阅读说明：本技术 快速检测阿尔茨海默病相关神经丝蛋白AD7c-NTP的免疫层析检测卡及其制备方法 (Immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP and preparation method thereof ) 是由 林斯 柴诗缘 樊涛 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阿尔茨海默病相关神经丝蛋白AD7c-NTP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AD7c-NTP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AD7c-NTP的免疫层析检测卡,其可用于尿液的检测,用于阿尔兹海默病的诊断。(The invention relates to an immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP, which comprises a test strip, wherein the test strip comprises a detection line and a quality control line, a mouse anti-human AD7c-NTP monoclonal antibody is coated on the detection line, and a goat anti-rabbit polyclonal antibody is coated on the quality control line. The immunochromatography detection card for rapidly detecting AD7c-NTP provided by the invention can be used for urine detection and Alzheimer disease diagnosis.)

1. An immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP comprises a test strip, wherein the test strip comprises a detection line and a quality control line, and is characterized in that a mouse anti-human AD7c-NTP monoclonal antibody is coated on the detection line, and a goat anti-rabbit polyclonal antibody is coated on the quality control line.

2. the immunochromatographic test card for rapidly detecting Alzheimer's disease-associated neurofilament protein AD7c-NTP according to claim 1, wherein the test strip further comprises a PVC plate, a sample pad, a marker pad, a coating pad and a water absorption pad which are sequentially connected are fixed on the PVC plate, a detection line and a quality control line are sequentially arranged on the coating pad, and the sample pad and the marker pad are integrally connected.

3. The immunochromatographic test card for rapidly detecting Alzheimer's disease-associated neurofilament protein AD7c-NTP according to claim 2, wherein the end of the coated pad near the detection line is connected with a label pad, and the end near the quality control line is connected with a water absorption pad.

4. The immunochromatographic detection card for rapidly detecting Alzheimer's disease-related neurofilament protein AD7c-NTP according to claim 3, wherein the label pad is coated with another mouse anti-human AD7c-NTP monoclonal antibody-labeled surface-activated fluorescent latex microsphere and a rabbit IgG-labeled surface-activated fluorescent latex microsphere, and the molar ratio of the another mouse anti-human AD7c-NTP monoclonal antibody-labeled surface-activated fluorescent latex microsphere to the rabbit IgG-labeled surface-activated fluorescent latex microsphere is 1: 0.2-4.

5. The immunochromatographic test card for rapid detection of Alzheimer's disease-associated neurofilament protein AD7c-NTP according to claim 4, wherein the immunochromatographic test card for rapid detection of AD7c-NTP further comprises a card shell for holding a test strip.

6. the immunochromatographic detection card for rapidly detecting Alzheimer's disease-associated neurofilament protein AD7c-NTP according to claim 5, wherein the card shell comprises:

A bottom trough connected to the PVC sheet;

The upper cover is connected with the bottom groove, and a sample adding hole for adding samples to the sample pad is formed in the upper cover;

And the observation window is arranged on the upper cover and is used for data acquisition of the detection line and the quality control line.

7. A method for preparing an immunochromatographic test card for rapidly detecting Alzheimer's disease-associated neurofilament protein AD7c-NTP according to any one of claims 1 to 6, comprising the steps of:

1) preparation of the coating pad: respectively coating a mouse anti-human AD7c-NTP monoclonal antibody and a goat anti-rabbit polyclonal antibody on a nitrocellulose membrane, and drying for later use;

2) Preparation of the marker pad: mixing the surface-activated fluorescent latex microspheres marked by the other mouse anti-human AD7c-NTP monoclonal antibody and the surface-activated fluorescent latex microspheres marked by rabbit IgG, spraying the mixture on a glass cellulose membrane, and drying the mixture for later use;

3) Assembling the test strip: bonding a coating pad on a PVC plate, overlapping a water absorption pad at one end close to a quality control line on the coating pad, and overlapping a marker pad and a sample pad connected with the marker pad at one end close to a detection line on the coating pad; then cut into test strips with required width, and then put the test strips into the card shell.

