阅读说明：本技术 一种快速检测总Tau蛋白的免疫层析检测卡及其制备方法 (A kind of immunochromatographydetection detection card and preparation method thereof of the total Tau albumen of quick detection ) 是由 林斯 柴诗缘 杨法辰 于 2019-09-26 设计创作，主要内容包括：本发明涉及一种快速检测总Tau蛋白的免疫层析检测卡,包括试纸条,所述试纸条包括检测线及质控线,所述检测线上包被有一株鼠抗人总Tau蛋白单克隆抗体,所述质控线上包被有羊抗兔多克隆抗体。本发明所提供的快速检测总Tau蛋白的免疫层析检测卡,其可用于血清、血浆和全血的检测,以用于阿尔兹海默病的诊断。(The present invention relates to the immunochromatographydetection detection cards that one kind quickly detects total Tau albumen, including test strips, the test strips include detection line and nature controlling line, and the anti-human total Tau protein monoclonal antibody of one plant of mouse is coated in the detection line, is coated with goat-anti rabbit polyclonal antibody on the nature controlling line.The immunochromatographydetection detection card of the total Tau albumen of quick detection provided by the present invention, can be used for the detection of serum, blood plasma and whole blood, with the diagnosis for Alzheimer's disease.)

1. one kind quickly detects the immunochromatographydetection detection card of total Tau albumen, including test strips, the test strips include detection line and Nature controlling line, which is characterized in that the anti-human total Tau protein monoclonal antibody of one plant of mouse, the nature controlling line are coated in the detection line On be coated with goat-anti rabbit polyclonal antibody.

2. quickly detecting the immunochromatographydetection detection card of total Tau albumen as described in claim 1, which is characterized in that the test paper Item further includes PVC board, and sequentially connected sample pad, labeling pad, coating pad and water absorption pad are fixed in the PVC board, described Packet, which is covered with, is successively arranged detection line and nature controlling line, and the sample pad and labeling pad are connected as one.

3. quickly detecting the immunochromatographydetection detection card of total Tau albumen as claimed in claim 2, which is characterized in that the coating It pads and is connected with labeling pad close to one end of detection line, one end close to nature controlling line is connected with water absorption pad.

4. quickly detecting the immunochromatographydetection detection card of total Tau albumen as claimed in claim 3, which is characterized in that the label Fluorescent latex microballoon and the rabbit of the surface active of the anti-human total Tau protein monoclonal antibody label of another plant of mouse are coated on object pad The fluorescent latex microballoon of the surface active of IgG label, the surface of the anti-human total Tau protein monoclonal antibody label of another plant of mouse The molar ratio of the fluorescent latex microballoon of the surface active of fluorescent latex microballoon and the rabbit igg label of activation is 1:0.2~4.

5. quickly detecting the immunochromatographydetection detection card of total Tau albumen as claimed in claim 4, which is characterized in that described quick The immunochromatographydetection detection card for detecting AD7c-NTP further includes that getting stuck for test strips is set for card.

6. quickly detecting the immunochromatographydetection detection card of total Tau albumen as claimed in claim 5, which is characterized in that described to get stuck Include:

Kerve is connected to the PVC board；

Upper cover is connected to the kerve, the well for being loaded in the sample pad is provided on the upper lid；

Observation window is set on lid and for detection line and the acquisition of the data of nature controlling line.

7. a kind of preparation method of the immunochromatographydetection detection card of the quick total Tau albumen of detection described in any one of claims 1-6, Characterized by comprising the following steps:

1) preparation of coating pad: the anti-human total Tau protein monoclonal antibody of one plant of mouse and goat-anti rabbit polyclonal antibody are coated with respectively Onto nitrocellulose filter, drying for standby；

2) preparation of labeling pad: by the fluorescence cream of the surface active of the anti-human total Tau protein monoclonal antibody label of another plant of mouse After the fluorescent latex microballoon mixing of the surface active of glue microballoon and rabbit igg label, it is sprayed on glass fibre element film, drying is standby With；

3) test strips are assembled: the bonding coating pad in PVC board, and in the overlap joint water suction of the one end for the nature controlling line being covered with close to the packet Pad, overlap joint labeling pad and its sample pad of connection in the one end for the detection line being covered with close to the packet；Then it is cut into institute The test strips of width are needed, the reagent strip is put into gets stuck later.

