A kind of preparation method of H-FABP and cTnI bigeminy efficient detection test paperTechnical field
The invention belongs to biological technology applications, and in particular to a kind of cardic fatty acid binding protein H-FABP(Heart-typeFattyAcidBindingProtein, H-FABP) and cTnI (cardiactroponinI,CTnI) cTnI bigeminy efficient detection test paper.
Background technology
H-FABP is present in myocardial cell cytoplasm, rich content.Myocardial damage early stage, cardiac muscle cell's film integrality are brokenBad, H-FABP enters blood.H-FABP 1-2h after myocardial damage can be detected in blood.Since H-FABP cardiac muscles are specialProperty higher than current clinically used myoglobins, therefore H-FABP can become a kind of important detection Acute myocardial tissue damageEarlier specificity marker.Currently, the detection of biochemical marker is widely used to acute myocardial infarction AMI, the instability mode heart twistsBitterly, the diagnosis of the myocardial injury diseases such as acute heart failure, myocarditis, openheart surgery lesion assessment, Newborns with Asphyxia myocardial damage, dangerDangerous degree layering and prognosis are judged.The U.S. every year because suspect property coronary syndrome emergency treatment medical treatment patient up to 80,000,000, wherein45% or so is related to heart, and 15% is diagnosed as acute coronary syndrome.The incidence of China's acute coronary syndrome is about50/100000, the number for dying of acute myocardial infarction AMI every year is more than 1,000,000.Therefore, the application of H-FABP detections has important societyMeeting and economic benefit.
When cardiac muscle cells go to pot because of factors such as hypoxic-ischemics, sequestered cTnI and cTnT can be first rapidly from cellsIt is released into blood, is gradually decomposed later in conjunction with the mating type cTnI and cTnT in myocardial structural albumen, is slowly released into circulatingIn, to explain with the disease that myocardial cell damage is performance, such as acute myocardial infarction AMI (AMI), cTnI and cTnT are being recycledIt is relatively early in blood the reason of simultaneously energy last longer occur.
So H-FABP and cTnI is very important health index parameters for myocardium health.The prior art cannotTwo kinds of substances of efficient detection simultaneously, substance interferes with each other when detection, instead reduces the detection sensitivity of gold-labelled pad.
Invention content
Goal of the invention：For the deficiencies in the prior art, the object of the present invention is to provide a kind of H-FABP and cTnIBigeminy efficient detection test paper, make it have easy to operate, sensitivity ＜ 5ng/mL, result stablize the advantages that.
Invention content：The present invention provides a kind of H-FABP and cTnI bigeminy efficient detection test paper, including are located at fixed plateOn the sample pad, gold-labelled pad, nitrocellulose filter and the absorption pad that are sequentially connected；Detection line antibody is equipped on nitrocellulose filterWith nature controlling line antibody；Gold labeling antibody is equipped in gold-labelled pad, the sample pad is pad, the gold made of glass fibre cottonMark pad is pad made of carboxylated cellulose film, and the gold labeling antibody is colour developing antibody, and detection line antibody is to capture antibody, colour developingAntibody and capture antibody are that 3 plants of gold marks to be formed described in sandwich anti-human H-FABP and cTnI monoclonal antibodies that can mutually match resistBody is marked with 40nm nanometers of golden shells, and the gold-labelled pad is equipped with functional layer, and the gold-labelled pad passes through sodium bicarbonate, bovine serum albuminIn vain, casein and polyoxy alkene bay ether mixed liquid dipping.
Further, the functional layer based on mass fraction by 18% pure silver nitrate, 70% gelatin, 8% department's sheet 80 withAnd 4% cyclodextrin forms.
Further, the gold-labelled pad by 80% sodium bicarbonate solution, 8% bovine serum albumin(BSA), 8% casein and4% polyoxy alkene bay ether mixed liquid dipping.
