CN110412266A - For detecting the colloidal gold strip of IL-1 β, IL-1Ra and IL-1Ra/IL-1 β ratio simultaneously - Google Patents

Abstract

The present invention provides a kind of for detecting IL-1 β, the colloidal gold strip of IL-1Ra content and IL-1Ra/IL-1 β ratio and its application simultaneously, is related to technical field of immunoassay；The colloidal gold strip, including fiberglass packing, nitrocellulose filter and blotting paper；In conjunction with the anti-il-i-beta -1Ra-2 rabbit source polyclonal antibody of IL-1 β and IL-1Ra while being coated with colloid gold label on the glass fibre element pad；The nitrocellulose filter successively includes detection line T1, detection line T2 and nature controlling line；The cavy source polyclonal antibody in conjunction with the anti-il-i-beta -1 of IL-1 β is coated in the detection line T1；The cavy source polyclonal antibody of the anti-IL-1Ra-1 in conjunction with IL-1Ra is coated on the detection line T2；Goat anti-rabbit antibody is coated on the nature controlling line.Colloidal gold strip provided by the invention has many advantages, such as special, convenient, at low cost, reproducible.

Description

For detecting the colloidal gold examination of IL-1 β, IL-1Ra and IL-1Ra/IL-1 β ratio simultaneously Paper slip

Technical field

The present invention relates to technical field of immunoassay, and in particular to one kind is used for while detecting IL-1 β, IL-1Ra and IL- The colloidal gold strip of 1Ra/IL-1 β ratio.

Background technique

Cell factor emerges one after another as inflammatory disease research hotspot, detection method.The detection method of IL-1 β and IL-1Ra Mainly there are immunological detection, intracellular cytokine detection method, bioassay, molecular biology method etc..By stream The cytokines measurement method of formula cell art, it is cumbersome expensive, it is unfavorable for the popularization and application of on-site test and base.It is raw Object activity detection sensitivity is higher but poor specificity, and more demanding to external environments such as temperature, sample compositions.

The current detection method most common on the market is immunological method, and highest utilization rate is integrated enzyme reaction kit, It operates relatively simple.But existing commercial kit can only detect IL-1 β and IL-1Ra respectively, cannot detect the two quantity simultaneously And ratio calculated.

Summary of the invention

In view of this, be used for the purpose of the present invention is to provide one kind while detecting IL-1 β, IL-1Ra content and IL- The colloidal gold strip of 1Ra/IL-1 β ratio and its application, colloidal gold strip provided by the invention have it is special, convenient, at The advantages that this is low, reproducible.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention provides a kind of for detecting the colloid of IL-1 β, IL-1Ra content and IL-1Ra/IL-1 β ratio simultaneously Gold test paper strip, including fiberglass packing, nitrocellulose filter and blotting paper；Colloidal gold mark is coated on the glass fibre element pad In conjunction with the anti-il-i-beta -1Ra-2 rabbit source polyclonal antibody of IL-1 β and IL-1Ra while note；The nitrocellulose filter successively wraps Include detection line T1, detection line T2 and nature controlling line；The cavy source in conjunction with the anti-il-i-beta -1 of IL-1 β is coated in the detection line T1 Polyclonal antibody；The cavy source polyclonal antibody of the anti-IL-1Ra-1 in conjunction with IL-1Ra is coated on the detection line T2；It is described Goat anti-rabbit antibody is coated on nature controlling line.

Preferably, the dosage of the rabbit source polyclonal antibody of the anti-il-i-beta -1Ra-2 of the colloid gold label is 1.0~1.4 μ g/100μL。

Preferably, in the detection line T1 cavy source polyclonal antibody of coated anti-il-i-beta -1 concentration be 1.8~ 2.2mg/mL。

Preferably, on the detection line T2 cavy source polyclonal antibody of coated anti-IL-1Ra-1 concentration be 0.8~ 1.2mg/mL。

Preferably, the concentration of coated goat anti-rabbit antibody is 0.6~1.0mg/mL on the nature controlling line.

The present invention also provides the colloidal gold strips described in above-mentioned technical proposal in preparation test sample IL-1 β and IL- It is applied in the kit of the concentration of 1Ra.

Preferably, comprising the following steps:

(1) sample to be tested is added drop-wise to fiberglass packing；

(2) after nature controlling line colour developing, whether observation detection line from colourless becomes red；Detection line becomes red, to test sample Product contain IL-1 β and IL-1Ra；Detection line is non-discolouring, and IL-1 β and IL-1Ra content is lower than detection limit in sample to be tested.

The present invention also provides the colloidal gold strips described in above-mentioned technical proposal in preparation test sample IL-1Ra and IL- It is applied in the kit of the ratio of 1 β.

Preferably, the sample includes brucellosis correlation blood sample, including brucellosis infection animal blood sample, Bu Lu Animal blood sample is immunized in Salmonella disease vaccine, brucellosis vaccine animal blood sample is not immunized for health.

The present invention provides a kind of for detecting the colloid of IL-1 β, IL-1Ra content and IL-1Ra/IL-1 β ratio simultaneously Gold test paper strip and its application.Colloidal gold strip provided by the invention use gold labeling antibody of the same race, can measure simultaneously IL-1 β with The numerical value of IL-1Ra simultaneously makes ratio, can exclude sample detection error between different batches.Colloid gold test paper provided by the invention The detection of IL-1 β is limited to 0.7ng/mL, and the detection of IL-1Ra is limited to 1.0ng/mL.

The detection antibody that the present invention is arranged is that can identify and resist simultaneously in conjunction with the IL-1 β -1Ra-2 of IL-1 β and IL-1Ra Body.The present invention is when being arranged double T line positions, using the capture antibody of IL-1 β as T1 line, marks in the position close to gold-labelled pad, Using the capture antibody of IL-1Ra as detection line T2 wire tag in nitrocellulose filter (NC) film middle position, so that being integrated to The colloidal gold probe of IL-1 β can be integrated to the colloidal gold probe meeting and detection line of IL-1Ra first by the capture antibody capture of IL-1 β The capture antibody of T2 line IL-1Ra combines, and the ratio that such IL-1Ra and IL-1 β makes influences result smaller.

Detailed description of the invention

Fig. 1 is relevant recom-binant protein SDS-PAGE result figure, 1 expression IL-1 β -1 induction in A；2 expression IL-1 β -1 are not lured It leads；3 indicate IL-1Ra-1 induction in B；4 expression IL-1Ra-1 are not induced；5 indicate IL-1 β -1Ra-2 induction in C；6 indicate IL-1 β -1Ra-2 is not induced；7 expression IL-1 β are not induced in D；8 indicate that IL-1 is beta induced；9 indicate IL-1Ra induction in E；10 indicate IL- 1Ra is not induced；M indicates ProteinMarker；

Fig. 2 is the Westernblot result figure of relevant recom-binant protein, and 1 expression IL-1 β -1 is not induced；2 indicate IL-1 β -1 Induction；3 expression IL-1Ra-1 are not induced；4 indicate IL-1Ra-1 induction；5 expression IL-1 β -1Ra-2 are not induced；6 indicate IL-1 β- 1Ra-2 induction；7 expression IL-1 β are not induced；8 indicate IL-1 β -1 induction；9 expression IL-1Ra are not induced；10 expression IL-1Ra are lured It leads；M indicates Marker；

Fig. 3 is that relevant recom-binant protein purifies SDS-PAGE result figure, and 1 indicates IL-1Ra purifying；2 indicate IL-1 β purifying；3 Indicate IL-1 β -1 purifying；4 indicate IL-1Ra-1 purifying；5 indicate IL-1 β -1Ra-2 purifying；M indicates Marker；

Fig. 4 is purified antibodies electrophoretogram, and 1 indicates that -1 cavy source of anti-il-i-beta is mostly anti-；2 indicate anti-IL-1Ra-1 cavy source It is mostly anti-；3 indicate that anti-il-i-beta -1Ra-2 rabbit source is mostly anti-；