8. preparation method as claimed in claim 7, which is characterized in that the fluorescent latex microballoon of the surface active passes through following Step is made:

1) take surfactant be added 0.1~0.5mol/L, pH value be 8~10 boric acid-borax buffer solution in, add two Methylformamide, N, N '-dicyclohexylcarbodiimide and n-hydroxysuccinimide, are stirred to react；The boric acid-borax buffering Solution includes the PEG2000 of 0.5wt%~3wt%；

2) take surface with the dispersion liquid of the fluorescent latex microballoon of carboxyl, with alkali borate buffer solution tune pH value to 8~10 Afterwards, it is added in the resulting product of step 1), 1~5h is stirred to react at 25 DEG C, after completion of the reaction, centrifugation removal supernatant, The fluorescent latex microballoon of surface active is obtained, is redissolved with alkali borate buffer solution spare；The alkali borate buffering is molten Liquid includes the PEG2000 of 0.5wt%~3wt%.

9. preparation method as claimed in claim 8, which is characterized in that the surfactant includes N, the bis- lauroyl of N'- Base ethylenediamine diacrylate sodium, polyethyleneglycol moon esters of silicon acis, dodecyl benzene sulfonate and lauroyl glutamate, four Weight ratio is (0.5~6): (4~9): (0.8~3): (5~14)；The fluorescence of the surfactant and amino surface cream The weight ratio of glue microballoon is (0.5~120): 1.

10. preparation method as claimed in claim 8 or 9, which is characterized in that the anti-human total Tau albumen Dan Ke of another plant of mouse The fluorescent latex microballoon of the surface active of grand antibody label is made by following steps:

It takes the fluorescent latex microballoon of surface active to be added in carbodiimides and n-hydroxysuccinimide and 2~6h is stirred at room temperature, Then the anti-human total Tau protein monoclonal antibody of another plant of mouse is added, stirs 1~4h at room temperature, adds 10~50mg BSA envelope Liquid is closed, 1~4h of stirring is continued；At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, removes supernatant；Finally, Solid sediment is redissolved with the phosphate buffer solution of 0.01M~0.5M, pH=7.4, Proclin300 is added and is protected at 4 DEG C It deposits stand-by；

Wherein, the mass ratio of the fluorescent latex microballoon of the surface active and the anti-human total Tau protein monoclonal antibody of another plant of mouse Example is 1:(0.01~4).

Technical field

The invention belongs to in-vitro diagnosis field, be related to a kind of immunochromatographydetection detection card for quickly detecting total Tau albumen and its Preparation method.

Background technique

Microtubule system is the framework ingredient of nerve cell, is made of tubulin and microtubule associated protein, and total Tau albumen is The highest microtubule associated protein of content.Many neurodegenerative diseases, including Alzheimer's disease (Alzheimerg Disease, AD), Parkinson's disease (Parkinson gdisease, PD), amyotrophic lateral sclerosis (Amyotro.phiclateral sclerosis, ALS), prions disease (prion disease) etc. has a common disease Feature is managed, i.e. it is different to form neurofibrillary tangles (neurofibrillary, NFTs) for the aggregation due to microtubule associated protein Tau It is often deposited on intracerebral, and then generates neurodegeneration, this kind of disease is referred to as Tau lesion.

Total Tau albumen is the constitutive protein of tangle, has been reported that within 1993 display, total Tau egg in AD patient CSF White level increases, 2~3 times higher than normal person.ANDREASEN etc. compares 2400 AD patients and 1250 healthy person discoveries, The susceptibility about 82.0% of total Tau protein diagnostic AD, specificity are 88.0%.Abnormal phosphoric acid is presented in total Tau albumen of AD patient Change, losing makes tubulin assemble the ability to form micro-pipe, and always assembles at neurofibrillary tangles.More and more Evidence shows, in CSF total Tau albumen (hyperphosphorylated Tau, p-Tau) of phosphorylation can by AD patient with Healthy person, Frontotemporal dementia, dementia with Lewy body, vascular dementia distinguish diagnosis.