The preparation method of above-mentioned H-FABP and cTnI bigeminy efficient detection test paper, includes the following steps：
(1) it uses using trisodium citrate as stabilizer and reducing agent, prepares monodispersed silver nano-grain as sacrificeTemplate, by the 40nm gold nanoshells for replacing reaction acquisition colloid, high dispersive；
(2) nanogold shell solution pH8.0~8.5 are adjusted, colour developing antibody is added and is reacted with nanogold shell solution, centrifuges heavyIt forms sediment, precipitation is resuspended, obtains gold labeling antibody solution；
(3) after carboxylated cellulose film being pre-processed, drying, by that will be matched respectively by infrared spraying equipment in vacuum environment18% pure silver nitrate, 70% macromolecule dispersant, 8% department's sheet 80 and 4% cyclodextrin made is sprayed into carboxylation fiberPlain film forms well gold-labelled pad, then uses gold labeling antibody solution spray printing；
(4) immersion of gold-labelled pad：Gold-labelled pad by 80% sodium bicarbonate solution, 8% bovine serum albumin(BSA), 8% casein withAnd 4% polyoxy alkene bay ether mixed liquid dipping, then dry.
(5) it is sprayed on having activated nitrocellulose filter with capture antibody and draws a detection line, vacuum drying；Then sheep anti mouse is usedIgG sprays on having activated nitrocellulose membrane draws nature controlling line, vacuum drying；
(6) it is pasted onto on the plastic plate of single side pressure sensitive adhesive, cuts by sample pad, gold-labelled pad, nitrocellulose membrane, absorption pad sequenceIt cuts, is packed into plastic clip, packaging；Wherein, colour developing antibody is 2 plants of anti-human H-FABP that can form sandwich pairing mutually with antibody is capturedAntibody.
Advantageous effect：H-FABP the and cTnI bigeminy efficient detection test paper of the present invention, contains specific function by specialThe gold-labelled pad and soaking technology of layer so that gold mark rate of release slows down, and is buffered, to make analyte in sample be marked with goldThe concentration proportion of antibody increases, to improve the sensitivity of ELISA test strip.
In addition, by the gold-labelled pad and soaking technology of particular functional layer, enable to avoid mutually between sample when detectionInterference, can greatly improve accuracy of detection.
Description of the drawings
Fig. 1 is the structural schematic diagram of test strips.
Specific implementation mode
With reference to specific embodiment, the present invention is described further.
As shown in Figure 1, a kind of H-FABP and cTnI bigeminy efficient detection test paper, including it is located at and is sequentially connected in fixed plateSample pad (1), gold-labelled pad (2), nitrocellulose filter (3) and absorption pad (4)；Detection line is equipped on nitrocellulose filter (3)Antibody (5) and nature controlling line antibody (6)；Gold labeling antibody is equipped in gold-labelled pad (2), the sample pad (1) is glass fibre cottonManufactured pad, the gold-labelled pad (2) are pad made of carboxylated cellulose film, and the gold labeling antibody is colour developing antibody, detectionLine antibody is to capture antibody, and colour developing antibody is 3 plants with capture antibody can mutually match to form sandwich anti-human H-FABP and cTnIGold labeling antibody described in monoclonal antibody is marked with 40nm nanometers of golden shells, and the gold-labelled pad is equipped with functional layer, the gold-labelled padPass through sodium bicarbonate, bovine serum albumin(BSA), casein and polyoxy alkene bay ether mixed liquid dipping.The functional layer is with quality pointSeveral meters are made of 18% pure silver nitrate, 70% gelatin, 8% department's sheet 80 and 4% cyclodextrin, and the gold-labelled pad is by 80% carbonSour hydrogen sodium solution, 8% bovine serum albumin(BSA), 8% casein and 4% polyoxy alkene bay ether mixed liquid dipping.