8. The method of claim 7, wherein the surface-activated fluorescent latex microspheres are prepared by:

1) adding a surfactant into 0.1-0.5 mol/L boric acid-borax buffer solution with the pH value of 8-10, adding dimethylformamide, N' -dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stirring for reaction; the boric acid-borax buffer solution contains 0.5-3 wt% of PEG 2000;

2) Taking a dispersion liquid of the fluorescent latex microspheres with carboxyl on the surfaces, adjusting the pH value to 8-10 by using a boric acid-borax buffer solution, adding the dispersion liquid into the product obtained in the step 1), stirring and reacting at 25 ℃ for 1-5 hours, centrifuging and removing supernatant after the reaction is finished to obtain fluorescent latex microspheres with activated surfaces, and re-dissolving the fluorescent latex microspheres with the boric acid-borax buffer solution for later use; the boric acid-borax buffer solution contains 0.5 wt% -3 wt% of PEG 2000.

9. the method of claim 8, wherein the surfactant comprises N, N' -dilauroyl ethylenediamine diacrylate, polyethylene glycol monolaurate, dodecylbenzene sulfonate, and lauroyl glutamate, in a weight ratio of (0.5-6): 4-9): 0.8-3): 5-14; the weight ratio of the surfactant to the fluorescent latex microspheres on the amino surface is 0.5-120: 1.

10. the method of claim 8 or 9, wherein the another strain of mouse anti-human AD7c-NTP monoclonal antibody-labeled surface-activated fluorescent latex microspheres are prepared by the steps of:

Adding the surface-activated fluorescent latex microspheres into carbodiimide and N-hydroxysuccinimide, stirring for 2-6 h at room temperature, adding another mouse anti-human AD7c-NTP monoclonal antibody, stirring for 1-4 h at room temperature, adding 10-50 mg BSA confining liquid, and continuing stirring for 1-4 h; centrifuging at 2-8 ℃, and removing supernatant; finally, redissolving the solid precipitate by using 0.01-0.5M phosphate buffer solution with pH of 7.4, adding Proclin300, and storing at 4 ℃ for later use;

Wherein the mass ratio of the surface-activated fluorescent latex microsphere to another mouse anti-human AD7c-NTP monoclonal antibody is 1: 0.01 to 4.

Technical Field

The invention belongs to the field of in-vitro diagnosis, and particularly relates to an immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP and a preparation method thereof.

background

alzheimer's Disease (AD), also known as senile dementia, is a common degenerative Disease of the central nervous system, which is characterized by memory impairment, cognitive decline, dyskinesia, etc., accompanied by a series of mental symptoms, and finally resulting in complete loss of the patient's ability to live and care. With the progress of aging of the social population, AD seriously threatens human health, and a huge patient group brings heavy mental and economic burden to families and society, but the pathogenesis of the disease is not clear at present. Necropsy studies have demonstrated that Senile Plaques (SP) and neurofibrillary tangles (NFT) formed by deposition of β amyloid peptide (β) are two of the major pathological features of AD and have been included as diagnostic criteria for AD.

At present, the diagnosis of AD is mainly based on the clinical manifestations of patients, the imaging data of skull CT or MRI, the psychoneurosis examination scale and other comprehensive evaluations, and the clinical diagnosis rate is low. Therefore, high-sensitivity, specific and noninvasive biological indexes are searched for improving the early diagnosis rate of AD, and help is provided for early life style intervention, medicine intervention and disease course delay.

Alzheimer's disease associated neurofilament protein (AD7c-NTP) is a transmembrane phosphoprotein expressed in neurons with a molecular weight of about 41 kDa. The fact that AD7c-NTP is increased in brain expression level of AD patients and appears in degenerated neurons which are complete in histology is found for the first time by professor de la Monte in general hospital of Massachusetts in 1996, and the fact that abnormal AD7c-NTP expression is increased is proved to be an early event of AD neurodegeneration. A large number of subsequent researches from different national populations prove that AD7c-NTP exists in NFTs, is mainly related to induction of neuroinflammation and nerve cell death, and is an AD biomarker which can reflect AD pathological characteristics and has higher sensitivity and specificity. Based on the fact that the AD7c-NTP in urine has the same molecular mass with the protein in brain tissues and cerebrospinal fluid, and the detection result has similarity, the AD7c-NTP in urine can specifically reflect cerebral nerve damage, and has the same effect with the AD7c protein. AD7c-NTP is specifically distributed in frontal lobe and temporal lobe of brain, and only reflects cerebral neuron damage; AD7c-NTP is a neuron axon transmembrane protein and is co-localized with AD7c protein; the specificity of urine AD7c-NTP protein reflects brain neuron injury, so the clinical development of urine AD7c-NTP detection can assist the early detection and diagnosis and treatment of AD patients.

