阅读说明：本技术 一种测定人体神经丝轻链蛋白含量的磁微粒化学发光检测试剂盒及其制备方法 (A kind of magnetic microparticle chemiluminescence detection kit and preparation method thereof measuring human nerve silk light chain protein content ) 是由 王国武 郭健夫 王鹏 于 2019-08-30 设计创作，主要内容包括：本发明公开了一种测定人体神经丝轻链蛋白含量的磁微粒化学发光试剂盒及其制备方法,试剂盒包括：神经丝轻链蛋白测定R1试剂、R2试剂、磁分离试剂、校准品液系列和化学发光底物液。本发明采用的夹心法的反应模式,利用化学发光检测技术与磁微粒免疫分离技术相结合的原理,定量检测人血清、血浆或脑脊液等多种样本中的神经丝轻链蛋白含量,确保了检测的灵敏度,本试剂盒与传统试剂盒相比灵敏度高、无污染、特异性强、操作简单、并且对样品的前处理要求低、可检测样本类型广泛、可快速高通量检测大批量样本,便于临床试剂应用。本发明为临床检测人血清中的神经丝轻链蛋白提供了一种更准确、精确、方便、快捷和简单的方法。(The invention discloses a kind of magnetic microparticle chemiluminescence kit and preparation method thereof for measuring human nerve silk light chain protein content, kit includes: neurofilament light chain protein determination R1 reagent, R2 reagent, Magneto separate reagent, calibration object liquid series and Chemoluminescent substrate.The reaction pattern for the sandwich method that the present invention uses, the principle that isolation technics combines is immunized with magnetic particle using chemiluminescence detection technology, neurofilament light chain protein content in a variety of samples such as quantitative detection human serum, blood plasma or cerebrospinal fluid, ensure the sensitivity of detection, this kit compared with conventional reagents box high sensitivity, pollution-free, high specificity, it is easy to operate and to the pre-treatment of sample require low, detectable sample type extensively, can fast high-flux detect high-volume sample, be convenient for clinical reagent application.The present invention provides a kind of more acurrate, accurate, convenient, fast and simple method for the neurofilament light chain albumen in clinical detection human serum.)

1. a kind of magnetic microparticle chemiluminescence detection kit for measuring human nerve silk light chain protein content, it is characterised in that: packet It includes: R1 reagent, R2 reagent, Magneto separate reagent, calibration object liquid series and Chemoluminescent substrate；

Wherein, R1 reagent is the anti-neurofilament light chain protein monoclonal antibody dilution of marked by fluorescein isothiocyanate, R2 reagent For the anti-neurofilament light chain protein antibodies dilution of alkali phosphatase enzyme mark, Magneto separate reagent is anti-fluorescein isothiocynate Dan Ke The grand coated magnetic particle diluent of antibody, calibration object liquid series are the antigen diluent containing various concentration neurofilament light chain albumen Liquid, Chemoluminescent substrate are the substrate solution of alkaline phosphatase catalytic luminescence.

2. kit as described in claim 1, it is characterised in that: the R1 reagent is that concentration made of being diluted as buffer is The anti-neurofilament light chain protein monoclonal antibody dilution of the marked by fluorescein isothiocyanate of 0.3~0.9 μ g/mL；

Wherein, it is preferred that be used to prepare R1 reagent pH of cushioning fluid be 7.2~8.0, buffer include concentration be 12.0~ Sodium chloride that Sodium azide that the Tris of 12.3g/L, concentration are 1.98~1.99g/L, concentration are 5.7~5.9g/L, molar concentration 0.8~1.2mL/L of liquor zinci chloridi, the concentration 5- for being 0.1M for 0.8~1.2mL/L of magnesium chloride solution of 1M, molar concentration The newborn bovine serum that the bovine serum albumin(BSA) and concentration that the fishskin gelatin of 20g/L, concentration are 2~5g/L are 10~50g/L, Remaining is deionized water.

3. kit as described in claim 1, it is characterised in that: the R2 reagent is that concentration made of being diluted as buffer is The anti-neurofilament light chain protein antibodies dilution of 0.5~2.0 μ g/mL alkali phosphatase enzyme mark；

Wherein, it is preferred that the pH value for being used to prepare the buffer of R2 reagent is 7.2~8.0, the AP being more preferably commercialized Conjugate Stabilizer。

4. kit as described in claim 1, it is characterised in that: the Magneto separate reagent is dense made of being diluted as buffer Degree is the coated magnetic particle solution of anti-fluorescein isothiocynate monoclonal antibody of 0.5~2mg/mL；

Wherein, it is preferred that be used to prepare the buffer of Magneto separate reagent pH value be 7.5~9.0, buffer composition be include dense Spend the chlorination that the Tris for being 10.5~11.3g/L, the Sodium azide that concentration is 1.91~1.95g/L, concentration are 5.5~5.7g/L 0.8~1.2mL/ of liquor zinci chloridi that 0.8~1.2mL/L of magnesium chloride solution that sodium, molar concentration are 1M, molar concentration are 0.1M L, the superfine horse serum that the bovine serum albumin(BSA) and concentration that concentration is 4.7~4.9g/L are 4.8~5.0g/L, remaining group are divided into Deionized water.

