, and 100 L/ holes, L ~ 200 are placed in 37 DEG C, the CO of 5%2Incubator is cultivated；Cultivate 10 ~ 14 days, carry out positive hole sizer choosing with indirect ELISA and indirect competitive ELISA method, the hole of selection is carried out 2 ~ 3 limited dilution clonings, after the amplification culture of positive hole, sets up hybridoma cell strain；Use in Mice Body and induce ascites method and obtain ascites, more purified obtain Mus resource monoclonal antibody.
5. according to the immune chromatography test paper described in any one of claim 1-4, it is characterized in that: wherein the preparation method of the KTI Mus resource monoclonal antibody of Au colloidal nanoparticles labelling is as follows:
In the chlorauric acid solution of the 0.01 ~ 0.02% of boiling, addition 1% newly prepares sodium citrate solution, it is thus achieved that the colloidal gold solution of diameter 15 ~ 20nm, with the K of 0.1mol/L2CO3Regulation pH value, to 8.5 ~ 9.5, is put 2 ~ 8 DEG C and is saved backup；With the labelling of 1:2000 ratio KTI Mus resource monoclonal antibody to be marked added in the colloidal gold solution of pH 8.5 ~ 9.5, after labelling 30min, add the BSA of 10%, stand 30min, 4 DEG C, 13000rpm is centrifuged 30min and abandons supernatant；Precipitate and redissolve with the 0.02mol/L sodium tetraborate solution containing 2% BSA, 4 DEG C, 13000rpm is centrifuged 30min and abandons supernatant；By resuspended by the TB re-suspension liquid of 25 mL for Au colloidal nanoparticles precipitation, it is thus achieved that the KTI Mus resource monoclonal antibody of colloid gold label；Wherein TB re-suspension liquid is the sodium tetraborate solution of 0.02 mol/L, wherein containing the sodium azide of the BSA and 0.03% that weight ratio is 1%.
6. the preparation method of the immune chromatography test paper described in claim 1, is characterized in that: the method comprises the following steps:
First KTI rabbit source polyclonal antibody is prepared; then KTI Mus resource monoclonal antibody is prepared; blend compounds body gold nano grain is marked; pad is prepared with the KTI Mus resource monoclonal antibody of Au colloidal nanoparticles labelling; line is detected with the Anti-TNF-α liquid solution specking stealth of KTI rabbit source; with goat anti-mouse igg antibody or rabbit anti-mouse IgG antibody solution specking stealth nature controlling line; then stack gradually on supporting layer and paste sample pad, pad, cellulose membrane and adsorptive pads; finally add up-protective layer, prepare immune chromatography test paper.
7. the preparation method of immune chromatography test paper as claimed in claim 6, is characterized in that: wherein the preparation method of KTI rabbit source polyclonal antibody is as follows:
Taking cleaning grade 3 monthly age new zealand white rabbit, carry out immunity with KTI solution, subcutaneous point 4 ~ 6 injections of cervical region, first immunisation dosage is 200 g/, mixes with equivalent Freund's complete adjuvant with aseptic PBS dilution KTI, the fully emulsified 10min of 25000r/min；Booster immunization dosage only increases to 300 g/, immunity 4 times, KTI is diluted with aseptic PBS, mix with equivalent incomplete Freund's adjuvant, the fully emulsified 10min of 25000r/min, altogether immunity 4 times, immunization interval 3 weeks every time, last immunity measured titer with ELISA method after 30 days, and identified its Sensitivity and Specificity, and Culling heart blood also separates and collects serum；With saturated ammonium sulfate salting out method purified rabbit source polyclonal antibody, i.e. take the PBS that 1 part of serum adds 2 parts of pH7.2, mixing, add the mixing of equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerator 12h, 4 DEG C, 10000rpm is centrifuged 30min, abandon supernatant, again with the PBS dissolution precipitation of appropriate pH7.2, add saturated ammonium sulfate solution to final concentration of 33%, put 4 DEG C of refrigerator 12h, 4 DEG C, 10000rpm is centrifuged 30min, abandon supernatant, PBS dissolution precipitation with appropriate pH7.2, put the PBS 48 ~ 72h of 4 DEG C of interior pH7.2 of refrigerator, liquid is changed 6 ~ 8 times in centre, 4 DEG C, 12000rpm is centrifuged 15min, collect supernatant, obtain the KTI rabbit source polyclonal antibody of preliminary purification；
Then Protein G affinity column is used to be further purified, first balance chromatographic column with the PBS start buffer of 10 times of column volumes, after the KTI rabbit source polyclonal antibody of preliminary purification is diluted with start buffer 1:1, it is loaded after being filtered by the disposable sterilized filter of 0.22 μm, washes with the start buffer stream of 10 times of column volumes subsequently, remove uncombined antibody, finally with the elution buffer eluting of 2 times of column volumes, collect with the centrifuge tube filling neutralizer, more fully dialyse with PBS, standby；Described elution buffer is the glycine-HCI of 0.1mol/L, and pH value is 2.5；Neutralizer be Tris-HCl, pH value of 0.1mol/L be 9.0.