disclosure of Invention

In view of the above, the main objective of the present invention is to provide an immunochromatography detection card for rapidly detecting Alzheimer's disease-associated neurofilament protein AD7c-NTP and a preparation method thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

An immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP comprises a test strip, wherein the test strip comprises a detection line and a quality control line, a mouse anti-human AD7c-NTP monoclonal antibody is coated on the detection line, and a goat anti-rabbit polyclonal antibody is coated on the quality control line.

In a specific scheme of the invention, the test strip further comprises a PVC plate, a sample pad, a marker pad, a coating pad and a water absorption pad which are sequentially connected are fixed on the PVC plate, a detection line and a quality control line are sequentially arranged on the coating pad, and the sample pad and the marker pad are connected into a whole.

In one embodiment of the invention, one end of the coating pad close to the detection line is connected with a marker pad, and one end close to the quality control line is connected with a water absorption pad.

In a specific embodiment of the invention, the marker pad is coated with another strain of mouse anti-human AD7c-NTP monoclonal antibody labeled surface-activated fluorescent latex microsphere and a rabbit IgG labeled surface-activated fluorescent latex microsphere, and the molar ratio of the another strain of mouse anti-human AD7c-NTP monoclonal antibody labeled surface-activated fluorescent latex microsphere to the rabbit IgG labeled surface-activated fluorescent latex microsphere is 1: 0.2-4.

In one embodiment of the present invention, the surface-activated fluorescent latex microspheres are prepared by the following steps:

1) Adding a surfactant into 0.1-0.5 mol/L boric acid-borax buffer solution (containing 0.5-3 wt% of PEG2000) with the pH value of 8-10, adding dimethylformamide, N' -dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stirring for reaction;

2) taking a dispersion liquid of the fluorescent latex microspheres with carboxyl on the surfaces, adjusting the pH value to 8-10 by using a boric acid-borax buffer solution (containing 0.5-3 wt% of PEG2000), adding the dispersion liquid into the product obtained in the step 1), stirring and reacting for 1-5 hours at 25 ℃, and centrifuging to remove supernatant after the reaction is finished to obtain the fluorescent latex microspheres with activated surfaces, and redissolving the fluorescent latex microspheres with the boric acid-borax buffer solution for later use.

In one embodiment of the present invention, the surfactant comprises sodium N, N' -dilauroyl ethylenediamine diacrylate, polyethylene glycol monolaurate, dodecylbenzene sulfonate and lauroyl glutamate, wherein the weight ratio of the four is (0.5-6): (4-9): (0.8-3): (5-14); the weight ratio of the surfactant to the fluorescent latex microspheres on the amino surface is (0.5-120): 1.

in a specific embodiment of the present invention, the another strain of mouse anti-human AD7c-NTP monoclonal antibody labeled surface-activated fluorescent latex microspheres are prepared by the following steps:

adding the surface-activated fluorescent latex microspheres into carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and stirring for 2-6 h at room temperature, then adding another mouse anti-human AD7c-NTP monoclonal antibody, stirring for 1-4 h at room temperature, adding 10-50 mg BSA confining liquid, and continuing stirring for 1-4 h; centrifuging at 2-8 ℃, and removing supernatant; finally, the solid precipitate was redissolved with 0.01M to 0.5M phosphate buffer (pH 7.4), and Proclin300 was added and stored at 4 ℃ until use.