5. kit as described in claim 1, it is characterised in that: the calibration object liquid series is made of being diluted as buffer Antigenic dilution containing various concentration neurofilament light chain albumen；

Wherein, it is preferred that it is the Tris of 8.5~10.2g/L, concentration that the buffer for being used to prepare calibration object liquid series, which includes concentration, For the sodium chloride of 11.8~14.5g/L, concentration be 0.008~0.028g/L tetracycline hydrochloride, concentration be 0.008~ Casein that Sodium azide that the neomycinsulphate of 0.028g/L, concentration are 1.5~2.0g/L, concentration are 6.0~10.0g/L and Concentration is the polysorbas20 of 2.6-~3.4g/L, remaining group is divided into deionized water.

6. kit as claimed in claim 5, it is characterised in that: the concentration range of the calibration object liquid series be 0~ 500pg/ml, pH of cushioning fluid are 7.0~8.0；

Wherein, it is preferred that the calibration object liquid series include respectively containing concentration be 0pg/ml, 10pg/ml, 50pg/ml, The antigenic dilution of 100pg/ml, 250pg/ml, 500pg/ml neurofilament light chain albumen.

7. kit as described in claim 1, it is characterised in that: the Chemoluminescent substrate is diluted by buffer The Chemoluminescent substrate containing 0.2~0.4mg/mL alkaline phosphatase catalytic luminescence substrate；

Wherein, it is preferred that the buffer is the Tris-HCl buffering that the molar concentration that pH value is 8~10 is 0.1-0.3M Liquid, the alkaline phosphatase catalytic luminescence substrate are dioxane compound (APCL).

8. kit as claimed in claim 7, it is characterised in that: it is 9.3 to rub that the Chemoluminescent substrate, which is by pH value, Dioxane compound (APCL) containing 0.3mg/mL made of the Tris-HCl buffer dilution that your concentration is 0.2M Chemoluminescent substrate.

9. the described in any item kits of claim 1-8 neurofilament light chain protein content reagent in preparation measurement human sample In purposes.

10. purposes as claimed in claim 9, it is characterised in that: the sample is human serum, blood plasma or cerebrospinal fluid.

Technical field

Human nerve silk is detected using magnetic microparticle chemiluminescence technology and Ag-Ab combination technology the present invention relates to a kind of Magnetic microparticle chemiluminescence detection kit of light chain protein content and preparation method thereof.The invention belongs to medical diagnosis necks Domain.

Background technique

Neurofilament protein is the main cytoskeletal protein of neuron, respectively by neurofilament protein light chain, middle chain and heavy chain Composition, is specifically expressed in axon and aixs cylinder.The major function of neurofilament protein is to provide structural support for aixs cylinder and regulate and control The growth diameter of neural axon affects the rate and accuracy of neural traffic.Neurofilament protein light chain (neurofilament Light, NFL) it is most important constituent in neurofilament protein, molecular weight 68KDa is the marker of axonal injury.Mind Organizine light chain constitutes the skeleton of heavy chain, and heavy chain aggregation forms neural silk fiber.Due to direct wound or slowly move back Change process, impaired nerve cell content are discharged to the compartment on periphery, it is possible to quantitative detection neuronin.It is moved back a variety of The neurofilament of the row disease such as patient of cerebral injury, multiple sclerosis, frontotemporal dementia and other a variety of nervus retrogression diseases is light Catenin level can be increased.

Alzheimer disease (Alzheimer's disease, AD) is a kind of Neuro-degenerative for carrying out sexual development Disease often shows as essential characteristic with dementias such as memory disorders, personality and behavior changes in clinic, and that falls ill after 65 years old is referred to as For Delayed onset AD.With population in the world aging, the number of AD patient is quicklyd increase, it is contemplated that the year two thousand thirty, global AD patient will Reach 6,6,000,000 people, China will be more than 1,6,000,000 people.Compared with normal aging people, AD minimal invasive treatment's self-care ability is poor, simultaneously It is often accompanied by mental act exception, great burden is brought to family and society, has taken over myocardial infarction, cancer, apoplexy Summation, to society and family exert heavy pressures on.The cause of disease of the disease is unknown at present, once studies have reported that AD and cerebrospinal fluid neutralize Neurofilament protein content in blood has certain connection, especially with light chain protein therein (neurofilamentlight, NFL) in close relations.Therefore, research and development neurofilament light chain protein diagnostic kit has very important clinical value.