Fig. 5 is that Westernblot analyzes antibody combining target protein characteristic result figure, and 1 indicates anti-il-i-beta -1Ra in (A) Antibody and IL-1 β recombinant protein, 2 indicate anti-il-i-beta -1Ra antibody and IL-1Ra recombinant protein；(B) 1 indicates anti-il-i-beta -1 in Antibody and IL-1Ra recombinant protein, 2 indicate -1 antibody of anti-il-i-beta and IL-1 β recombinant protein；(C) 1 indicates that anti-IL-1Ra-1 is anti-in Body and IL-1Ra recombinant protein, 2 indicate anti-IL-1Ra-1 antibody and IL-1 β recombinant protein；M indicates Marker；

Fig. 6 is colloidal gold solution ultraviolet scanning atlas；

Fig. 7 is colloidal gold probe qualification result figure, and 1 indicates negative findings；2 indicate that IL-1Ra is positive, and IL-1 β is negative；3 tables Show that IL-1 β is positive, IL-1Ra is negative；4 indicate full positive findings；

Fig. 8 is gold mark probe pH value figure；

Fig. 9 is gold mark probe antibody spirogram；

Figure 10 is the result figure of NC film, and 1 indicates sartorius95；2 indicate millipore135；3 indicate millipore180；4 indicate sartorius140；

Figure 11 is antibody concentration result figure；

Figure 12 is the canonical plotting of IL-1 β；

Figure 13 is the canonical plotting of IL-1Ra；

Figure 14 is the present invention and commercial reagent box sample detection IL-1Ra/IL-1 β ratio comparison diagram, wherein left side bar shaped Figure is kit of the present invention, and right bars figure is commercial reagent box；

Figure 15 is part serum sample rose bengal precipitation test result figure, and wherein A is that cloth disease vaccine sheep is not immunized for health； B is immune cloth disease vaccine sheep；C is that brucellosis infects sheep；D is negative control and positive control；

Figure 16 is sheep immune group, IL-1Ra/IL-1 β ratio difference analysis chart in healthy group and infected group serum sample；

Figure 17 is the ROC curve analysis chart of sheep blood serum sample IL-1Ra/IL-1 β ratio diagnosis index, wherein A is health Group and the ROC of immune group IL-1Ra/IL-1 β ratio are analyzed；B is ROC points of health group and infected group IL-1Ra/IL-1 β ratio Analysis；C is the ROC analysis of immune group and infected group IL-1Ra/IL-1 β ratio.

Specific embodiment

The present invention provides a kind of for detecting the colloid of IL-1 β, IL-1Ra content and IL-1Ra/IL-1 β ratio simultaneously Gold test paper strip, including fiberglass packing, nitrocellulose filter and blotting paper；Colloidal gold mark is coated on the glass fibre element pad In conjunction with the anti-il-i-beta -1Ra-2 rabbit source polyclonal antibody of IL-1 β and IL-1Ra while note；The nitrocellulose filter successively wraps Include detection line T1, detection line T2 and nature controlling line；The cavy source in conjunction with the anti-il-i-beta -1 of IL-1 β is coated in the detection line T1 Polyclonal antibody；The cavy source polyclonal antibody of the anti-IL-1Ra-1 in conjunction with IL-1Ra is coated on the detection line T2；It is described Goat anti-rabbit antibody is coated on nature controlling line.

The present invention is not particularly limited the source of the fiberglass packing, using conventional commercial product.In this hair In bright, the fiberglass packing is as loading pad.Colloid gold label is preferably coated on glass fibre element pad of the present invention Anti-il-i-beta -1Ra-2 rabbit source polyclonal antibody, the anti-il-i-beta -1Ra-2 rabbit source polyclonal antibody be detection antibody, institute Stating anti-il-i-beta -1Ra-2 antibody titer is 1:64000.

In the present invention, the dosage of the anti-il-i-beta -1Ra-2 rabbit source polyclonal antibody of the colloid gold label is preferably 1.0 ~1.4 μ g/100 μ L, more preferably 1.2 μ g/100 μ L.

The present invention is not particularly limited the type of the nitrocellulose filter, preferably include sartorius95, Millipore135, millipore180 or sartorius140.The present invention is to the source of the nitrocellulose filter without spy Different limit uses conventional commercial product.

In the present invention, the concentration of the cavy source polyclonal antibody of coated anti-il-i-beta -1 is preferred in the detection line T1 For 1.8~2.2mg/mL, more preferably 2mg/mL.The cavy source Anti-TNF-α of coated anti-IL-1Ra-1 on the detection line T2 The concentration of body is preferably 0.8~1.2mg/mL, more preferably 1mg/mL.In the present invention, the cavy source of the anti-il-i-beta -1 is more The cavy source polyclonal antibody of clonal antibody and anti-IL-1Ra-1 are preferably capture antibody, -1 antibody titer of anti-il-i-beta For 1:128000, anti-IL-1Ra-1 antibody titer is 1:128000.

In the present invention, the concentration of coated goat anti-rabbit antibody is preferably 0.6~1.0mg/mL on the nature controlling line, more excellent It is selected as 0.8mg/mL.

The present invention also provides the colloidal gold strips described in above-mentioned technical proposal in preparation test sample IL-1 β and IL- It is applied in the kit of the concentration of 1Ra.It preferably includes following steps to be detected: (1) sample to be tested being added drop-wise to glass fibre Pad；(2) after nature controlling line colour developing, whether observation detection line from colourless becomes red；Detection line becomes red, and sample to be tested contains IL-1 β and IL-1Ra；Detection line is non-discolouring, and IL-1 β and IL-1Ra content is lower than detection limit in sample to be tested.

Sample to be tested of the present invention flows under Sidestream chromatography effect, when flowing through double T (T1 and T2) lines, gold mark probe respectively with IL-1 β develops the color in conjunction with IL-1Ra capture antibody, and excessive gold mark probe develops the color in conjunction with goat anti-rabbit antibody at C line, sample Middle detectable substance content is bigger, and T line red is deeper.C line is as nature controlling line, regardless of whether target substance is detected in the sample, C line It will colour developing.If C line does not develop the color, colloidal gold probe preparation failure cannot form colloidal gold strip.

The present invention also provides the colloidal gold strips described in above-mentioned technical proposal in preparation test sample IL-1Ra and IL- It is applied in the kit of the ratio of 1 β.The sample preferably includes brucellosis correlation blood sample, including brucellosis infection Brucellosis vaccine animal blood sample is not immunized for animal blood sample, brucellosis vaccine immunity animal blood sample, health.

Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention Example is applied, all other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to In the scope of protection of the invention.

Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to In the scope of protection of the invention.

Embodiment 1

The expression and preparation of IL-1 β and IL-1Ra overall length and domain protein

1.1 material

1.1.1 consumptive material

Mini-scale plasmid extracts kit is purchased from love and pursues progress (Hangzhou) biotech company；Agarose is purchased from Spain Biowest company；Glycine, acrylamide, ammonium persulfate, chloroform, dehydrated alcohol, glacial acetic acid, are purchased from Beijing Chemical Plant； Peptone, yeast powder are purchased from OXOID company, Britain；ProteinMarker is purchased from Genstar Kang Run biology Co., Ltd.

1.1.2 instrument and equipment

Double Vertial electrophorestic tanks are purchased from Beijing and like general magnificent Biotechnology Co., Ltd；Gel imager analysis system, is purchased from UVP company, the U.S.；Horizontal shaker is purchased from Liuyi Instruments Plant, Beijing；Constant incubator is purchased from the limited public affairs of Shanghai laboratory apparatus factory Department；Low-temperature and high-speed centrifuge is purchased from DNR company, the U.S.；Electronic balance, Denver's acidometer, are purchased from instrument company, Denver；AKTA egg White purification system, is purchased from General Electric Company.

1.1.3 prepared by main agents

LB liquid medium: yeast powder: peptone: NaCl ratio is 1:2:2, when scale base is 1g, adds distilled water fixed Hold to 200mL.After high pressure steam sterilization, 4 DEG C of short-term preservations after room temperature cooling.

