CN109187981A

CN109187981A - A kind of quantum dot immune chromatograph test strip of quick detection beta-amyloid protein and application

Info

Publication number: CN109187981A
Application number: CN201810865711.3A
Authority: CN
Inventors: 万东山; 郭兰英
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-08-01
Filing date: 2018-08-01
Publication date: 2019-01-11

Abstract

The invention belongs to technical field of immunoassay, and in particular to one kind quickly quantum dot immune chromatograph test strip of detection beta-amyloid protein and application.The quantum dot immune chromatograph test strip of the quick detection beta-amyloid protein, comprising being equipped with the nitrocellulose filter of detection zone and quality control region, glass fibre membrane, bottom plate, sample pad and the water absorption pad of coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β.The quantum dot immune chromatograph test strip of quick detection beta-amyloid protein provided by the invention passes through quantum dot immunochromatography technique, primary first-order equation can be achieved and detect A β 40,42 antigen index of A β simultaneously, it avoids different albumen and leads to the problem of intersection mutually, detection time is short, high sensitivity is in 10 times of enzyme-linked immunoassay method or more, high specificity, and reaction speed is fast, accuracy is high, quantitative accurate.

Description

A kind of quantum dot immune chromatograph test strip of quick detection beta-amyloid protein and application

Technical field

The invention belongs to technical field of immunoassay, and in particular to the quantum dot that one kind quickly detects beta-amyloid protein is exempted from Epidemic disease chromatograph test strip and application.

Background technique

Alzheimer disease (Alzheimer disease, AD), by Bavarian neuropathologist's Alzheimer It is a kind of central nervous system degeneration, insidious onset, the course of disease is in chronic in first discovery in 1907, and with its naming Progressive is mainly shown as the psychoneurals disease such as gradual memory obstacle, cognition dysfunction, personality change and aphasis Shape seriously affects social, occupation and vital function.Alzheimer disease is senile dementia one of the most common type type, accounts for about institute Have dull-witted 50~60%, according to statistics in western countries, more than or equal to 85 years old age group illness rate between 24~33%, Although developing country lacks authoritative and representative number statistics, estimate that in the world 60% dementia patients are present in hair It is national in exhibition.The cause of disease and pathogenesis of AD not yet illustrates, characteristic pathology change into beta-amyloid protein deposit to be formed it is thin Neurofibrillary tangles and neuron loss companion in the nerve cell that extracellular senile plaque and Protein tau Hyperphosphorylationof are formed Glial cells hyperplasia etc..Therefore it is representative with the characteristics of blood vessel hyperplasia that beta-amyloid protein, which can be used as Alzheimer disease, Medical diagnosis on disease index.

Nineteen thirty, Divry can dye with the Congo red lesion area in patient's AD brain, are successfully made to be deposited on The coloring of extracellular senile plaque, and then find that the main component of senile plaque is a kind of thermophilic Congo red amyloid protein, 1984 Glenner etc. has successfully completed the separation and examining order to this albumen respectively, it is found that this albumen is by 39~43 ammonia Base acid residue composition names beta-amyloid protein, abbreviation A β because it has the secondary structure of a β lamella then.The source intracerebral A β In its precursor substance amyloid beta-protein precursor (amyloid precursor protein, APP).APP is a kind of transmembrane protein Matter, various tissues are widely present in vivo, and in the expression highest of brain tissue, in physiological conditions, most APP are by A2 secretase It is cracked into soluble App peptide, App peptide is further generating the few part APP of P3 in cytoplasm lysosome by C2 secretion enzymatic lysis It is cracked into A β through 2 secretase of β and C2 secretion enzyme effect, be metabolized as amyloid beta process from App and be divided into two steps: β 2 divides first The N-terminal for secreting enzyme in A β cracks APP, generates the APP derivative APPS2 β of soluble secretory and the C-terminal piece through film component Disconnected, C-terminal segment is further cracked into A β by C2 secretase, and A β has 2 kinds of forms of 40 amino acid and 42 amino acid, wherein A β 42 easily cause aggregation, have stronger cytotoxicity, and under certain pathological conditions, APP is mainly secreted through 2 secretase of β and C2 Enzyme sequence shearing, which generates excessive A β, causes AD sick, and C2 secretase plays a very important role in A β generation, determines generation A β 42 in wherein shared ratio.

Beta-amyloid protein (amyloid β-protein, A β) molecular weight about 4KDa, by amyloid beta-protein precursor (β- Amyloid precursor protein, APP) hydrolysis, be circulated in blood, cerebrospinal fluid and brain interstitial fluid, mostly with companion Companion's protein molecular combines, and minority exists with free state.The most common hypotype of A β is A β 40 and A β 42 in human body.In human cerebrospinal fluid In blood, 10 times higher than the contents level of A β 42 and 1.5 times respectively of A β 40, A β 42 has stronger toxicity, and is easier to assemble, To form the core of A β precipitating, cause neurotoxic effect.The deposition of A β is not only related with the retrogression pathological changes of neuron, and And a series of pathology affairs can be activated, the broken ring of activation, blood-brain barrier including star spongiocyte and microglia and The variation etc. of microcirculation, the main reason for being the denaturation of patient's AD intracerebral senile plaque peripheral nerve member and is dead.Researcher thinks, Accumulation shows do not occur beta-amyloid protein also outside cranial nerve cell early stage beta-amyloid protein is inside cranial nerve cell When accumulation, Alzheimer's disease is just had already appeared.This discovery helps to diagnose ahead of time, treats Alzheimer's disease, To delay as far as possible, sb.'s illness took a turn for the worse.

Quantum dot is widely used in biological and medical field as a kind of novel inorganic nano fluorescent material, in cell Label, biomolecule detection, immunoassay etc. all achieve many important achievements.Compared with traditional organic fluorescent dye, Quantum dot there is exciting light spectrum width and it is continuous, emission spectrum is narrow and symmetrical, anti-light Bleachability strong, fluorescence intensity height etc. is significant excellent Point.To meet the clinical needs quickly detected, a variety of easy, quick immunological detection methods, quantum dot are developed in recent years Chromatographic technique be one of which, the quantum dot of nearly new development in 2 years quickly measure test strips be primarily adapted for use in emergency treatment examine, it is small From surveying in type laboratory or family, for traditional detection means there are cumbersome, the time is long, and expensive instrument and equipment etc. is needed to lack It falls into, quantum dot is led in researchs such as biochemistry, cell biology, molecular biology in recent years due to its excellent fluorescence property Domain all shows extremely wide application prospect.