The neurofilament light chain method of protein detection being currently known is mainly enzyme-linked immunosorbent assay, fluoroimmunoassay Method.But these methods there are still sensitivity it is low, the range of linearity is narrow, unstable result the defects of.

Therefore, it is still necessary to a kind of high sensitivity, easy to operate, high degree of automation, result stabilizations, low-cost mind at present Organizine light chain protein detection kit and application method.

Summary of the invention

It is stable, low-cost the present invention is directed to develop a kind of high sensitivity, easy to operate, high degree of automation, result A kind of magnetic microparticle chemiluminescence detection kit and preparation method thereof measuring human nerve silk light chain protein content.

Based on above-mentioned purpose, the present invention provides a kind of magnetic microparticle chemiluminescence for measuring human nerve silk light chain protein content Detection kit, comprising: R1 reagent, R2 reagent, Magneto separate reagent, calibration object liquid series and Chemoluminescent substrate；Wherein, R1 Reagent is the anti-neurofilament light chain protein monoclonal antibody dilution of marked by fluorescein isothiocyanate, and R2 reagent is alkaline phosphatase The anti-neurofilament light chain protein antibodies dilution of label, Magneto separate reagent are that anti-fluorescein isothiocynate monoclonal antibody is coated Magnetic particle diluent, calibration object liquid series are the antigenic dilution containing various concentration neurofilament light chain albumen, chemiluminescence bottom Thing liquid is the substrate solution of alkaline phosphatase catalytic luminescence.

Wherein, it is preferred that the R1 reagent is the different sulphur cyanogen that concentration made of being diluted as buffer is 0.3~0.9 μ g/mL The fluorescein-labeled anti-neurofilament light chain protein monoclonal antibody dilution of acid.

Wherein, it is preferred that the pH of cushioning fluid for being used to prepare R1 reagent is 7.2~8.0, and buffer includes that concentration is 12.0 It is sodium chloride that Sodium azide that the Tris of~12.3g/L, concentration are 1.98~1.99g/L, concentration are 5.7~5.9g/L, mole dense Spending the 0.8~1.2mL/L of magnesium chloride solution for being 1M, 0.8~1.2mL/L of liquor zinci chloridi that molar concentration is 0.1M, concentration is The newborn bovine serum that the bovine serum albumin(BSA) and concentration that the fishskin gelatin of 5-20g/L, concentration are 2~5g/L are 10~50g/L, Remaining is deionized water.

Wherein, it is preferred that the R2 reagent is that concentration made of being diluted as buffer is 0.5~2.0 μ g/mL alkaline phosphatase The anti-neurofilament light chain protein antibodies dilution of enzyme label.

Wherein, it is preferred that the pH value for being used to prepare the buffer of R2 reagent is 7.2~8.0, the AP being preferably commercialized Conjugate Stabilizer。

Wherein, it is preferred that the Magneto separate reagent is that concentration made of being diluted as buffer is that resisting for 0.5~2mg/mL is different The coated magnetic particle solution of thiocyanic acid fluorescein monoclonal antibody.

Wherein, it is preferred that the pH value for being used to prepare the buffer of Magneto separate reagent is 7.5~9.0, and buffer composition is packet Include the chlorine that Sodium azide, concentration that Tris, concentration that concentration is 10.5~11.3g/L are 1.91~1.95g/L are 5.5~5.7g/L Change liquor zinci chloridi 0.8 that 0.8~1.2mL/L of magnesium chloride solution, molar concentration that sodium, molar concentration are 1M are 0.1M~ The superfine horse serum that the bovine serum albumin(BSA) and concentration that 1.2mL/L, concentration are 4.7~4.9g/L are 4.8~5.0g/L, remaining Group is divided into deionized water.

Wherein, it is preferred that the calibration object liquid series is light containing various concentration neurofilament made of being diluted as buffer The antigenic dilution of catenin.

Wherein, it is preferred that be used to prepare calibration object liquid series buffer include concentration be 8.5~10.2g/L Tris, Tetracycline hydrochloride that sodium chloride that concentration is 11.8~14.5g/L, concentration are 0.008~0.028g/L, concentration is 0.008~ Casein that Sodium azide that the neomycinsulphate of 0.028g/L, concentration are 1.5~2.0g/L, concentration are 6.0~10.0g/L and Concentration is 2.6-~3.4g/L polysorbas20, remaining group is divided into deionized water.

Wherein, it is preferred that the concentration range of the calibration object liquid series be 0~500pg/ml, pH of cushioning fluid be 7.0~ 8.0。

Wherein, it is preferred that the calibration object liquid series include respectively containing concentration be 0pg/ml, 10pg/ml, 50pg/ml, The antigenic dilution of 100pg/ml, 250pg/ml, 500pg/ml neurofilament light chain albumen.