LB solid medium: yeast powder: peptone: NaCl: agar powder ratio is that 1:2:2:3 adds when scale base is 1g Distilled water is settled to 200mL.High pressure steam sterilization.It pours into plate, room temperature is cooled to solidification.After sealed membrane sealing, 4 DEG C short-term It saves.Plate Procedure should be between sterile working.

10% ammonium persulfate: ammonium persulfate 0.3g, two distilled water 1mL are mixed.Matching while using.

Coomassie brilliant blue R_250 dyeing liquor: 250mL isopropanol dissolves 1gR-250 as solvent, uses after adding 100mL acetic acid Deionized water is settled to 1L.

SDS-PAGE electrophoretic buffer: weighing Tris15.1g, SDS5.0g, Glycine94g, water-soluble with 800mL deionization 1L is settled to after solution.

Film transfer buffer: weighing Glycine2.9g, Tris5.8g, SDS0.37g respectively, is dissolved in 600mL deionization It is settled to 800mL after in water, methanol 200mL is added.

TBST:8.8g sodium chloride, 20mLTris-HCl (1M, pH8.O), is dissolved in 800mL deionized water, is added 0.5mLTween20 is settled to 1L using deionized water.

Block buffer: 5g skimmed milk power is dissolved in 100mLTBST solution.

1.2 method

1.2.1 plasmid extracts

Overall length IL-1 β, overall length IL-1Ra and IL-1 β -1 segment, IL-1Ra-1 segment, IL-1 β -1Ra-2 segment are constructed (referring to (Guo Xing master thesis, Jilin University, in June, 2015)；(the ring master thesis of water one, Jilin University, 2018 Year December)) expression vector, be stored in DH5 α competence.All recombinant proteins mergeLabel, IL-1 β -1 Section, IL-1Ra-1 segment, IL-1 β -1Ra-2 segment composition MBP albumen.To obtain the plasmid in DH5 α competence, 5mL is taken respectively The strain of 100 μ L preservation is added in every culture medium test tube for LB liquid medium.IL-1 β, IL-1Ra strain culture medium in point Be not added 5 μ L kanamycins, IL-1 β -1, IL-1Ra-1, IL-1 β -1Ra-2 strain culture medium in be separately added into 5 μ L ammonia benzyls west Woods cultivates 8h in 37 DEG C of shaking tables, and precipitating thallus is taken to extract plasmid after centrifuge 8000rpm1min.Kit extracts plasmid procedure It is as follows.

1. taking the bacterium solution of 5mL37 DEG C of culture 8h, it is centrifuged 1min, revolving speed 12000rpm (subsequent step revolving speed is herewith), it is heavy to take The 250 μ LBufferS1 of addition that form sediment uniformly are resuspended.

2. 250 μ LBufferS2 are added in re-suspension liquid, soft overturning 4-6 times, when bacterium solution is bright, thallus, which be can be obtained, fills Division solution.This step should operate rapidly, can not place more than 5min, movement must softly, otherwise will lead to DNA pollution and BufferS2 inactivation.

3. 350 μ LBufferS3 are added in the phage solution sufficiently cracked, centrifugation after soft overturning 6-8 times 10min.Otherwise this step rotary movement softly must will lead to DNA pollution.

4. centrifugation gained supernatant, addition in step 3 is taken to prepare in pipe, prepare pipe and be placed in 1.5mL centrifuge tube, it is centrifuged 1min abandons filtrate.

5. 500 μ LBufferW1 are added in preparing in pipe for step 4, filtrate is abandoned after being centrifuged 1min.

6. BufferW2700 μ L is added in preparing in pipe for step 5, it is centrifuged 1min, abandons filtrate.Repeat this step twice, Ensure that salinity thoroughly removes.

7. the pipe for preparing in step 6 is taken to be placed in new centrifuge tube, Eluent to 65 DEG C is preheated in advance, 50 μ L is taken to be added in system Standby tube hub, being stored at room temperature 1min reacts it sufficiently, is centrifuged 1min.Filtrate is gained plasmid.

1.2.2 the expression of recombinant protein

The plasmid that upper step is obtained is transformed into respectively in expression bacterium.IL-1 β, IL-1Ra plasmid are transformed into BL21conden Plus is expressed in bacterium, and IL-1 β -1, IL-1Ra-1, IL-1 β -1Ra-2 plasmid are transformed into BL21 expression bacterium, is turned Change method is as follows.

1. BL21/BL21condenPlus is taken to express bacterium from -80 DEG C of ultra low temperature freezers, it is put into ice bath 5min in ice chest.

2. taking 5 μ L of plasmid that respectively expression strain is added respectively, mix gently, ice-water bath 30min.Addition is few in ice in ice chest Water is measured, EP pipe is allowed to come into full contact with ice water.

3. 42 DEG C of electrobath 90s of product.

4. ice bath 10min.

5. LB culture medium 450 μ L, 37 DEG C of electrobath 10min are added in product.

6. product is put into 37 DEG C of shaking tables, 85rpm cultivates 20min.Speed-raising rotates 10min to 100rpm.Speed-raising is extremely 120rpm rotates 10min.

7. it is flat to take 100 μ LIL-1 β, IL-1Ra product to be coated on the LB solid medium containing 0.1% kanamycins respectively On plate, IL-1 β -1, IL-1Ra-1, IL-1 β -1Ra-2 product are coated in the LB solid medium tablets containing 0.1% ammonia benzyl. 37 DEG C of culture 12h.

8. picking them separately monoclonal colonies, after expanding culture, send to Changchun department, Ku Mei Technology Co., Ltd. and be sequenced. Confirmation is compared in sequencing acquired results.

It takes and accurate bacterium solution is sequenced in 1.2.2, IL-1 β, IL-1Ra are inoculated in the training of the LB liquid containing 0.1% kanamycins It supports in base, IL-1 β -1, IL-1Ra-1, IL-1 β -1Ra-2 are inoculated in the LB liquid medium containing 0.1% ammonia benzyl.37 DEG C are shaken 2h, revolving speed 170rpm are shaken in bed.IPTG inducer is added in 0.1% ratio, continues to shake 6h in 37 DEG C of shaking tables.It takes out Bacterium solution is centrifuged 10min through refrigerated centrifuge 8000rmp, takes bacterial sediment.Expression albumen, step are verified whether with SDS-PAGE electrophoresis It is rapid as follows.

A) processing of electrophoresis Sample: taking induction bacterium solution 1mL and does not induce a group bacterium solution 1mL as control, is centrifuged 1min, abandons Supernatant takes bacterial sediment.The bacterial sediment of 1mL bacterium solution is hanged with 80 μ LPBS, and after pressure-vaccum is uniform, 20 μ L sample-loading buffers are added. Centrifugation nozzle package sealed membrane prevents from popping, and 10min is boiled in boiling water, low-speed centrifugal 30s takes supernatant as sample.

B) match glue: the gel slab for taking cleaning dry is mounted in electrophoresis tank, after tight no leakage is installed in confirmation, is added Lower layer's separation gel of 9mL12% concentration, addition 1mL isopropanol is depressed into bubble collapse and liquid level is smooth, is stored at room temperature, about 30min After solidify.Isopropanol is poured out after solidification and is carefully exhausted liquid with blotting paper, and blotting paper not touch lower layer's micelle colloid.It fills it up with Glue is concentrated in upper layer, is inserted into mating stripping fork, and room temperature lays flat standing, about 30min to solidification.

C) electrophoresis: electrophoresis liquid will be poured into electrophoresis tank to that can form access, is added 8 in every hole after careful extraction stripping fork μ L sample.Positive and negative the two poles of the earth are paid attention to when energization, 90V voltage 30min observes bromophenol blue band, runs to separation gel, adjusts voltage extremely 120V, observation band are run to board bottom 1cm, can close power supply.