Compared with traditional organic fluorescent dye, quantum dot has incomparable superiority: 1. exciting light as fluorescence probe Spectrum width and it is continuous, traditional organic fluorescence reagent excitation spectrum is relatively narrow, and different organic fluorescent dyes needs different wave length exciting light Excitation.And quantum dot can absorb all light more shorter wavelengths of than its first launch wavelength, therefore the quantum dot of different colours can be used Same light excites simultaneously, enormously simplifies actually detected process, reduces the requirement to excitation light source.2. emission peak is narrow and right Claiming, the fluorescence emission spectrum peak shape of quantum dot is narrow and symmetrical, and peak width at half height (fwhm) usually only 40nm is even more small, and Long wave direction does not have apparent trailing phenomenon, can use the quantum dot of different emission peaks simultaneously, and emission peak is not overlapped. This property makes quantum dot be possible to be used as colorful coding application in biological fluorescent labelling.3. photostability is strong, continuing Under light excitation, the fluorescence decay of organic fluorescent dye is rapid, is easy to be bleached, which greatly limits them in biology Application on fluorescent marker.And quantum dot is due to belonging to inorganic nanoparticles, is a kind of highly stable fluorescent material, It is anti-light it is Bleachability will be far better than organic dyestuff.4. launch wavelength " can tune ", in organic fluorescent dye reagent, only only a few Compound launch wavelength up to 700nm or more (such as cyanine fluorochrome), and quantum dot is by changing its size and chemical group At can make its emission spectrum cover far ultraviolet to near-infrared wavelength region, to compensate for common fluorescent molecular in near-infrared The deficiency of spectral regions less varieties, the controllability and continuity of quantum dot light emitting property, for its every field application ten Divide important.5. fluorescence intensity is high, quantum dot has very high excitation overlay region (excitation cross-sections), This means that they can absorb a large amount of exciting light；Quantum dot also has very high quantum yield, therefore they can emit greatly Measure the light that they are absorbed.The brightness that the two factors result in quantum dot is very high, and single quantum dot also can be easily detected Measure, opposite organic dyestuff improves the sensitivity of detection, this be investigate single biomolecule activity condition and they it Between interaction provide advantageous tool.

Summary of the invention

For overcome the deficiencies in the prior art and disadvantage, the primary purpose of the present invention is that providing a kind of quickly detection β-shallow lake The quantum dot immune chromatograph test strip of powder sample albumen, the different spy of the emission wavelength of quantum dot of the test strips based on different-grain diameter Point detects beta-amyloid protein using quantum point methods, primary to test, and can measure beta-amyloid protein 40 (A β 40) and β-simultaneously Two subunits of amyloid protein 42 (A β 42), the test strips have high sensitivity, and high specificity, reaction speed is fast, can be used as house The quick testing product in front yard, has broad application prospects.

Another object of the present invention is to provide the quantum dot immune chromatographic test papers of above-mentioned quick detection beta-amyloid protein The preparation method of item.

A further object of the present invention is to provide the quantum dot immune chromatographic test papers of above-mentioned quick detection beta-amyloid protein The application of item.

The purpose of the invention is achieved by the following technical solution:

A kind of quantum dot immune chromatograph test strip of quick detection beta-amyloid protein, comprising being equipped with detection zone and quality control region Nitrocellulose filter, coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β glass fibre membrane；

The nitrocellulose filter equipped with detection zone and quality control region is successively arranged 40 monoclonal antibody of A β, 42 Dan Ke of A β Grand antibody and DNP antibody form detection zone T1, detection zone T2 and quality control region C；

Spacing between the detection zone T1 and detection zone T2 is preferably 0.5cm；

Spacing between the detection zone T2 and quality control region C is preferably 0.5cm；

The preparation method of the nitrocellulose filter for being equipped with detection zone and quality control region, comprises the following steps:

(1) 40 monoclonal antibody of A β, 42 monoclonal antibody of A β are diluted respectively with the phosphate buffer containing trehalose It is 1.0~2.5mg/mL to concentration, it is 0.75~2.5mg/mL that DNP antibody, which is diluted to concentration, obtains 40 monoclonal antibody work of A β Make liquid, 42 monoclonal antibody working solution of A β and DNP antibody working solution；

(2) by 40 monoclonal antibody working solution of A β made from step (1), 42 monoclonal antibody working solution of A β and DNP antibody Working solution draws film on nitrocellulose (NC) film respectively, forms detection zone T1, detection zone T2 and quality control region C, dry, is set There is the nitrocellulose filter of detection zone and quality control region；

It is 1% trehalose that phosphate buffer containing trehalose described in step (1), which preferably contains mass fraction, 0.01M phosphate buffer, pH7.5~8.5；PH is preferably 8.0；

40 monoclonal antibody of A β described in step (1), 42 monoclonal antibody of A β are preferably with the phosphate containing trehalose Buffer is diluted to 1.5mg/mL respectively；

It is 1.0mg/mL that DNP antibody described in step (1), which is preferably diluted to concentration,；

Nitrocellulose filter described in step (2) is preferably CN 140NC film (Sartorius (Sai Duolisi))；

The aperture of nitrocellulose filter described in step (2) is preferably 8 μm, creep speed 110S/4cm；

It is preferably 1 μ L/cm that film speed is drawn described in step (2)；

The condition of drying described in step (2) is preferably 37 DEG C of dry 3~4h；

The preservation condition of the nitrocellulose filter for being equipped with detection zone and quality control region is preferably 1~30 DEG C；

The preparation method of the glass fibre membrane of the coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β, packet Containing following steps:

(1)QD₆₅₅Quantum dot microsphere marks 40 polyclonal antibody of A β, QD₅₆₅Quantum dot microsphere marks 42 polyclonal antibody of A β

By QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere is washed and is diluted with activation buffer respectively, obtains QD₆₅₅Amount Son point microspheres solution, QD₅₆₅Quantum dot microsphere solution；By 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and QD₆₅₅Quantum dot microsphere solution, QD₅₆₅Quantum dot microsphere solution mixes respectively, and 25 DEG C of ± 0.1 DEG C of oscillations 30~ Supernatant is abandoned in 60min, centrifugation, is then washed with coupling liquid, the QD after being washed₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere； Then the proportion of 10 μ g antibody proteins, QD after washing are added according to every milligram of quantum dot microsphere₆₅₅Quantum dot microsphere, QD₅₆₅ 40 polyclonal antibody of A β (label QD is separately added into quantum dot microsphere₆₅₅), 42 polyclonal antibody of A β (label QD₅₆₅), 2~8 DEG C Oscillation 18~for 24 hours；25 DEG C of ± 0.1 DEG C of 60~120min of oscillation of confining liquid are added, supernatant is abandoned in centrifugation, is then washed with preservation liquid And dilute, respectively obtain QD₆₅₅40 polyclonal antibody of A β, the QD of quantum dot microsphere label₅₆₅The A β more than 42 of quantum dot microsphere label Clonal antibody；

(2)QD₆₅₅Quantum dot microsphere or QD₅₆₅Quantum dot microsphere marks DNP-BSA (haptens)

By QD₆₅₅Quantum dot microsphere or QD₅₆₅Quantum dot microsphere is washed and is diluted with activation buffer, obtains QD₆₅₅Quantum dot Microspheres solution or QD₅₆₅Quantum dot microsphere solution；By 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) with QD₆₅₅Quantum dot microsphere solution or QD₅₆₅The mixing of quantum dot microsphere solution, 25 DEG C of ± 0.1 DEG C of 30~60min of oscillation are centrifuged, in abandoning Clearly, it is then washed with coupling liquid, the QD after being washed₆₅₅Quantum dot microsphere or QD₅₆₅Quantum dot microsphere；Then according to every milligram The proportion of 10 μ g albumen, QD after washing is added in quantum dot microsphere₆₅₅Quantum dot microsphere or QD₅₆₅It is added in quantum dot microsphere DNP-BSA, 2~8 DEG C of oscillations 18~for 24 hours；25 DEG C of ± 0.1 DEG C of 60~120min of oscillation of confining liquid are added, centrifugation abandons supernatant, so It is washed and is diluted with preservation liquid afterwards, obtain QD₆₅₅The DNP-BSA (haptens) or QD of quantum dot microsphere label₅₆₅Quantum dot microsphere The DNP-BSA (haptens) of label；

(3) by QD made from step (1)₆₅₅40 polyclonal antibody of A β, the QD of quantum dot microsphere label₅₆₅Quantum dot microsphere mark QD made from 42 polyclonal antibody of A β and step (2) of note₆₅₅The DNP-BSA (haptens) or QD of quantum dot microsphere label₅₆₅Amount The DNP-BSA (haptens) of son point microballoon label is mixed in equal volume, obtains mixture；Mixture is coated in glass fibre membrane On, it is dry, obtain the glass fibre membrane of coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β；

QD described in step (1)₆₅₅Quantum dot microsphere solution and QD₅₆₅The concentration of quantum dot microsphere solution is preferably 1mg/ mL；

(3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 1- described in step (1) and QD₆₅₅Quantum dot is micro- Ball solution or QD₅₆₅The mass ratio of quantum dot microsphere solution is preferably 1:30；

QD described in step (1)₆₅₅40 polyclonal antibody of A β or QD of quantum dot microsphere label₅₆₅Quantum dot microsphere label 42 polyclonal antibody of A β in the concentration of quantum dot microsphere be preferably 1mg/mL；

QD described in step (2)₆₅₅Quantum dot microsphere solution or QD₅₆₅The concentration of quantum dot microsphere solution is preferably 1mg/ mL；

(3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 1- described in step (2) and QD₆₅₅Quantum dot is micro- Ball solution or QD₅₆₅The mass ratio of quantum dot microsphere solution is preferably 1:30；

QD described in step (2)₆₅₅The DNP-BSA (haptens) or QD of quantum dot microsphere label₅₆₅Quantum dot microsphere mark The concentration of quantum dot microsphere is preferably 1mg/mL in the DNP-BSA (haptens) of note；

QD described in step (3)₆₅₅40 polyclonal antibody of A β, the QD of quantum dot microsphere label₅₆₅Quantum dot microsphere label 42 polyclonal antibody of A β preferably and QD₆₅₅The DNP-BSA of quantum dot microsphere label is mixed in equal volume；

Activation buffer described in step (1) and (2) is preferably 0.05M MES；

Coupling liquid described in step (1) and (2) is preferably 0.01M P Β S, pH7.2~7.4；

Confining liquid described in step (1) and (2) is preferably the 0.01M P Β S of glycine containing 40mM, 1wt% Β SA, PH7.2~7.4；

Preservation liquid described in step (1) and (2) preferably contains 0.5wt%casine, 1wt%P300 (ProClin300) 0.01M P Β S, pH7.2~7.4；

It washed, washed with coupling liquid or is washed with preservation liquid specific with activation buffer described in step (1) and (2) After preferably microballoon is resuspended with activation buffer, coupling liquid or preservation liquid in operation, supernatant is abandoned in centrifugation；Repeat aforesaid operations 2~3 It is secondary；The centrifugal condition is preferably 10000 turns of centrifugation 30min；

The QD of the label of 40 polyclonal antibody of A β described in step (1)₆₅₅Quantum dot microsphere, 42 polyclonal antibody of A β label QD₅₆₅The storage temperature of quantum dot microsphere is preferably 2~8 DEG C；

QD described in step (2)₆₅₅The DNP-BSA (haptens) or QD of quantum dot microsphere label₅₆₅Quantum dot microsphere mark The storage temperature of the DNP-BSA (haptens) of note is preferably 2~8 DEG C；