The preparation method of above-mentioned test paper is as follows：
(1) preparation of nanogold shell solution：
Using trisodium citrate as stabilizer and reducing agent, prepares monodispersed silver nano-grain and be used as " seed "：At 70 DEG CWater-bath under 80mL deionized waters are heated to constant temperature, the citric acid three sodium solution of 20mL1% (wt) is added, stirs evenly, addsEnter the AgNO of 1.7mL10mg/mL3Solution is vigorously stirred lower addition 2mL1%NaHB4Solution, then continuous heating and stirring 1h,It is settled to 100mL after room temperature cooling.
" seed " of silver nano-grain is grown：2mL1% sodium citrate solutions are added into 80mL deionized waters and are heated toFluidized state, the silver-colored seed solution that certain volume is then added are added 1.7mL10mg/mL's in being vigorously stirred processAgNO3Solution continues to keep stirring and heat 30min, and constant volume is to 100mL after room temperature cooling.
It is to sacrifice template with silver nano-grain, colloid, high monodispersed gold nanoshell is prepared by replacing reaction：It takes30mL silver nanoparticle solutions centrifuge 20min at 12500g, pour out supernatant, then with deionized water constant volume to 100mL.It willThe silver nanoparticle solution of this 100mL is heated to fluidized state, and 2mM HAuCl are added with vigorous stirring4Solution 2mL, reactionLiquid color is gradually changed into blackish green, room temperature cooling by yellow.
(2) the gold label of colour developing antibody：
Nanogold shell solution pH8.0~8.5 are adjusted, colour developing antibody is added and is reacted with nanogold shell solution, centrifuges to obtain precipitation,Precipitation is resuspended, obtains gold labeling antibody solution.
(3) prepared by gold-labelled pad and spray is padded：
After carboxylated cellulose film is pre-processed, drying, by that will be prepared respectively by infrared spraying equipment in vacuum environment18% pure silver nitrate, 70% macromolecule dispersant, 8% department's sheet 80 and 4% cyclodextrin got well are sprayed into carboxylated celluloseFilm forms well gold-labelled pad, then uses gold labeling antibody solution spray printing.
(4) immersion of gold-labelled pad：Gold-labelled pad by 80% sodium bicarbonate solution, 8% bovine serum albumin(BSA), 8% casein withAnd 4% polyoxy alkene bay ether mixed liquid dipping, then dry.
(5) prepared by detection line：
1 plant of anti-human H-FABP monoclonal antibodies BT01 is taken to do detection line antibody, with buffer solution (10% trehalose, 6% firstAlcohol) it is diluted to 1mg/mL；Antibody is drawn on nitrocellulose filter with film instrument is drawn, a concentration of 1ul/cm forms detection line, vacuumIt is dry.
(6) prepared by nature controlling line：
Sheep anti-mouse igg is diluted to 0.5mg/mL with buffer solution (10% trehalose, 6% methanol)；Antibody is drawn with film instrument is drawnOn nitrocellulose filter, a concentration of 0.5ul/cm forms nature controlling line, vacuum drying.
(7) test strips are assembled：
Sample pad 1, gold-labelled pad 2, nitrocellulose filter 3 and blotting paper 4 are pasted onto to the plastics of single side pressure sensitive adhesive in orderOn plate, as shown in Figure 1, being equipped with detection line antibody 5 and nature controlling line antibody 6 on nitrocellulose filter 3；6cm width is cut to pastePlastic plate afterwards is packed into detection card；It is packed with the heat-sealing Fresco Bag with drier, room temperature preservation.
(8) it detects：
10000 μ l of sample are divided into 100 groups and are added dropwise to well respectively, when 15min is observed, interpretation ELISA test stripAs a result standard is：The positive has colour developing band at detection line and at nature controlling line；Feminine gender, without colour developing band, Quality Control at detection lineThere is colour developing band at line；And developing sensitivity ＜ 3ng/mL, no broken string phenomenon.
The method of reference standard sample detection is detected 300 physical examination human normal plasma's samples, wherein 298 5 are dividedClock i.e. detect as a result, and developing sensitivity ＜ 3ng/mL, no broken string phenomenon.
The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the artFor member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answeredIt is considered as protection scope of the present invention.