But many limitations of CSF sampling limit the application of total Tau albumen.

Summary of the invention

In view of this, the main purpose of the present invention is to provide the immunochromatography detections that one kind quickly detects total Tau albumen Card and preparation method thereof.

To achieve the goals above, the present invention provides the following technical scheme that

A kind of immunochromatographydetection detection card of the total Tau albumen of quick detection, including test strips, the test strips include detection line And nature controlling line, the anti-human total Tau protein monoclonal antibody of one plant of mouse is coated in the detection line, is coated with sheep on the nature controlling line Anti-rabbit polyclonal antibody.

In a concrete scheme of the invention, wherein the test strips further include PVC board, it is fixed in the PVC board Sequentially connected sample pad, labeling pad, coating pad and water absorption pad, the packet, which is covered with, is successively arranged detection line and nature controlling line, The sample pad and labeling pad are connected as one.

In a concrete scheme of the invention, wherein the coating pads and is connected with marker close to one end of detection line Pad, one end close to nature controlling line are connected with water absorption pad.

In a concrete scheme of the invention, wherein being coated with the anti-human total Tau egg of another plant of mouse in the labeling pad The fluorescent latex microballoon of the surface active of fluorescent latex microballoon and the rabbit igg label of the surface active of white labeling of monoclonal antibody, What the fluorescent latex microballoon and rabbit igg of the surface active of the anti-human total Tau protein monoclonal antibody label of another plant of mouse marked The molar ratio of the fluorescent latex microballoon of surface active is 1:0.2~4.

In a concrete scheme of the invention, wherein the immunochromatographydetection detection card of the total Tau albumen of the quick detection is also Including setting getting stuck for test strips for card.

In a concrete scheme of the invention, wherein described get stuck includes:

Kerve is connected to the PVC board；

Upper cover is connected to the kerve, the well for being loaded in the sample pad is provided on the upper lid；

Observation window is set on lid and for detection line and the acquisition of the data of nature controlling line.

A kind of preparation method of the immunochromatographydetection detection card of the total Tau albumen of quick detection, comprising the following steps:

1) preparation of coating pad: the anti-human total Tau protein monoclonal antibody of one plant of mouse and goat-anti rabbit polyclonal antibody are distinguished It is coated on nitrocellulose filter, drying for standby；

2) preparation of labeling pad: by the glimmering of the surface active of the anti-human total Tau protein monoclonal antibody label of another plant of mouse After the fluorescent latex microballoon mixing of the surface active of light latex beads and rabbit igg label, it is sprayed on glass fibre element film, it is dry It is spare；

3) test strips are assembled: the bonding coating pad in PVC board, and overlapped in the one end for the nature controlling line being covered with close to the packet Water absorption pad, overlap joint labeling pad and its sample pad of connection in the one end for the detection line being covered with close to the packet；Then it is cut At the test strips of required width, the reagent strip is put into gets stuck later.

In a concrete scheme of the invention, wherein the fluorescent latex microballoon of the surface active passes through following steps system :

1) take surfactant that 0.1~0.5mol/L is added, boric acid-borax buffer solution that pH value is 8~10 (includes The PEG2000 of 0.5wt%~3wt%) in, add dimethylformamide, N, N '-dicyclohexylcarbodiimide and N- hydroxyl amber Amber acid imide, is stirred to react；

2) it takes surface to have the dispersion liquid of the fluorescent latex microballoon of carboxyl, (includes with boric acid-borax buffer solution The PEG2000 of 0.5wt%~3wt%) adjust pH value to after 8~10, it is added in the resulting product of step 1), is stirred at 25 DEG C 1~5h is reacted, after completion of the reaction, centrifugation removal supernatant obtains the fluorescent latex microballoon of surface active, slow with boric acid-borax It is spare to rush solution redissolution.