The fiberglass packing of preferably every milliliter of mixture 8 square centimeters of coating described in step (3)；

The temperature of drying described in step (3) is preferably 37 DEG C of dry 3~4h；

The quantum dot immune chromatograph test strip of the quick detection beta-amyloid protein preferably also includes bottom plate, sample Pad and water absorption pad；

The glass fibre membrane of the sample pad, coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β is equipped with The nitrocellulose filter and water absorption pad of detection zone and quality control region are successively pasted on bottom plate；

The preparation method of the quantum dot immune chromatograph test strip of the quick detection beta-amyloid protein includes following step It is rapid:

The nitrocellulose filter for being equipped with detection zone and quality control region is pasted in the middle part of bottom plate (quality control region is as end), coating Quantum dot microsphere is coupled A β 40 and the glass fibre membrane of 42 polyclonal antibody of A β is close to nitrocellulose filter front end, and sample pad is placed in Glass fibre membrane front end；Water absorption pad is placed in nitrocellulose filter end；

The glass fibre membrane of the sample pad, coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β is equipped with It is overlapped between detection zone and the nitrocellulose filter and water absorption pad of quality control region；

The quantum dot immune chromatograph test strip of the quick detection beta-amyloid protein is in detection beta-amyloid protein Application；

The quantum dot immune chromatograph test strip of the quick detection beta-amyloid protein is in detection beta-amyloid protein Application, preferably specifically include the following steps:

Sample pad is added in sample to be tested, after placing 7~10min, with the fluorescence of quantum dot analyzer measurement quantum dot；

The volume of the sample to be tested is preferably 75 μ L；

The time of the placement is preferably 8min；

The application does not include treatment and the diagnostic purpose of disease；

The principle of the present invention:

A β 40,42 polyclonal antibody of A β are coupled on the quantum dot microsphere of different-grain diameter by the present invention respectively, after coupling A β 40,42 monoclonal antibody of A β NC film cross, quantum dot fluorescence test strips are assembled into, by the mark for establishing A β 40, A β 42 Directrix curve, quantitative detection A β 40, A β 42.Detailed process is as follows: sample to be tested being added in sample fiber, passes through quantum dot immune Chromatography reacts, and is formed in conjunction with quantum dot-labeled A β 40,42 polyclonal antibody of A β in sample containing A β 40,42 albumen of A β immune Compound, due to chromatography action-reaction compound along nitrocellulose membrane move forward, by detection line on NC film draw film A β 40, 42 monoclonal antibody of A β captures, and the A β 40,42 protein content of A β in sample are directly proportional to quantum dot signal strength, is examined by quantum dot The detection for surveying instrument, can detect the concentration of A β 40, A β 42 respectively.

The present invention has the following advantages and effects with respect to the prior art:

(1) the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein provided by the invention is exempted from by quantum dot Epidemic disease chromatographic technique is, it can be achieved that primary first-order equation detects A β 40,42 antigen index of A β simultaneously.

(2) the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein provided by the invention avoids different eggs It is white it is mutual lead to the problem of intersection, detection time is short, and high sensitivity is in 10 times of enzyme-linked immunoassay method or more, high specificity, reaction Speed is fast, and accuracy is high, quantitative accurate.

(3) present invention uses 40 monoclonal antibody of phosphate buffered saline A β containing trehalose, 42 monoclonal of A β Antibody still keeps the advantage that potency is high, specificity is good after drawing film.

(4) A β 40 and 42 polyclonal antibody of A β use EDC method covalent coupling quantum dot microsphere, connection effect in the present invention It is good, still keep the advantage that potency is high, specificity is good.

(5) the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein provided by the invention, without large-scale instrument Device can be used easily using simplicity in general mini clinic or family, be provided the diagnosis of patients with Alzheimer disease A kind of method prevents senile dementia for the mankind and provides strong detection instrument, has broad application prospects.

Specific embodiment

Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.

(NC film, model sartor μ gs CN 140, aperture are 8 μm to nitrocellulose filter, creep speed in embodiment 110S/4cm) it is purchased from Sartorius, QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere is opened purchased from Wuhan Ka source technology of quantum dots Send out Co., Ltd, beta-amyloid protein 40 (A β 40) monoclonal antibody, beta-amyloid protein 42 (A β 42) monoclonal antibody, β-shallow lake (the A β 40) polyclonal antibody of powder sample albumen 40 and (the A β 42) polyclonal antibody of beta-amyloid protein 42 are purchased from Invitrogen, β-shallow lake Powder sample albumen 40 (A β 40) antigen, beta-amyloid protein 42 (A β 42) antigen, DNP-BSA are purchased from Sigma, and chemical reagent etc. is purchased from Sigma。

Embodiment 1

(1) it is equipped with the preparation of the nitrocellulose filter of detection zone and quality control region

1. by 40 monoclonal antibody of A β, 42 monoclonal antibody of the A β 0.01M phosphoric acid for containing mass fraction being 1% trehalose Salt buffer (pH=8 uses 0.22 μm to be filtered using preceding) is diluted to concentration respectively as 1.5mg/mL, and DNP antibody dilutes It is 1mg/mL to concentration, obtains 40 monoclonal antibody working solution of A β, 42 monoclonal antibody working solution of A β and DNP antibody working solution；

2. by 40 monoclonal antibody working solution of A β made from step (1), 42 monoclonal antibody working solution of A β and DNP antibody work Make liquid and draw film on NC film respectively with film instrument is drawn, drawing film speed is 1 μ L/cm, wherein 40 monoclonal antibody of A β is drawn at 0.8cm Film, 42 monoclonal antibody of A β draw film at 1.3cm, and DNP antibody draws film at 1.8cm, form detection zone T1, detection zone T2 and matter Area C is controlled, then 37 DEG C of dry 3.5h, obtain the nitrocellulose filter equipped with detection zone and quality control region, which protects Depositing condition is 20 DEG C；

(2) preparation of the glass fibre membrane of coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β