D) it dyes: dismantling gel slab finishing and remove upper layer concentration glue, retain lower layer's separation gel, be immersed in dyeing liquor, shake Bed concussion dyeing 3h.

E) it decolourizes: after the gel addition distilled water after dyeing washes away loose colour, adding distillation water decolorization, microwave ingle adds Hot 5min rejoins distilled water after pouring out liquid.It repeats the above steps to showing clear protein band on gel.Analysis knot Fruit.

Bacterial sediment is resuspended with PBS buffer solution, and Ultrasonic Cell Disruptor is crushed thallus.Select No. 6 ultrasonic heads, power 30W, ultrasound 3s, interval 4s, total 40min.Heat can be generated when ultrasonic, influence protein active, therefore pay attention to bacterium solution ice bath when ultrasound.Ultrasound knot Shu Hou, 4 DEG C of centrifugations 30min, revolving speed 12000rmp in refrigerated centrifuge obtain supernatant and precipitating, are inclusion body in precipitating.

1.2.3 the purifying of recombinant protein

Supernatant inclusion body is verified by SDS-PAGE electrophoresis and is expressed.Ni-sepharose purification method purifies the egg expressed in supernatant White, protein electrophoresis cuts glue purification method and purifies the albumen expressed in inclusion body, and is analyzed after purification by Western-blot Recombinant protein.

1. steps are as follows for ni-sepharose purification:

A) BufferA is cleaned twice of nickel column, each 10mL.

B) supernatant 10mL is added and mixes, stands 30min.

C) lid is opened after nickel column gel precipitation flows out liquid naturally, and 10 drops are followed by flowing through liquid to liquid level above gel At 1cm.

D) BufferA is cleaned twice of nickel column, each 5mL, collects washing lotion.

E) add 40mM imidazole buffer and mix, stand 30min to gel precipitation, the natural drop of trickle 10 is followed by eluting Liquid is to liquid level above the gel at 0.5cm.

F) add 80mM imidazole buffer and mix, stand 30min to gel precipitation, the natural drop of trickle 10 is followed by eluting Liquid is to liquid level above the gel at 0.5cm.

G) add 150mM imidazole buffer and mix, stand 30min to gel precipitation, the natural drop of trickle 10 is followed by washing De- liquid is to liquid level above the gel at 0.5cm.

H) 20% ethyl alcohol cleans twice of nickel column, and clean nickel column is placed 4 DEG C of preservations.

2. the albumen in inclusion body cuts glue purification step

A) after cleaning inclusion body 2 times with PBS, inclusion body, 800 μ LPBS re-suspension liquids the processing of electrophoresis Sample: are resuspended with PBS 200 μ L sample-loading buffers of middle addition.Centrifugation nozzle package sealed membrane prevents from popping, and is used as sample after 10min is boiled in boiling water.

B) match glue: the gel slab for taking cleaning dry is mounted in electrophoresis tank, after tight no leakage is installed in confirmation, is added Lower layer's separation gel of 9mL12% concentration, addition 1mL isopropanol is depressed into bubble collapse and liquid level is smooth, is stored at room temperature, about 30min After solidify.Isopropanol is poured out after solidification and is carefully exhausted liquid with blotting paper, this process not touch lower layer's glue, and upper layer is added Glue to plate mouth 1cm place is concentrated, 1mL isopropanol is added, and to be depressed into liquid level smooth, and room temperature lays flat standings, and the upper layer about 30min, which is gelled, to be consolidated, Except isopropanol.

C) electrophoresis: electrophoresis liquid will be poured into electrophoresis tank to access is formed, 8 μ L samples are added in every hole after careful extraction stripping fork Product.90V voltage runs electrophoresis, observes bromophenol blue band, runs to separation gel, adjusts voltage to 120V, observes band and run to board bottom 1cm Place, can close power supply.

D) it dyes: dismantling gel slab, modify gel upper layer glue with blade, retain lower layer's separation gel, be immersed in 0.4mMKCl 3min in solution.KCl solution needs to be stored in advance in 4 DEG C.

E) cut glue: the salt ion of upper step middle and high concentration can make albumen show the single band of white, and pocket knife cuts white bars Band.

F) it collects: white ribbon slitting being put into the bag filter of molecular cut off 8000-14000Da, is placed on and fills In the nucleic acid electrophoresis tank of electrophoresis liquid, 90V voltage runs 10min.Fluid preservation in bag filter is taken out, discarding becomes transparent gel and cuts Item.

3.Western-blot step

A) sample preparation and electrophoresis process process are the same as SDS-PAGE electrophoresis.

B) 10min is activated in the sizeable pvdf membrane bubble methanol of clip.

C) dismantle gel slab and take out lower layer's separation gel, modify lower layer's separation gel size, in order by filter membrane, separation gel, Pvdf membrane is put into transferring film folder, cannot there is bubble between film and film.

D) transferring film is folded up in electrophoresis tank and is added buffer, notice that electrode is positive and negative, adjust electric current to 200mA transferring film 2h. Ice bath electrophoresis tank prevents During migration temperature excessively high, albuminous degeneration.

E) after transferring film, it is careful clamp film be put into confining liquid and 5% skimmed milk power in close, shaken on room temperature shaker 1h。

F) it takes corresponding antibodies to be diluted with confining liquid, the film after closing is put into wherein, 1h, TBST shake are incubated in 37 DEG C of incubators Swing washing 4 times, each 10min.

G) it takes secondary antibody skimmed milk power multiple to dilute, the film after closing is put into wherein, is incubated for 1h, TBST in 37 DEG C of incubators Concussion washing 4 times, each 10min.

H) developed with developer solution to pvdf membrane, chemiluminescence imaging system acquisition data.

1.3 result

1.3.1 the expression of recombinant protein

After IL-1 β, IL-1Ra, IL-1 β -1, IL-1Ra-1, the success of IL-1 β -1Ra-2 picking monoclonal colonies, expression Bacterium respectively in 16 DEG C, 25 DEG C, 37 DEG C induce 8h, 16h, IL-1 β, IL-1Ra expression bacterium in 37 DEG C of 8h expression quantity highest, IL-1 β -1, IL-1Ra-1, IL-1 β -1Ra-2 expression bacterium in 16 DEG C of 16h expression quantity highest.SDS-PAGE electrophoresis result point After analysis, IL-1 β, IL-1Ra, IL-1 β -1, IL-1Ra-1, IL-1 β -1Ra-2 recombinant protein express success.As a result such as Fig. 1 institute Show.

Westernblot detection is carried out to recombinant protein, using the antibody of anti-His label as primary antibody, in expection after development There is obvious band in position, it was demonstrated that albumen is purpose albumen in figure, is expressed successfully.Group weaker band of appearance is not induced in figure, is Background expression of the destination protein under non-inductive condition, as a result as shown in Figure 2.

1.3.2 the purifying of recombinant protein

IL-1 β, IL-1Ra full length recombinant protein concentration are 1.5mg/mL, IL-1 β -1, IL-1Ra-1 after purifying dialysis concentration Recombinant protein concentration is 2.0mg/mL, and IL-1 β -1Ra-2 recombinant protein concentration is 2.2mg/mL.The analysis of SDS-PAGE electrophoresis result Afterwards, as shown in figure 3, destination protein purifies successfully.

Embodiment 2

The preparation of IL-1 β and IL-1Ra polyclonal antibody

2.1 material

2.1.1 consumptive material

Freund's complete adjuvant, incomplete Freund's adjuvant are purchased from Sigma company.

2.1.2 key instrument equipment

Ultramicron microwell plate spectrophotometer, is purchased from Biotek company；Electronic balance is purchased from the U.S. DenverInstrament company.

2.1.3 prepared by main agents

1. coating buffer: 1.59g/LNa2CO3,2.93g/LNaHCO3 adjust pH to 9.6 after mixing.

2. confining liquid: 0.1MNH4Cl.

3.PBS:8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate, 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, 800mL Deionized water stirs evenly, and adjusting pH is 7.2-7.4, is settled to 1L using deionized water.