8. the preparation method of immune chromatography test paper as claimed in claim 6, is characterized in that: wherein the preparation method of KTI Mus resource monoclonal antibody is as follows:
Take SPF level 6 ~ 8 week old BALB/c mouse, immunity is carried out with KTI solution, dosage is 50 g/, dorsal sc divides 4 ~ 6 injections, immunity 4 times, first immunisation dilutes KTI with aseptic PBS, mixing with equivalent FCA, booster immunization dilutes KTI with aseptic PBS, mixes with equivalent FIA, the fully emulsified 10min of 25000r/min, immunization interval 3 weeks, after determining that antibody titer meets the requirements, is not added with adjuvant with 50 g/ dosage only and carries out superpower immunity, hole under immune mouse socket of the eye was taken a blood sample in 3 ~ 4 days afterwards, separate positive serum；De-neck is lethal, and with alcohol-pickled mice 5 ~ 10min sterilization body surface that volume ratio is 75%, sterile working takes its spleen, shredded by spleen and grind, and filters through 120 mesh nylon gauzes, and 1000rpm is centrifuged 10min, collects splenocyte, by 1 × 108Splenocyte mix in the ratio of 10:1 with NS0 myeloma cell, 1000rpm is centrifuged 10min, abandons supernatant, and cell precipitate is slowly added to the PEG1500 effect 1min of 0.7 ~ 1.0mL in 37 DEG C of water-baths, it is then slowly added into serum-free 1640 culture medium 15mL, to terminate the effect of PEG；37 DEG C of water-baths 5 ~ 10 min, 1000rpm are centrifuged 10min and abandon supernatant, are resuspended in HAT Selective agar medium by cell precipitate, and add 96 porocyte culture plates, and 100 L/ holes, L ~ 200 are placed in 37 DEG C, the CO of 5%2Incubator is cultivated；Cultivate 10 ~ 14 days, carry out positive hole sizer choosing with indirect ELISA and indirect competitive ELISA method, the hole of selection is carried out 2 ~ 3 limited dilution clonings, after the amplification culture of positive hole, sets up hybridoma cell strain；Use in Mice Body and induce ascites method and obtain ascites, more purified obtain Mus resource monoclonal antibody.
9. the preparation method of immune chromatography test paper as described in any one of claim 6-8, is characterized in that: wherein the preparation method of the KTI Mus resource monoclonal antibody of Au colloidal nanoparticles labelling is as follows:
In 0.01 ~ 0.02% chlorauric acid solution of boiling, addition 1% newly prepares sodium citrate solution, it is thus achieved that the colloidal gold solution of diameter 15 ~ 20nm, with the K of 0.1mol/L2CO3Regulation pH value, to 8.5 ~ 9.5, is put 2 ~ 8 DEG C and is saved backup；With the labelling of 1:2000 ratio KTI Mus resource monoclonal antibody to be marked added in the colloidal gold solution of pH 8.5 ~ 9.5, after labelling 30min, adds the BSA of 10%, stand 30min, 4 DEG C, 13000rpm be centrifuged 30min, abandon supernatant；Precipitate with containing 2% BSA 0.02mol/L sodium tetraborate solution redissolve, 4 DEG C, 13000rpm be centrifuged 30min, abandon supernatant；By resuspended by the TB re-suspension liquid of 25 mL for Au colloidal nanoparticles precipitation, it is thus achieved that the KTI Mus resource monoclonal antibody of colloid gold label；Wherein TB re-suspension liquid is the sodium tetraborate solution of 0.02 mol/L, wherein containing the sodium azide of the BSA and 0.03% that weight ratio is 1%.
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| US4959310A (en) * | 1987-09-28 | 1990-09-25 | The United States Of America As Represented By The Secretary Of Agriculture | Monoclonal antibodies to soybean kunitz trypsin inhibitor and immunoassay methods |
| CN204882573U (en) * | 2015-07-08 | 2015-12-16 | 河南省农业科学院 | Short -term test soybean kunitz trypsin inhibitory factor's colloidal gold immunoassay chromatography test paper |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US4959310A (en) * | 1987-09-28 | 1990-09-25 | The United States Of America As Represented By The Secretary Of Agriculture | Monoclonal antibodies to soybean kunitz trypsin inhibitor and immunoassay methods |
| CN204882573U (en) * | 2015-07-08 | 2015-12-16 | 河南省农业科学院 | Short -term test soybean kunitz trypsin inhibitory factor's colloidal gold immunoassay chromatography test paper |
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| Title |
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| Rapid and Sensitive Detection of the Food Allergen Glycinin in Powdered Milk Using a Lateral Flow Colloidal Gold Immunoassay Strip Test;Yao Wang, et al.;《Journal of Agricultural and Food Chemistry》;20150211;第63卷(第8期);权利要求1-3，实施例1-4 * |
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