Wherein, it is preferred that the Chemoluminescent substrate is to contain 0.2~0.4mg/mL alkali made of being diluted as buffer The Chemoluminescent substrate of acid phosphatase catalytic luminescence substrate；It is furthermore preferred that the buffer is mole that pH value is 8~10 Concentration is the Tris-HCl buffer of 0.1-0.3M, and the alkaline phosphatase catalytic luminescence substrate is dioxane chemical combination Object (APCL).

Wherein, it is preferred that the Chemoluminescent substrate is the Tris-HCl that the molar concentration for being 9.3 by pH value is 0.2M The Chemoluminescent substrate of dioxane compound (APCL) containing 0.3mg/mL made of buffer dilution.

Further, the invention also provides a kind of magnetic microparticle chemiluminescences for measuring human nerve silk light chain protein content The preparation method of detection kit, includes the following steps:

Reagent preparation R1:1) according to reagent R1 buffer composition content preparation buffer, adjust pH；2) isothiocyanic acid is prepared Fluorescein-labeled anti-neurofilament light chain protein monoclonal antibody；3) by the anti-neurofilament light chain egg of marked by fluorescein isothiocyanate White monoclonal antibody is diluted with R1 buffer.

Reagent preparation R2:1) according to reagent R2 buffer composition content preparation buffer, adjust pH；2) alkaline phosphatase is prepared The anti-neurofilament light chain protein antibodies of enzyme label；3) the anti-neurofilament light chain protein antibodies of alkali phosphatase enzyme mark are buffered with R2 Liquid is diluted.

It prepares magnetic particle: 1) preparing buffer according to magnetic particle buffer composition content；2) anti-isosulfocyanic acid fluorescence is prepared The plain coated magnetic particle of monoclonal antibody；3) magnetic particle is diluted with Tris-HCl buffer.

It prepares calibration object: 1) preparing buffer according to calibration object buffer composition content；2) neurofilament light chain albumen is resisted Original, which is dissolved in calibration object buffer, is configured to different concentration.

It prepares Chemoluminescent substrate: 1) preparing buffer according to Chemoluminescent substrate buffer composition content；2) will The chemiluminescent substrate of alkaline phosphatase catalytic luminescence is dissolved in buffer.

Further, the invention also provides a kind of magnetic particle chemistry hairs for measuring human nerve silk light chain protein content Light detection kit application method, includes the following steps:

(1) by sample to be tested or 37 DEG C of calibration object, reagent R1, reagent R2 mixing incubation 15min；

(2) magnetic particle is added after above-mentioned reaction system and continues to incubate 5min in 37 DEG C；

(3) it washs, luminous substrate is added after removing unbonded antibody and impurity；

(4) luminous substrate is added, ALP catalysis substrate measures relative luminous intensity (RLU) after shining.

RLU and neurofilament light chain proteantigen concentration are proportional in a certain range, can be from standard by interpolation method The neurofilament light chain protein content of sample to be tested is read on curve.

Methodology appraising datum when detection kit measurement human nerve silk light chain protein content above-mentioned using the present invention It can reach following index:

Sensitivity-minimum detectable activity is 0.1pg/ml；

Neurofilament light chain albumen linear detection range, 0.1~500pg/ml；

Precision is respectively less than 8% between precision and analysis in precision-analysis, meets national requirements, illustrates examination of the present invention Agent box has good repeatability during the experiment；

Accuracy-, which uses the serum of known neurofilament light chain protein concentration, goes hormone serum after different proportion dilutes The rate of recovery is 95%~115%；

If the interferent concentration contained in interference-sample meet it is claimed below, on testing result without influence: bilirubin≤ 400umol/L, hemoglobin≤5g/L, chyle≤0.30%, V_C≤ 0.5g/L, heparin sodium≤100IU/mL.

Compared to the prior art, the beneficial effects of the present invention are:

Isolation technics is immunized using chemiluminescence detection technology and magnetic particle in the reaction pattern for the sandwich method that the present invention uses The principle combined, the neurofilament light chain protein content in a variety of samples such as quantitative detection human serum, blood plasma or cerebrospinal fluid, it is ensured that The sensitivity of detection, this kit compared with conventional reagents box high sensitivity (traditional ELISA kits sensitivity only~ 0.1ng/ml), pollution-free, high specificity, easy to operate and require low, detectable sample type wide the pre-treatment of sample It is general, can fast high-flux detect high-volume sample, be convenient for clinical reagent application.The present invention is the nerve in clinical detection human serum Silk light chain protein provides a kind of more acurrate, accurate, convenient, fast and simple method.

Detailed description of the invention

Fig. 1 is concentration-luminous value curve graph of neurofilament light chain albumen in 2 kit of the embodiment of the present invention；

Specific embodiment

The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.