4.PBST:PBS mixes 0.1% tween.

5. terminate liquid: the 11.1mL concentrated sulfuric acid, 88.9mL deionized water, strong acid is extremely corrosive, and when configuration should be in a reservoir Deionized water is first added, after pour into strong acid, side edged, which rocks, prevents liquid from splashing.

2.2 method

2.2.1 animal immune

2.2.1.1 rabbit immunization

First immunisation takes Freund's complete adjuvant to mix with L-1 β -1Ra-2 albumen grade ratio, and exempting from protein content for the first time is 1mg.Verifying Whether complete standard is that for drop drop in the water surface, drop closely complete indiffusion takes emulsion back at this time after taking emulsification for emulsification New Zealand White Rabbit is immunized in multi-point injection.Ear edge vein exploitating blood gives over to immune preceding negative serum, flicking injection after being immunized before immune Point facilitates the diffusion and absorption of emulsion.Using two weeks as the immune period, two exempt from after emulsifier select Freund not exclusively help Agent, protein content and the total 2mg of each 1mg of Freund's incomplete adjuvant are emulsified after isometric mixing and are verified emulsification completely.After third time is immune, Ear edge vein exploitating blood was carried out to white rabbit in immune latter 5 days every time, measures serum titer, Culling heart blood is collected after potency reaches desired value Serum is with to be purified.

2.2.1.2 immunized guinea pigs

First immunisation takes Freund's complete adjuvant and IL-1 β -1 or IL-1Ra-1 albumen grade ratio to mix, protein content 1mg.It tests Card emulsifies complete method and is same as above, and takes emulsion back multi-point injection immune guinea pig.Blood sampling gives over to immune preceding negative serum before immune, Flicking injection point contributes to the absorption diffusion of emulsion after immune.Using two weeks as the immune period, two exempt from after emulsifier select With incomplete Freund's adjuvant, protein content 1mg is emulsified after isometric mixing and is verified emulsification completely.After third time is immune, every time It takes a blood sample within 5 days after immune, measures serum titer, Culling heart blood collects serum with to be purified after potency reaches desired value.

2.2.2 antibody titer detects

Take immune preceding gained serum of taking a blood sample as negative serum as compareing, gained serum of taking a blood sample after immune is examined as sample Survey potency.Blood sampling gained new blood should be immediately placed in 30min in 37 DEG C of incubators, relay the 2h in 4 DEG C of refrigerators, coagulation process is complete Cheng Hou, refrigerated centrifuge are centrifuged 5min, revolving speed 3000rpm, and supernatant is gained serum.Serum packing, is stored in -80 DEG C.

The conventional ELISA method detection potency of serum after packing.Steps are as follows:

1. peridium concentration is 1 μ g/mL, every hole using three kinds of immunogenes as antigen coat in ELISA ELISA Plate board bottom 100 μ L are added, are coated with 2h in 37 DEG C of incubators.

2. drying coating buffer, the closing of 200 μ L1% skimmed milk powers, 37 DEG C of closing 1h are added in every hole.

3. drying confining liquid, the Post-immunisation serum of different extension rates is separately added into sample well, in negative control hole Middle addition preimmune serum.Every 100 μ L of hole.1h is incubated in 37 DEG C of incubators.With the concussion of PBST solution washing elisa plate 3 times, every time 60s。

4. with PBS solution 4000 times of dilution HRP label secondary antibodies, every 100 μ L of hole is incubated for 1h in 37 DEG C of incubators.It is molten with PBST Liquid concussion washing elisa plate 4 times, each 60s.

5. tmb substrate buffer is added, every 100 μ L of hole is incubated for 10min in 37 DEG C of incubators.

6. 50 μ L terminate liquids are added in every hole.

7. opening microplate reader, enzyme-linked product OD value, wavelength selection 450nm are measured.

2.2.3 the purifying of polyclonal antibody

After serum titer is ideal, with AKATA protein purification system antibody purification.Steps are as follows.

1. with ultrafiltration instrument ultrafiltration BindingBuffer, 20% ethyl alcohol, 0.1M glycine (pH2.7) and TrisHCl (pH7.0), impurity and bubble are removed.

2. protein purification instrument is opened, it is steady to ion line with 20% ethyl alcohol cleaning robot, loading slot and affinity column.

3. using BindingBuffer balancing machine, loading slot and affinity column after after cleaning.Ion line balance 10 A column volume.

4. serum to be purified is added in loading slot, 200 μ LTrisHCl are added in connecing sample pipe.

5. eluting after steady 15 column volumes of ion line, and start to connect sample when there is ultraviolet peak.

6. steady to ion line with 20% ethyl alcohol cleaning robot, loading slot and affinity column after ultraviolet peak decline.

7. the bag filter of the appropriate length of clip pays attention to directly touching bag filter.Clean beaker is taken, bag filter is existed 0.5h is boiled in the distilled water of boiling, antibody to be dialysed is added after taking-up in bag filter, uses the dedicated envelope of bag filter after ejecting bubble Mouth clamp opening.The PBS of 0.01MpH8.0 is placed in 4 DEG C of chromatography cabinets, low speed rotation liquid as dialyzate, and dialyse 72h, every 8h Change a not good liquor.After the completion of dialysis, the concentration of PEG20000 compartment.Microplate reader measures OD value, and packing is stored in -80 DEG C of ultra low temperature freezer In.

2.2.4 antibody specificity is analyzed

It is special using specificity of the three kinds of antibody of Western Blot hybridization analysis in conjunction with its target protein and cross reaction Property, analyze whether three kinds of antibody can be applied to the foundation of double T line colloidal gold Sidestream chromatography test strips.The same 1.2.4 of step.

2.3 result

2.3.1 antibody titer detects

It is respectively capture antibody obtained by IL-1 β -1 and IL-1Ra-1 protein immunization cavy, with IL-1 β -1Ra-2 albumen It is detection antibody obtained by immunizing rabbit.- 1 antibody titer of anti-il-i-beta is 1:128000, and anti-IL-1Ra-1 antibody titer is 1: 128000, anti-il-i-beta -1Ra-2 antibody titer is 1:64000.

2.3.2 the purifying of antibody

By AKATA protein purification system obtain after purification anti-il-i-beta -1, IL-1Ra-1, three kinds of IL-1 β -1Ra-2 it is anti- Body, as shown in figure 4, being purified successfully after the analysis of SDS-PAGE electrophoresis result.

2.3.3 the specificity analysis of antibody

Animal gained after purification is immunized as immunogene in three kinds of IL-1 β -1, IL-1Ra-1, IL-1 β -1Ra-2 recombinant proteins Antibody, -1 antibody of anti-il-i-beta, anti-IL-1Ra-1 antibody and anti-il-i-beta -1Ra-2 antibody and overall length IL-1 β, IL-1Ra are carried out WesternBlot hybridization analysis.As shown in figure 5, -1 antibody of anti-il-i-beta can only be in conjunction with IL-1 β, anti-IL-1Ra-1 antibody can only IL-1Ra and combination, anti-il-i-beta -1Ra-2 antibody can combine two kinds of albumen of IL-1 β and IL-1Ra.Three kinds of antibody specificities are good It is good.Therefore, -1 antibody of anti-il-i-beta may be used as anti-il-i-beta cavy source capture antibody, anti-IL-1Ra-1 antibody may be used as resisting IL-1Ra cavy source capture antibody, anti-il-i-beta -1Ra-2 antibody may be used as while the rabbit source of anti-il-i-beta and IL-1Ra are detected Use antibody.

Embodiment 3

For detecting the colloidal gold strip preparation of IL-1 β, IL-1Ra content and IL-1Ra/IL-1 β ratio simultaneously

3.1 material

3.1.1 consumptive material

Gold chloride is purchased from Beijing traditional Chinese medicines；Bovine serum albumin(BSA) (BSA), is purchased from Sigma company；Goat anti-rabbit igg, doctor Moral biotech firm；Tween-20, PEG20000, trehalose, sucrose, Beijing Ding Guo biotech firm；SB06 fiberglass packing, DL42 Polyester fibers of mat, nitrocellulose membrane, DL42 blotting paper, PVC bottom plate, test strips pack case, desiccant, aluminium foil are purchased from Shanghai gold Mark biotech firm.