In a concrete scheme of the invention, wherein the surfactant includes N, the bis- lauroyl second two of N'- Amine diacrylate sodium, polyethyleneglycol moon esters of silicon acis, dodecyl benzene sulfonate and lauroyl glutamate, four weight ratio For (0.5~6): (4~9): (0.8~3): (5~14)；The fluorescent latex microballoon of the surfactant and the amino surface Weight ratio be (0.5~120): 1.

In a concrete scheme of the invention, wherein the anti-human total Tau protein monoclonal antibody label of another plant of mouse Surface active fluorescent latex microballoon by following steps be made:

The fluorescent latex microballoon of surface active is taken to be added in carbodiimides (EDC) and n-hydroxysuccinimide (NHS) 2~6h is stirred at room temperature, the anti-human total Tau protein monoclonal antibody of another plant of mouse is then added, stirs 1~4h at room temperature, adds 10~50mg BSA confining liquid, continues 1~4h of stirring；At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, is removed Remove supernatant；Finally, being redissolved solid sediment with the phosphate buffer solution (pH=7.4) of 0.01M~0.5M, add Proclin300 is saved for use at 4 DEG C.

In a concrete scheme of the invention, wherein the fluorescent latex microballoon of the surface active and another plant of mouse are anti-human The mass ratio of total Tau protein monoclonal antibody is 1:(0.01~4).

Beneficial effects of the present invention:

1, the immunochromatographydetection detection card of the total Tau albumen of quick detection provided by the present invention, can be used for serum, blood plasma and The detection of whole blood, with the diagnosis for Alzheimer's disease；

2, the immunochromatographydetection detection card of the total Tau albumen of quick detection provided by the present invention, can pass through serum, blood plasma With the total Tau albumen of whole blood test, the risk for puncturing and obtaining cerebrospinal fluid is avoided, is easy to be accepted by patients, detected in 5~20min Within can be completed, linear detection range 15pg/mL-2000pg/mL greatly improves detection efficiency.

3, the immunochromatographydetection detection card of the total Tau albumen of quick detection provided by the present invention, high sensitivity, stability By force, the range of linearity is wide, has excellent accuracy and precision.

4, the immunochromatographydetection detection card of the total Tau albumen of quick detection provided by the present invention, by using surface active Fluorescent latex microballoon, overcome the defect of fluorescent dye poor sensitivity on the market and wet type fluorescent microsphere stability difference, can Guarantee that intergranular relative distance is not easy to reunite, be not necessarily to buffer, can redissolve after sample to be tested is added and smoothly chromatograph at once.

Detailed description of the invention

Fig. 1 is the immuno-chromatographic test paper strip schematic diagram of the quick total Tau albumen of detection provided by the present invention；

Fig. 2A is a kind of inside of upper cover in the immunochromatographydetection detection card of the quick total Tau albumen of detection provided by the present invention Structural schematic diagram；

Fig. 2 B is a kind of inside of kerve in the immunochromatographydetection detection card of the quick total Tau albumen of detection provided by the present invention Structural schematic diagram；

Fig. 3 is the calibration curve of total Tau albumen in the embodiment of the present invention 1；

Fig. 4 is the calibration curve of total Tau albumen in the embodiment of the present invention 2；

Fig. 5 is the calibration curve of total Tau albumen in the embodiment of the present invention 3；

Fig. 6 is the calibration curve of total Tau albumen in the embodiment of the present invention 4；

Wherein, 1-PVC plate, 2- coating pad, 3- labeling pad, 4- water absorption pad, 5- detection line, 6- nature controlling line, 7- marker Junction, 8- sample, 9- sample pad, 11- upper cover, 12- kerve, 13- well, 14- observation window, 15- test strips placement region, 16- positioning column, 17- location hole, the first limited section 18-, the second limited section 19-, 20- third limited section.

Specific embodiment

For a further understanding of the present invention, the preferred embodiment of the invention is described below with reference to embodiment, still It should be appreciated that these descriptions are only further explanation the features and advantages of the present invention, rather than to the claims in the present invention Limitation.

Following material or reagent are unless stated otherwise commercially available.