①QD₆₅₅Quantum dot microsphere marks 40 polyclonal antibody of A β, QD₅₆₅Quantum dot microsphere marks 42 polyclonal antibody of A β

By QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere is washed with activation buffer (0.05M MES) (with activation respectively After microballoon is resuspended in buffer, 10000 turns of centrifugation 30min abandon supernatant；Repeat aforesaid operations 2 times) and it is diluted to 1mg/mL, it obtains QD₆₅₅Quantum dot microsphere solution, QD₅₆₅Quantum dot microsphere solution；By 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride Salt (EDC) and QD₆₅₅Quantum dot microsphere solution, QD₅₆₅Quantum dot microsphere solution is mixed according to mass ratio 1:30 respectively, 25 DEG C of oscillations Supernatant is abandoned in 30min, centrifugation, then 2 times (method is same as above) is washed with coupling liquid (0.01M P Β S, pH7.4), after obtaining washing QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere；Then matching for 10 μ g antibody proteins is added according to every milligram of quantum dot microsphere Than QD after washing₆₅₅Quantum dot microsphere, QD₅₆₅40 polyclonal antibody of A β (label QD is separately added into quantum dot microsphere₆₅₅)、 42 polyclonal antibody of A β (label QD₅₆₅), 4 DEG C of oscillation 18h；Confining liquid (the 0.01M of glycine containing 40mM, 1wt% Β SA is added P Β S, pH7.4) 25 DEG C of oscillation 60min, centrifugation abandons supernatant, then with save liquid (contain 0.5wt%casine, 1wt%P300's 0.01M P Β S, pH7.4) it washs 2 times (method is same as above) and is diluted to microballoon concentration as 1mg/mL, respectively obtain QD₆₅₅Quantum dot 40 polyclonal antibody of A β, the QD of microballoon label₅₆₅42 polyclonal antibody of A β of quantum dot microsphere label, storage temperature are 4 DEG C；

②QD₆₅₅Quantum dot microsphere marks DNP-BSA (haptens)

By QD₆₅₅Quantum dot microsphere washs 2 times with activation buffer (0.05M MES) and (microballoon is resuspended with activation buffer Afterwards, 10000 turns of centrifugation 30min abandon supernatant；Repeat aforesaid operations 2 times) and it is diluted to 1mg/mL, obtain QD₆₅₅Quantum dot microsphere Solution；By 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and QD₆₅₅Quantum dot microsphere solution is according to matter Amount is mixed than 1:30,25 DEG C of oscillation 30min, and supernatant is abandoned in centrifugation, is then washed 2 times with coupling liquid (0.01M P Β S, pH7.4) (method is same as above), the QD after being washed₆₅₅Quantum dot microsphere；Then 10 μ g albumen are added according to every milligram of quantum dot microsphere Proportion, QD after washing₆₅₅DNP-BSA, 4 DEG C of oscillation 18h are added in quantum dot microsphere；Confining liquid (the sweet ammonia containing 40mM is added 0.01M P the Β S, pH7.4 of acid, 1wt% Β SA) 25 DEG C of oscillation 60min, centrifugation abandons supernatant, then (contained with preservation liquid 0.01M P the Β S, pH7.4 of 0.5wt%casine, 1wt%P300) it washing 2 times (method is same as above) and is diluted to microballoon concentration and is 1mg/mL obtains QD₆₅₅The DNP-BSA (haptens) of quantum dot microsphere label, storage temperature are 4 DEG C；

3. by QD made from step (1)₆₅₅40 polyclonal antibody of A β, the QD of quantum dot microsphere label₅₆₅Quantum dot microsphere mark QD made from 42 polyclonal antibody of A β and step (2) of note₆₅₅The DNP-BSA (haptens) of quantum dot microsphere label is mixed in equal volume It closes, obtains mixture；Mixture is coated on glass fibre membrane, every milliliter of mixture coats 8 square centimeters of glass fibre Pad, 37 DEG C of dry 3.5h obtain the glass fibre membrane of coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β；

(3) nitrocellulose filter for being equipped with detection zone and quality control region is pasted in the middle part of bottom plate (quality control region is as end), The glass fibre membrane for being coated with quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β is close to nitrocellulose filter front end, sample Pad is placed in glass fibre membrane front end, and water absorption pad is placed in nitrocellulose filter end；Sample pad, coating quantum dot microsphere are coupled A β 40 And 42 polyclonal antibody of A β glass fibre membrane, between nitrocellulose filter and water absorption pad equipped with detection zone and quality control region mutually Overlapping obtains the quantum dot immune chromatograph test strip for quickly detecting beta-amyloid protein.

Embodiment 2

1. by 40 monoclonal antibody of A β, 42 monoclonal antibody of the A β 0.01M phosphoric acid for containing mass fraction being 1% trehalose Salt buffer (pH=7.5 uses 0.22 μm to be filtered using preceding) is diluted to concentration respectively as 1.0mg/mL, and DNP antibody is dilute Releasing to concentration is 0.75mg/mL, obtains 40 monoclonal antibody working solution of A β, 42 monoclonal antibody working solution of A β and DNP antibody work Make liquid；

2. by 40 monoclonal antibody working solution of A β made from step (1), 42 monoclonal antibody working solution of A β and DNP antibody work Make liquid and draw film on NC film respectively with film instrument is drawn, drawing film speed is 1 μ L/cm, wherein 40 monoclonal antibody of A β is drawn at 0.8cm Film, 42 monoclonal antibody of A β draw film at 1.3cm, and DNP antibody draws film at 1.8cm, form detection zone T1, detection zone T2 and matter Area C is controlled, then 37 DEG C of dry 3h, obtain the nitrocellulose filter equipped with detection zone and quality control region, which saves Condition is 4 DEG C；

By QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere is washed with activation buffer (0.05M MES) (with activation respectively After microballoon is resuspended in buffer, 10000 turns of centrifugation 30min abandon supernatant；Repeat aforesaid operations 2 times) and it is diluted to 1mg/mL, it obtains QD₆₅₅Quantum dot microsphere solution, QD₅₆₅Quantum dot microsphere solution；By 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride Salt (EDC) and QD₆₅₅Quantum dot microsphere solution, QD₅₆₅Quantum dot microsphere solution is mixed according to mass ratio 1:30 respectively, 25 DEG C of oscillations Supernatant is abandoned in 45min, centrifugation, then 2 times (method is same as above) is washed with coupling liquid (0.01M P Β S, pH7.2), after obtaining washing QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere；Then matching for 10 μ g antibody proteins is added according to every milligram of quantum dot microsphere Than QD after washing₆₅₅Quantum dot microsphere, QD₅₆₅40 polyclonal antibody of A β (label QD is separately added into quantum dot microsphere₆₅₅)、 42 polyclonal antibody of A β (label QD₅₆₅), 2 DEG C of oscillations are for 24 hours；Confining liquid (the 0.01M of glycine containing 40mM, 1wt% Β SA is added P Β S, pH7.2) 25 DEG C of oscillation 90min, centrifugation abandons supernatant, then with save liquid (contain 0.5wt%casine, 1wt%P300's 0.01M P Β S, pH7.2) it washs 2 times (method is same as above) and is diluted to microballoon concentration as 1mg/mL, respectively obtain QD₆₅₅Quantum dot 40 polyclonal antibody of A β, the QD of microballoon label₅₆₅42 polyclonal antibody of A β of quantum dot microsphere label, storage temperature are 2 DEG C；

②QD₆₅₅Quantum dot microsphere marks DNP-BSA (haptens)

By QD₆₅₅Quantum dot microsphere washs 2 times with activation buffer (0.05M MES) and (microballoon is resuspended with activation buffer Afterwards, 10000 turns of centrifugation 30min abandon supernatant；Repeat aforesaid operations 2 times) and it is diluted to 1mg/mL, obtain QD₆₅₅Quantum dot microsphere Solution；By 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and QD₆₅₅Quantum dot microsphere solution is according to matter Amount is mixed than 1:30,25 DEG C of oscillation 45min, and supernatant is abandoned in centrifugation, is then washed 2 times with coupling liquid (0.01M P Β S, pH7.2) (method is same as above), the QD after being washed₆₅₅Quantum dot microsphere；Then 10 μ g albumen are added according to every milligram of quantum dot microsphere Proportion, QD after washing₆₅₅DNP-BSA is added in quantum dot microsphere, 2 DEG C of oscillations are for 24 hours；Confining liquid (the sweet ammonia containing 40mM is added 0.01M P the Β S, pH7.2 of acid, 1wt% Β SA) 25 DEG C of oscillation 90min, centrifugation abandons supernatant, then (contained with preservation liquid 0.01M P the Β S, pH7.2 of 0.5wt%casine, 1wt%P300) it washing 2 times (method is same as above) and is diluted to microballoon concentration and is 1mg/mL obtains QD₆₅₅The DNP-BSA (haptens) of quantum dot microsphere label, storage temperature are 2 DEG C；

(3) sample pad is cut into length 30cm, width 28mm, coating quantum dot microsphere coupling A β 40 and 42 Anti-TNF-α of A β The glass fibre membrane of body is cut into length 30cm, width 10mm, and the nitrocellulose filter equipped with detection zone and quality control region is cut into length 30cm, width 25mm, water absorption pad cut into length 30cm, width 27mm, are then successively attached on lining card, wherein be equipped with Detection zone and the nitrocellulose filter of quality control region are pasted in the middle part of bottom plate (quality control region is as end), and coating quantum dot microsphere is even The glass fibre membrane for joining A β 40 and 42 polyclonal antibody of A β is close to nitrocellulose filter front end, before sample pad is placed in glass fibre membrane End, water absorption pad are placed in nitrocellulose filter end；Sample pad, coating quantum dot microsphere coupling A β 40 and A β 42 polyclonal antibody It is overlapped between glass fibre membrane, nitrocellulose filter and water absorption pad equipped with detection zone and quality control region, after forming big plate, cut The single person-portion test strips of length 8cm and width 4mm are cut into, test is fitted into and gets stuck, obtain quickly detecting beta-amyloid protein Quantum dot immune chromatograph test strip, by its equipped with desiccant hermetic bag in, deposit in room temperature.

Embodiment 3

1. by 40 monoclonal antibody of A β, 42 monoclonal antibody of the A β 0.01M phosphoric acid for containing mass fraction being 1% trehalose Salt buffer (pH=8.5 uses 0.22 μm to be filtered using preceding) is diluted to concentration respectively as 2.5mg/mL, and DNP antibody is dilute Releasing to concentration is 2.5mg/mL, obtains 40 monoclonal antibody working solution of β, 42 monoclonal antibody working solution of A β and the work of DNP antibody Liquid；

2. by 40 monoclonal antibody working solution of A β made from step (1), 42 monoclonal antibody working solution of A β and DNP antibody work Make liquid and draw film on NC film respectively with film instrument is drawn, drawing film speed is 1 μ L/cm, wherein 40 monoclonal antibody of A β is drawn at 0.8cm Film, 42 monoclonal antibody of A β draw film at 1.3cm, and DNP antibody draws film at 1.8cm, form detection zone T1, detection zone T2 and matter Area C is controlled, then 37 DEG C of dry 4h, obtain the nitrocellulose filter equipped with detection zone and quality control region, which saves Condition is 30 DEG C；

By QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere is washed with activation buffer (0.05M MES) (with activation respectively After microballoon is resuspended in buffer, 10000 turns of centrifugation 30min abandon supernatant；Repeat aforesaid operations 3 times) and it is diluted to 1mg/mL, it obtains QD₆₅₅Quantum dot microsphere solution, QD₅₆₅Quantum dot microsphere solution；By 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride Salt (EDC) and QD₆₅₅Quantum dot microsphere solution, QD₅₆₅Quantum dot microsphere solution is mixed according to mass ratio 1:30 respectively, 25 DEG C of oscillations Supernatant is abandoned in 60min, centrifugation, then 3 times (method is same as above) is washed with coupling liquid (0.01M P Β S, pH7.3), after obtaining washing QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere；Then matching for 10 μ g antibody proteins is added according to every milligram of quantum dot microsphere Than QD after washing₆₅₅Quantum dot microsphere, QD₅₆₅40 polyclonal antibody of A β (label QD is separately added into quantum dot microsphere₆₅₅)、 42 polyclonal antibody of A β (label QD₅₆₅), 8 DEG C of oscillation 20h；Confining liquid (the 0.01M of glycine containing 40mM, 1wt% Β SA is added P Β S, pH7.3) 25 DEG C of oscillation 120min, centrifugation abandons supernatant, then (contains 0.5wt%casine, 1wt%P300 with saving liquid 0.01M P Β S, pH7.3) washing 3 times (method is same as above) and be diluted to microballoon concentration be 1mg/mL, respectively obtain QD₆₅₅Quantum 40 polyclonal antibody of A β, the QD of point microballoon label₅₆₅42 polyclonal antibody of A β of quantum dot microsphere label, storage temperature are 8 DEG C；

②QD₆₅₅Quantum dot microsphere marks DNP-BSA (haptens)

By QD₆₅₅Quantum dot microsphere washs 2 times with activation buffer (0.05M MES) and (microballoon is resuspended with activation buffer Afterwards, 10000 turns of centrifugation 30min abandon supernatant；Repeat aforesaid operations 2 times) and it is diluted to 1mg/mL, obtain QD₆₅₅Quantum dot microsphere Solution；By 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and QD₆₅₅Quantum dot microsphere solution is according to matter Amount is mixed than 1:30,25 DEG C of oscillation 60min, and supernatant is abandoned in centrifugation, is then washed 2 times with coupling liquid (0.01M P Β S, pH7.3) (method is same as above), the QD after being washed₆₅₅Quantum dot microsphere；Then 10 μ g albumen are added according to every milligram of quantum dot microsphere Proportion, QD after washing₆₅₅DNP-BSA, 8 DEG C of oscillation 20h are added in quantum dot microsphere；Confining liquid (the sweet ammonia containing 40mM is added 0.01M P the Β S, pH7.3 of acid, 1wt% Β SA) 25 DEG C of oscillation 60min, centrifugation abandons supernatant, then (contained with preservation liquid 0.01M P the Β S, pH7.3 of 0.5wt%casine, 1wt%P300) it washing 3 times (method is same as above) and is diluted to microballoon concentration and is 1mg/mL obtains QD₆₅₅The DNP-BSA (haptens) of quantum dot microsphere label, storage temperature are 8 DEG C；

Effect example

(1) 75 μ L of sample to be tested (sample respectively the dilution containing A β 40,42 antigen of A β) is taken to be added made from embodiment 1 Sample pad in test paper, 25 DEG C of room temperature are placed 8 minutes, and sample is reacted by immunochromatography, measure quantum with quantum dot analyzer The fluorescence of point.

(2) dose-response relationship: be measured in parallel A β 40,42 calibration object of A β, with the β of A containing 2000pg/mL 40 be diluted for Calibration object, calibration object concentration (0,1.0,10,50,200,1000pg/mL), is diluted with the β of A containing 2000pg/mL 42, calibration Product concentration (0,0.5,5,50,100,500pg/mL), is fitted curve tracing with log (X)-log (Y) mathematical model.It calculates Its linearly dependent coefficient, 40 related coefficient of A β is 0.995,42 related coefficient of A β is 0.997.

(3) sensitivity for analysis: being measured in parallel the quantum dot fluorescence intensity of 10 hole zero calibration objects, and it is strong to calculate quantum dot fluorescence The standard deviation (SD) and average value (Mean) of degree calculate M=Mean+2 × SD, and calculated result M is substituted into formula log (M/ Mean/ (1-M/Mean)), wherein Mean is S₀The average value of luminous value, then by the calculating of log (M/Mean/ (1-M/Mean)) As a result substitute into linear formula in Y value, by the value substitute into linear formula calculate X value, then calculate Power i.e. (Mean+2SD) when pair The concentration value answered is sensitivity for analysis.40 sensitivity for analysis of A β of the test paper is 0.033pg/mL, and 42 sensitivity for analysis of A β is 0.048pg/mL。

(4) 10 hole A β 40 sample (2.5pg/mL, 50pg/mL) Quality Control sample, 42 sample of A β accuracy: are measured in parallel (2.5pg/mL, 50pg/mL) calculates the mean concentration (Mean) and standard deviation (SD) of measurement result, calculates A β 40,42 essence of A β Density (CV%)=SD/Mean × 100%.40 coefficient of variation of A β is respectively 6.34%, 6.00%；42 coefficient of variation of A β is respectively 5.05%, 4.21%.

5) accuracy rate: measuring A β 40, A β 42 definite value calibration object, calculates the measurement concentration of calibration object and the phase of mark concentration To deviation.The mean relative deviation of A β 40, the sign value of 42 calibration object of A β and concentration value are respectively less than 5%, and linear regression is related Coefficient (r) >=0.99.

6) sample measures: measurement is compared to 83 parts of AD clinical samples, and is compared with enzyme-linked immunization measurement.It surveys Fixed 83 AD samples, are compared, A amyloid beta (40) coincidence rate 95.3%, wherein Clinical Sensitivity with enzyme-linked immunization 96.4%, clinical specificity 94.2%；Amyloid beta (42) coincidence rate 94.7%, wherein Clinical Sensitivity 93.3%, clinical Specificity 96.1%.Linearly dependent coefficient r is respectively 0.987,0.958.

In addition, compared with the DNP-BSA (haptens) of QD655 quantum dot microsphere label, using QD565 quantum dot microsphere mark Test strips made from the DNP-BSA (haptens) of note are in chromatography process, because there is quantum dot interval, actual observation is easier.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Claims

1. a kind of quantum dot immune chromatograph test strip of quickly detection beta-amyloid protein, it is characterised in that comprising being equipped with detection zone The glass fibre membrane of A β 40 and 42 polyclonal antibody of A β are coupled with nitrocellulose filter, the coating quantum dot microsphere of quality control region；

It is anti-that the nitrocellulose filter equipped with detection zone and quality control region is successively arranged 40 monoclonal antibody of A β, 42 monoclonal of A β Body and DNP antibody form detection zone T1, detection zone T2 and quality control region C.

2. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein according to claim 1, feature exist In:

Spacing between the detection zone T1 and detection zone T2 is 0.5cm；

Spacing between the detection zone T2 and quality control region C is 0.5cm.

3. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein according to claim 1, feature exist In:

(1) 40 monoclonal antibody of A β, 42 monoclonal antibody of A β are diluted to respectively with the phosphate buffer containing trehalose dense Degree is 1.0~2.5mg/mL, and it is 0.75~2.5mg/mL that DNP antibody, which is diluted to concentration, obtains 40 monoclonal antibody working solution of β, A 42 monoclonal antibody working solution of β and DNP antibody working solution；

(2) by 40 monoclonal antibody working solution of β made from step (1), 42 monoclonal antibody working solution of A β and DNP antibody working solution Film is drawn on nitrocellulose filter respectively, forms detection zone T1, detection zone T2 and quality control region C, it is dry, obtain being equipped with detection zone and The nitrocellulose filter of quality control region.

4. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein according to claim 3, feature exist In:

Phosphate buffer containing trehalose described in step (1) is to contain the 0.01M phosphorus that mass fraction is 1% trehalose Phthalate buffer, pH7.5~8.5.

5. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein according to claim 1, feature exist In:

The preparation method of the glass fibre membrane of the coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β, comprising such as Lower step:

By QD₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere is washed and is diluted with activation buffer respectively, obtains QD₆₅₅Quantum dot Microspheres solution, QD₅₆₅Quantum dot microsphere solution；By 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and QD₆₅₅Amount Son point microspheres solution, QD₅₆₅Quantum dot microsphere solution mixes respectively, 25 DEG C of ± 0.1 DEG C of 30~60min of oscillation, and supernatant is abandoned in centrifugation, Then it is washed with coupling liquid, the QD after being washed₆₅₅Quantum dot microsphere, QD₅₆₅Quantum dot microsphere；Then according to every milligram of quantum The proportion of 10 μ g antibody proteins, QD after washing is added in point microballoon₆₅₅Quantum dot microsphere, QD₅₆₅Add respectively in quantum dot microsphere Enter 40 polyclonal antibody of A β, 42 polyclonal antibody of A β, 2~8 DEG C of oscillations 18~for 24 hours；25 DEG C of ± 0.1 DEG C of oscillations 60 of confining liquid are added Supernatant is abandoned in~120min, centrifugation, is then washed and is diluted with preservation liquid, respectively obtains QD₆₅₅The A β 40 of quantum dot microsphere label Polyclonal antibody, QD₅₆₅42 polyclonal antibody of A β of quantum dot microsphere label；

(2)QD₆₅₅Quantum dot microsphere or QD₅₆₅Quantum dot microsphere marks DNP-BSA

By QD₆₅₅Quantum dot microsphere or QD₅₆₅Quantum dot microsphere is washed and is diluted with activation buffer, obtains QD₆₅₅Quantum dot microsphere Solution or QD₅₆₅Quantum dot microsphere solution；By 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and QD₆₅₅Quantum Point microspheres solution or QD₅₆₅The mixing of quantum dot microsphere solution, 25 DEG C of ± 0.1 DEG C of 30~60min of oscillation, centrifugation abandon supernatant, then It is washed with coupling liquid, the QD after being washed₆₅₅Quantum dot microsphere or QD₅₆₅Quantum dot microsphere；Then according to every milligram of quantum dot The proportion of 10 μ g albumen, QD after washing is added in microballoon₆₅₅Quantum dot microsphere or QD₅₆₅DNP-BSA is added in quantum dot microsphere, 2~8 DEG C of oscillations 18~for 24 hours；25 DEG C of ± 0.1 DEG C of 60~120min of oscillation of confining liquid are added, supernatant is abandoned in centrifugation, then with preservation Liquid is washed and is diluted, and obtains QD₆₅₅The DNP-BSA or QD of quantum dot microsphere label₅₆₅The DNP-BSA of quantum dot microsphere label；

(3) by QD made from step (1)₆₅₅40 polyclonal antibody of A β, the QD of quantum dot microsphere label₅₆₅Quantum dot microsphere label QD made from 42 polyclonal antibody of A β and step (2)₆₅₅The DNP-BSA or QD of quantum dot microsphere label₅₆₅Quantum dot microsphere label DNP-BSA mix in equal volume, obtain mixture；Mixture is coated on glass fibre membrane, it is dry, obtain coating quantum dot The glass fibre membrane of microballoon coupling A β 40 and 42 polyclonal antibody of A β.

6. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein according to claim 5, feature exist In:

QD described in step (1)₆₅₅40 polyclonal antibody of A β or QD of quantum dot microsphere label₅₆₅The A β of quantum dot microsphere label The concentration of quantum dot microsphere is 1mg/mL in 42 polyclonal antibodies；

QD described in step (2)₆₅₅The DNP-BSA or QD of quantum dot microsphere label₅₆₅In the DNP-BSA of quantum dot microsphere label The concentration of quantum dot microsphere is 1mg/mL.

7. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein according to claim 5, feature exist In:

QD described in step (3)₆₅₅40 polyclonal antibody of A β, the QD of quantum dot microsphere label₅₆₅The A β of quantum dot microsphere label 42 polyclonal antibodies and QD₆₅₅The DNP-BSA of quantum dot microsphere label is mixed in equal volume.

8. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein according to claim 1, feature exist In also comprising bottom plate, sample pad and water absorption pad.

9. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein according to claim 8, feature exist In:

The glass fibre membrane of the sample pad, coating quantum dot microsphere coupling A β 40 and 42 polyclonal antibody of A β is equipped with detection The nitrocellulose filter and water absorption pad of area and quality control region are successively pasted on bottom plate.

10. the quantum dot immune chromatograph test strip of quick detection beta-amyloid protein described in claim 1~9 is in detection β-shallow lake Application in powder sample albumen.