3.1.2 key instrument equipment

XYZ3060 three-dimensional point sprays platform, is purchased from Biodot company；Electronic balance is purchased from Sai Duolisi company；Vacuum Package Machine is purchased from Golden Bridge's electric appliance；Constant incubator is purchased from Shanghai laboratory apparatus factory；Centrifuge is purchased from Thermo company；Refrigerator is purchased from Company, Haier.

3.1.3 main solution is prepared

1. 5% dichlorodimethylsilane solution: 50mL dichlorodimethylsilane, 950mL chloroform being uniformly mixed, room temperature It is kept in dark place.

2. 0.005%NaCl solution: weighing 200mgNaCl with distilled water and be settled to 4L, 4 DEG C of preservations.

3. 10%NaCl solution: weighing 2gNaCl with distilled water and be settled to 20mL, 0.22 μm of aperture membrane filtration, 4 DEG C of guarantors It deposits.

4. 1% sodium citrate solution: weighing 500mg sodium citrate with pure water and be settled to 50mL, 0.22 μm of aperture filter membrane Filtering, matching while using.

5. 0.2mol/LK₂CO₃Solution: 1.38gK is weighed₂CO₃50mL, 0.22 μm of aperture filter membrane mistake are settled to pure water Filter, 4 DEG C of preservations.

6. 1%BSA solution: weighing 2gBSA with pure water and be settled to 200mL, 0.22 μm of aperture membrane filtration, -20 DEG C of guarantors It deposits, packing uses.

7. 5%PEG20000 solution: weighing 500mgPEG20000 with pure water and be settled to 10mL, 0.22 μm of aperture filter membrane Filtering, -20 DEG C of preservations, packing use.

8. 0.01mol/LPB solution: weighing 2.86gNa₂HPO₄·12H2O、0.272gKH₂PO₄, it is settled to pure water 1L adjusts pH to 7.4,0.22 μm of aperture membrane filtration, 4 DEG C of preservations.

9. 0.01mol/LPBS solution: weighing 0.8gNaCl, 0.29gNa₂HPO₄·12H₂O, 0.02gKCl and 0.2g KH₂PO₄, it is settled to 100mL using pure water, adjusts PH to 7.4,0.22 μm of aperture membrane filtration, 4 DEG C of preservations.

10. re-suspension liquid: 15% sucrose, 2.5% trehalose, 1%BSA, 0.1%PEG20000, solvent 0.01mol/L PB, 0.22 μm of aperture membrane filtration, -20 DEG C of preservations, packing use.

11. sample-loading buffer: 0.2%PEG20000,2% sucrose, 2%BSA, solvent 0.01mol/LPB, aperture 0.22 μm membrane filtration, -20 DEG C of preservations, packing use.

3.2 method

3.2.1 colloidal gold solution preparation and identification

During firing colloidal gold, the colloid gold particle diameter of Nano grade is smaller, is liable to stick to glassware table Face, it is therefore desirable to which glassware is used in first silicidation test.Silicide step is as follows:

1. thoroughly cleaning glassware with liquid detergent, sour cylinder soaked overnight after drying；

2. rinsing the vessel after acid soak with tap water, then with distilled water flushing 3 times, 4h is impregnated in distilled water；

It is dried 3. the vessel impregnated take out, the silicidation in draught cupboard, fills 5% dichloro-dimethyl in glassware Solution of silane stands 0.5h in draught cupboard；

4. recycling silication liquid, vessel standing and drying 1h in draught cupboard after silication；

5. glassware is kept in dark place after drying with distilled water flushing 5 times.Dichlorodimethylsilane toxicity is violent, answers small The heart uses, and takes care.

It takes and pure water 100mL and 10% gold chloride, 100 μ L is added after silication in conical flask is uniformly mixed, on universal electric furnace It is heated to boiling completely.It is rapidly added 1% sodium citrate 2.8mL, shaking conical flask makes its uniformly sufficiently reaction, the visible liquid of naked eyes Body color is gradually deepened from faint yellow.After colloidal gold solution becomes claret, low boiling state is kept to continue to fire 5min.Naturally cold But to after room temperature, constant volume colloidal gold solution to 100mL, in the blue mouth bottle after the membrane filtration to silication of 0.22 μm of aperture, 4 DEG C of low temperature It saves.

Colloidal gold is visually observed, if limpid bright free from admixture, color is in claret, then best in quality, suitable label tracer. Ultraviolet spectrometry scans the colloidal gold solution prepared, and calculates colloidal gold partial size.

3.2.2 IL-1 β -1Ra2 polyclonal antibody removes salt treatment

Colloid gold particle reaches stable bond by surface charge adsorbed proteins and does not influence protein active, and excessively high Salt ionic concentration will affect colloid gold particle charge condition, the unstable Percentage bound that will affect with albumen of colloidal gold influences glue The sensibility of body Au probe.Because before marking colloidal gold probe, generally first carrying out dialysis except salt treatment, reducing ion influences. The bag filter of the appropriate length of clip first pays attention to directly touching bag filter.Clean beaker is taken, by bag filter in boiling 0.5h is boiled in distilled water, antibody to be dialysed is added after taking-up in bag filter, uses bag filter special-purpose sealing clamp after ejecting bubble Mouthful, it is placed in 4 DEG C light rotation dialysis, every 8h in 0.01% PBS solution and replaces dialyzate, replace 7 times.Low-temperature centrifugation after dialysis 15min, revolving speed are that 12000rpm removes the albumen precipitation occurred in dialysis procedure, and dilution antibody is standby to 5mg/mL after protein concentration With.

3.2.3 prepared by colloidal gold probe

Specific preparation process is as follows for colloidal gold probe.

1. sequentially adding colloidal gold solution 1mL, 0.2mol/LK in 2mLEP pipe₂CO₃10 μ L are put into 37 DEG C of constant-temperature tables Shake 30min, revolving speed 170rpm；

2. 10 μ gIL-1 β -1Ra-2 polyclonal antibodies are added, 37 DEG C of constant-temperature tables shake 30min, revolving speed 170rpm；

3. continuously adding 10%BSA100 μ L, 37 DEG C of constant-temperature table 170rpm shake 30min；

4. low-temperature centrifugation 10min abandons impurity, revolving speed 1000rpm；

5. 14 000rpm low-temperature centrifugation 40min abandon supernatant；

6. 1%BSA1mL, which is added, is resuspended sediment, 14000rpm low-temperature centrifugation 40min abandons supernatant；100 μ of re-suspension liquid is added L, 4 DEG C of preservations.

Since colloid gold particle partial size is smaller, not easy cleaning is easily attached on glassware, therefore pH meter cannot be selected to survey Measure colloidal gold solution pH numerical value.For the pH value for determining label colloidal gold probe, 0.2mol/LK is arranged in gradient₂CO₃Dosage, in 100 μ 0.6 μ L, 0.7 μ L, 0.8 μ L, 0.9 μ L, 1.0 μ L, 1.1 μ L are separately added into L colloidal gold solution.Height is utilized after sufficient antibodies are added Salt destroys method and tests colloidal gold probe optimum pH, and 10 μ L10%NaCl are added in above-mentioned solution, and vortex oscillation 60s makes it sufficiently It mixes, is stored at room temperature 4h.Solution colour variation is visually observed, minimum K when select colour stable be red₂CO₃Dosage is determined as Mark colloidal gold probe pH.

Determine that antibody dosage is arranged in the antibody dosage for preparing colloidal gold probe, gradient, divide in 100 μ L colloidal gold solutions It Jia Ru not antibody 0,0.2 μ g, 0.4 μ g, 0.6 μ g, 0.8 μ g, 1.0 μ g, 1.2 μ g, 1.4 μ g, 1.6 μ g, 1.8 μ g.It is broken using with high salt Bad method, condition are same as above, and visually observe solution colour variation, and minimum amount of antibody when select colour stable be red is determined as marking Colloidal gold probe antibody dosage.