In a specific embodiment of the present invention, the mass ratio of the surface-activated fluorescent latex microsphere to another mouse anti-human AD7c-NTP monoclonal antibody is 1: (0.01-4).

In a specific embodiment of the invention, the immunochromatographic test card for rapidly detecting AD7c-NTP further comprises a card shell for holding a test strip.

In one embodiment of the present invention, wherein the card case comprises:

A bottom trough connected to the PVC sheet;

The upper cover is connected with the bottom groove, and a sample adding hole for adding samples to the sample pad is formed in the upper cover;

and the observation window is arranged on the upper cover and is used for data acquisition of the detection line and the quality control line.

A preparation method of an immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP comprises the following steps:

1) preparation of the coating pad: respectively coating a mouse anti-human AD7c-NTP monoclonal antibody and a goat anti-rabbit polyclonal antibody on a nitrocellulose membrane, and drying for later use;

2) Preparation of the marker pad: mixing the surface-activated fluorescent latex microspheres marked by the other mouse anti-human AD7c-NTP monoclonal antibody and the surface-activated fluorescent latex microspheres marked by rabbit IgG, spraying the mixture on a glass cellulose membrane, and drying the mixture for later use;

3) Assembling the test strip: bonding a coating pad on a PVC plate, overlapping a water absorption pad at one end close to a quality control line on the coating pad, and overlapping a marker pad and a sample pad connected with the marker pad at one end close to a detection line on the coating pad; then cut into test strips with required width, and then put the test strips into the card shell.

The invention has the beneficial effects that:

1. The immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP can be used for urine detection and diagnosis of Alzheimer disease.

2. The immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP can detect AD7c-NTP through urine, avoids the risk of obtaining cerebrospinal fluid through puncture, is easy to accept by a patient, can complete detection within 5-20 min, has a linear detection range of 0.25ng/mL-10ng/mL, and greatly improves the detection efficiency.

3. the immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP provided by the invention has the advantages of high sensitivity, strong stability, wide linear range and excellent accuracy and precision.

4. According to the immunochromatography detection card for rapidly detecting Alzheimer disease related neurofilament protein AD7c-NTP, the surface-activated fluorescent latex microspheres are adopted, so that the defects of poor sensitivity of fluorescent dye and poor stability of wet fluorescent microspheres on the market are overcome, the relative distance between particles is ensured not to be easily aggregated, a buffer solution is not needed, and the particles can be instantly redissolved and chromatographed smoothly after being added into a urine sample.

Drawings

FIG. 1 is a schematic diagram of an immunochromatographic test strip for rapidly detecting Alzheimer's disease-associated neurofilament protein AD7c-NTP provided by the invention;

FIG. 2A is a schematic diagram of the internal structure of an upper cover of the immunochromatography detection card for rapidly detecting Alzheimer's disease-associated neurofilament protein AD7c-NTP provided by the present invention;

FIG. 2B is a schematic diagram of the internal structure of a bottom groove of the immunochromatography detection card for rapidly detecting Alzheimer's disease-associated neurofilament protein AD7c-NTP provided by the present invention;

FIG. 3 is a calibration curve of Alzheimer's disease-associated neurofilament protein AD7c-NTP according to example 1 of the present invention;

FIG. 4 is a calibration curve of Alzheimer's disease-associated neurofilament protein AD7c-NTP according to example 2 of the present invention;

FIG. 5 is a calibration curve of Alzheimer's disease-associated neurofilament protein AD7c-NTP according to example 3 of the present invention;

FIG. 6 is a calibration curve of Alzheimer's disease-associated neurofilament protein AD7c-NTP according to example 4 of the present invention;

The test strip detection device comprises a PVC plate 1, a coating pad 2, a marker pad 3, a water absorption pad 4, a detection line 5, a quality control line 6, a marker joint 7, a sample 8, a sample pad 9, a top cover 11, a bottom groove 12, a sample adding hole 13, an observation window 14, a test strip placement area 15, a positioning column 16, a positioning hole 17, a first limiting part 18, a second limiting part 19 and a third limiting part 20.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.

The following materials or reagents, unless otherwise specified, are commercially available.