3.2.4 colloidal gold strip assembles

By NC film, fiberglass packing, blotting paper, carefully linking is pasted on PVC board bottom plate in order.Colloidal gold strip Assembling flow path is as follows:

1. nitrocellulose filter (NC film) is fixed on PVC bottom plate；

2. clip 10cm fiberglass packing (loading pad), side is aligned with bottom plate side, and other side 1mm Chong Die with NC film is attached to T1 line side.

3. clip 10cm blotting paper, side are aligned with bottom plate side, side 1mm Chong Die with NC film C line side is attached on bottom plate.

After pasting assembling, test strips, width 3mm are cut.1mL sample and 10 μ L colloidal gold probes premix, Jin Biao Probe can be with the IL-1 β in sample in conjunction with IL-1Ra, and 14000rpm is centrifuged 20min, abandons supernatant and with 100 μ L sample-loading buffers Precipitating is resuspended, sample mixed liquor is added on loading pad, sample flows under Sidestream chromatography effect, and when flowing through double T lines, gold mark is visited Needle develops the color in conjunction with IL-1Ra capture antibody with IL-1 β respectively, and excessive gold mark probe is shown in conjunction with goat anti-rabbit antibody at C line Color.Detectable substance content is bigger in sample, and T line red is deeper.C line is as nature controlling line, regardless of whether detecting target in the sample Substance, C line can all develop the color.If C line does not develop the color, colloidal gold probe preparation failure cannot form colloidal gold strip.

3.2.5 NC film

Tetra- kinds of NC films of sartorius95, millipore135, millipore180 and sartorius140 are chosen, respectively Colloidal gold strip is successively assembled after spray film scribing line, sample is added and observes color change at scribing line, selects NC film.

3.2.6 double T line antibody working concentrations

It is 0,1mg/mL, 2mg/mL that double T line antibody concentrations, which are arranged, successively assembles colloidal gold strip after spray film scribing line, adds Enter sample and observes color change at scribing line.

3.2.7 the foundation of test strips standard curve

After 1mL standard items are premixed with 10 μ L colloidal gold probes and sufficiently combined, 14000rpm is centrifuged 20min, abandons supernatant simultaneously With 100 μ L sample-loading buffers be resuspended precipitate, be diluted to 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL, 7.813ng/mL, 3.906ng/mL, 1.953ng/mL, 0.976ng/mL totally 9 concentration are used as standard curve Foundation.Sample is added in test strips sample pad, is parched to test strips, is taken pictures and with ImageJ software analysis and processing result.With Sample-loading buffer is that blank sample does blank control, using the re-suspension liquid concentration log value obtained after sample pretreatment as abscissa, with It is ordinate that T line, which measures gray value, draws standard curve.To determination data regression analysis, and show that regression equation and standard are bent Line.

3.2.8 the determination of rate of recovery experiment and minimum detection limit

IL-1 β and IL-1Ra standard items are diluted to 50ng/mL, 100ng/mL, 200ng/mL by above-mentioned steps, by IL- 1Ra/IL-1 β is that 4:1,2:1,1:1,1:2,1:4 mix loading, for detecting the rate of recovery of semiquantitative test paper detection method.

According to standard curve, resuspension sample concentration value when with test strips T line being macroscopic bottom line is determined as Minimum detection limit.

3.2.9 repetitive test

IL-1 β standard items are handled to 10ng/mL with sample-loading buffer, and IL-1Ra standard items to 100ng/mL are arranged three Repeat control group, test strip repeatability.

3.3 result

3.3.1 colloidal gold solution is analyzed

Colloidal gold solution clear is in claret, is placed in 4 DEG C environment 4 months, and colloid gold particle is stablized, and color is without changing Become, bottom is without precipitating.UV scanning is illustrated in figure 6 as a result, colloidal gold has simple spike, and has absorption peak at 520nm, it can Know that colloid gold particle size is about 20nm.

3.3.2 colloidal gold probe is identified

Colloidal gold probe solution clear is in claret, and low-speed centrifugal is without Precipitation, therefore colloidal gold probe is stablized And it can be used for preparing test strips.Assemble test strips result such as Fig. 7, IL-1 β and the IL-1Ra mark of sample-loading buffer and 100ng/mL Quasi- product are loaded after mixing in equal volume, and double T lines and C line develop the color；Sample-loading buffer and distilled water mix sample-adding in equal volume, T1 line, T2 line does not develop the color, the colour developing of C line；The IL-1 β standard items of sample-loading buffer and 100ng/mL are loaded after mixing in equal volume, T1 line and C line develops the color, and T2 does not develop the color；The IL-1Ra standard items of sample-loading buffer and 100ng/mL are loaded after mixing in equal volume, T2 line and C Line develops the color, and T1 does not develop the color.Result above proof has successfully obtained the complete colloidal gold probe of label.

High concentration salt solutions will affect charge condition in solution, and colloid gold particle is made to have precipitating, and sufficiently be wrapped up by antibody The colloid gold particle of label not will receive the influence of concentration salting liquid.Therefore, colloidal gold probe pH is marked using destruction method with high salt With amount of antibody in colloidal gold probe.As a result as shown in figure 8,0.9 μ LK is added in 100 μ L mixed liquors₂CO₃When be colour stable be red K when color₂CO₃Minimum amount is determined as marking colloidal gold probe pH.

As a result as shown in figure 9, polyclonal antibody 1.0 μ g in anti-il-i-beta -1Ra-2 rabbit source is added in 100 μ L colloidal gold solutions When, colloidal gold color is stable pink.During real marking, since antibody activity is unstable, and to reach antibody Excess marker, therefore on the basis of amount of antibody increase by 2 gradient values, i.e. 100 μ L colloidal gold solutions antibody label dosage be 1.2μg。

3.3.3NC film

Tetra- kinds of NC films of sartorius95, millipore135, millipore180 and sartorius140 are chosen, respectively Colloidal gold strip is successively assembled after spray film scribing line, sample is added and observes color change at scribing line.As shown in Figure 10.

3.3.4 double T line antibody working concentration optimizations

It is 0mg/mL, 1mg/mL, 2mg/mL that double T line antibody concentrations, which are arranged, successively assembles colloid gold test paper after spray film scribing line Item is added sample and observes color change at scribing line.As shown in figure 11, when two kinds of capture antibody concentrations are 1mg/mL, double T lines It is high-visible with C line color.But since in actual sample detection, β concentration is too low, therefore select high concentration antibody scribing line.Therefore IL-1 β capture antibody concentration is that 2mg/mLIL-1Ra capture antibody concentration is 1mg/mL.

3.3.5 the foundation of test strips standard curve

After 1mL standard items are premixed with 10 μ L colloidal gold probes and sufficiently combined, 14000rpm is centrifuged 20min, abandons supernatant simultaneously With 100 μ L sample-loading buffers be resuspended precipitate, be diluted to 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL, 7.813ng/mL, 3.906ng/mL, 1.953ng/mL, 0.976ng/mL totally 9 concentration are used as standard curve Foundation, set up blank control.

Re-suspension liquid is added in test strips sample pad, parches to test strips, takes pictures and with ImageJ software analysis and processing result. Blank control is done by blank sample of sample-loading buffer, the re-suspension liquid concentration log value obtained after pre-processing with standard items is horizontal seat Mark to determination data regression analysis, and obtains regression equation and standard curve using T line measurement gray value as ordinate.

As shown in figure 12, the calibration curve equation for finally determining IL-1 β is y=24701x+9679.9, R2 0.9726. As shown in figure 13, the calibration curve equation for finally determining IL-1Ra is y=35117x+3247.9, R2 0.9753.

3.3.6 the determination of rate of recovery experiment and minimum detection limit

As shown in table 1, the average recovery rate of IL-1 β is the average recovery rate 82.44% of 83.52%, IL-1Ra.Detection limit Re-suspension liquid concentration when bottom line macroscopic for test strips, IL-1 β detection are limited to 0.7ng/mL, and IL-1Ra detection is limited to 1.0ng/mL。

1 IL-1 β of table and the IL-1Ra rate of recovery

3.3.7 repetitive test

IL-1 β standard items are handled to 10ng/mL with sample-loading buffer, and IL-1Ra standard items to 100ng/mL are arranged three Repeat control group, test strip repeatability.As shown in table 2, the coefficient of variation being calculated to obtain, the IL-1 β coefficient of variation is 9.36%, The IL-1Ra coefficient of variation is that 4.18%, the IL-1Ra/IL-1 β coefficient of variation is 9.63%.The coefficient of variation is respectively less than 10%, it was demonstrated that weight Renaturation is good.

2 IL-1 β of table and IL-1Ra repetitive test

3.3.8 compared with the box of commercial reagent

Using 10 sample IL-1Ra/IL-1 β ratios of test strips detection method sampling Detection of the invention, by commercially available ELISA kit detection same sample IL-1Ra and IL-1 β content simultaneously asks ratio as control.The result shows that glue of the invention For body gold test paper strip detection method compared with the box of commercial reagent, ratio difference is not significant (p=0.92, > 0.05), as shown in figure 14, Illustrate that the colloidal gold strip method of the invention result when measuring IL-1Ra/IL-1 β ratio is reliable.

Embodiment 4

The bis- T line colloidal gold immuno-chromatography test paper strip clinical practice sample detection applied analyses of IL-1Ra/IL-1 β

4.1 material

4.1.1 sample acquires

Brucella correlation sheep sample: being provided by Jilin Province, Yunnan Province, Liaoning Province some areas CDC relevant departments, wherein Non- 40 parts of vaccine immunity sheep blood serum sample of health, 40 parts of sample of brucella S2 vaccine immunity sheep blood serum, brucellosis infection 40 parts of positive sheep blood serum sample.

4.1.2 consumptive material, instrument and equipment

With embodiment 3.

4.2 method

4.2.1 sample treatment

Sheep blood serum sample 3mL is taken, 10 μ L colloidal gold probes, jog 20min after mixing, centrifugation 14000rpm centrifugation is added 20min abandons supernatant and uses 100 μ L sample-loading buffers that precipitating is resuspended as sample-loading buffer.

4.2.2 sample detection and gray analysis

Indoors under fluorescent lamp, at desktop 15cm, the test strips parched are shot.T line is analyzed with ImageJ software Locate gray value, and the re-suspension liquid concentration obtained in 4.2.1 is calculated by standard curve, acquires IL-1Ra/IL-1 β ratio.

4.2.3 statistical data is analyzed

With the non-immune group of SPSS software analysis brucellosis health, immune group and infected group IL-1Ra/IL-1 β ratio Statistical difference, and draw ROC curve and analyze its significance of differential diagnosis.

4.3 result

4.3.1 serum sample rose bengal precipitation test in part is as a result, such as Figure 15.

4.3.2 sheep blood serum IL-1 β and IL-1Ra and IL-1Ra/IL-1 β ratio tests and analyzes

IL-1Ra/IL-1 β ratio in the 3 non-vaccine immunity sheep blood serum of health of table

IL-1Ra/IL-1 β ratio in 4 brucella S2 vaccine immunity sheep blood serum of table

5 brucellosis of table infects IL-1Ra/IL-1 β ratio in positive sheep blood serum

4.3.3 the statistical analysis of clinical sample detection IL-1Ra/IL-1 β ratio

Statistical analysis health group, immune group, in infected group sheep sample IL-1Ra/IL-1 β ratio 99% confidence interval, Healthy group, immune group and infected group IL-1Ra/IL-1 β ratio difference are extremely significant (p < 0.01), illustrate to be aggregated by the red plate of tiger Test, IL-1Ra/IL-1 β ratio can be used as the effective means (being shown in Table 6, Figure 16) for distinguishing immune group and infected group.It draws respectively Immune group processed and healthy group, health group and IL-1Ra/IL-1 β ratio in infected group, immune group and infected group sheep blood serum sample ROC curve, as shown in figure 17, health group and immune group area under the curve AUC are 0.382, less than 0.5, illustrate to utilize IL-1Ra/ IL-1 β ratio does not have antidiastole reference significance when distinguishing health group with immune group.And immune group and infected group, health group with The area under the curve AUC of infected group is 1.000, is greater than 0.5, illustrates IL-1Ra/IL-1 β ratio in difference Brucella melitensis disease There is antidiastole reference value when weak poison immune sheep and natural infection sheep.It is analyzed by SPSS software, to exclude to greatest extent Infected group sample selects the difference 73.69 of immune group IL-1Ra/IL-1 β ratio median and three times standard deviation as identification base Directrix, i.e., it is actually detected in, screen to obtain positive sample (infection sheep and immune sheep) using rose bengal precipitation test, further Positive sample IL-1Ra/IL-1 β ratio is detected, it is immune group Healthy Sheep that ratio, which is greater than 73.69, and ratio suggests making less than 73.69 To slaughter target.

IL-1Ra/IL-1 β ratio statistical analysis in 6 sheep immune group of table, healthy group and infected group blood serum sample

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims (9)

1. a kind of for detecting the colloidal gold strip of IL-1 β, IL-1Ra content and IL-1Ra/IL-1 β ratio, feature simultaneously It is, including fiberglass packing, nitrocellulose filter and blotting paper；Colloid gold label is coated on the glass fibre element pad In combination with the anti-il-i-beta -1Ra-2 rabbit source polyclonal antibody of IL-1 β and IL-1Ra；The nitrocellulose filter successively includes inspection Survey line T1, detection line T2 and nature controlling line；More grams of the cavy source in conjunction with the anti-il-i-beta -1 of IL-1 β is coated in the detection line T1 Grand antibody；The cavy source polyclonal antibody of the anti-IL-1Ra-1 in conjunction with IL-1Ra is coated on the detection line T2；The Quality Control Goat anti-rabbit antibody is coated on line.

2. colloidal gold strip according to claim 1, which is characterized in that the anti-il-i-beta -1Ra- of the colloid gold label The dosage of 2 rabbit source polyclonal antibody is 1.0~1.4 μ g/100 μ L.

3. colloidal gold strip according to claim 1, which is characterized in that coated anti-il-i-beta-in the detection line T1 The concentration of 1 cavy source polyclonal antibody is 1.8~2.2mg/mL.

4. colloidal gold strip according to claim 1, which is characterized in that coated anti-IL- on the detection line T2 The concentration of the cavy source polyclonal antibody of 1Ra-1 is 0.8~1.2mg/mL.

5. colloidal gold strip according to claim 1, which is characterized in that coated goat anti-rabbit antibody on the nature controlling line Concentration be 0.6~1.0mg/mL.

6. colloidal gold strip described in Claims 1 to 5 any one is in the concentration of preparation test sample IL-1 β, IL-1Ra Kit in apply.

7. application according to claim 6, which comprises the following steps:

(1) sample to be tested is added drop-wise to fiberglass packing；

(2) after nature controlling line colour developing, whether observation detection line from colourless becomes red；Detection line becomes red, and sample to be tested contains There are IL-1 β and IL-1Ra；Detection line is non-discolouring, and IL-1 β and IL-1Ra content is lower than detection limit in sample to be tested.

8. colloidal gold strip described in Claims 1 to 5 any one is in the ratio of preparation test sample IL-1Ra and IL-1 β It is applied in the kit of (IL-1Ra/IL-1 β ratio).

9. application according to claim 8, which is characterized in that the sample includes brucellosis correlation blood sample, including The not immune brucellosis vaccine of brucellosis infection animal blood sample, brucellosis vaccine immunity animal blood sample, health is dynamic Object blood sample.