CN105938145A

CN105938145A - Biomacromolecule capable of being combined with beta-amyloid protein, encoding DNA, beta-amyloid protein detection kit and application of biomacromolecule

Info

Publication number: CN105938145A
Application number: CN201610120602.XA
Authority: CN
Inventors: 陶新博; 张守涛; 郭亚楠
Original assignee: Zhejiang Jukang Bioengineering Co Ltd
Current assignee: Zhejiang Jukang Bioengineering Co Ltd
Priority date: 2016-03-03
Filing date: 2016-03-03
Publication date: 2016-09-14
Anticipated expiration: 2036-03-03
Also published as: CN109655620A; CN105938145B; CN109655620B

Abstract

The invention relates to a biomacromolecule capable of being combined with a beta-amyloid protein, DNA encoding the macromolecule, a beta-amyloid protein detection kit and an application of the biomacromolecule. The beta-amyloid protein detection kit is prepared by the steps: firstly, immunizing a mouse by use of an antigen 43 peptide beta-amyloid protein, separating out mouse spleen and lymphonodus after confirming that immunization is successful, extracting total RNAs, performing reverse transcription on the total RNAs to obtain cDNAs, and amplifying to obtain light and heavy chain genes of an antibody by virtue of PCR; secondly, assembling and amplifying scFvDNA, and constructing a pCANTAB5E-scFv recombinant plasmid; thirdly, converting the plasmid to obtain a single-chain antibody phage library, and screening to obtain a target antibody; and finally, assembling to form the human beta-amyloid protein detection kit. The detection range of the screened antibody is relatively wide from 0 to 1000 pg/ml, the linear correlation coefficient of the kit is high, the detection sensitivity of the kit can reach 3.0 pg/ml, and the kit achieves high stability.

Description

The biomacromolecule being combined with amyloid beta and coding DNA, beta-amyloyd egg White detection kit and application thereof

Technical field

The present invention relates to technical field of molecular biology, particularly relate to the biomacromolecule being combined with amyloid beta And encode the DNA of this macromole, amyloid beta detection kit and application thereof.

Background technology

Aged tendency of population is the Social Events that world faces, and has caused the extensive pass of international community Note.By the end of the year 2014, more than 60 years old aging population of China have reached 2.12 hundred million, have accounted for the 15.5% of total population.Anticipated 2033 Front and back will be doubled to 400,000,000, to about the year two thousand fifty, aging population are up to 1/3rd of whole nation population, and " silver hair tide " will be to me The economy of state, society, politics, cultural development produce far-reaching influence.Psychology and the physical health issues of old people are increasingly becoming Whole society's focus of attention.Senile dementia is a kind of performance of old people's brain functional disorder, be with intellectual deterioration, behavior and Personality is changed to feature.Typical clinical symptom includes memory, abstract thought, disorientation etc., lives with society simultaneously Kinetic force goes down.Mainly show as forgetful, One's eyesight is restrained, ability to speak is blunt, then affect sensory nerve, lose self-care ability.It Become a big jinx of senior health and fitness.

Alzheimer disease (Alzheimer ' s disease, AD) it is senile dementia one common form.The most full generation Boundary about 25,000,000 AD patient.Patient mostly is old people, and AD there is no effective therapy at present.This sufferer not only patient is from general pain Hardship, and all can cause extremely negative effect for its family or even entire society.Up to now, the exact mechanism of AD morbidity Not yet it is fully apparent from.But numerous studies disclose: amyloid precursor protein (Amyloid precursor protein, APP) produces Amyloid (amyloid 1-Peptide, A1) on the pathology of AD, played important function.β starch in brain in patients There is abnormal accumulation in sample albumen, often formed in patient's brain cell brain fibrous tissue be entangled with (neurofibrillary tangle, NFT), senile plaque (senile plague, SP) is formed outside brain cell.Therefore, NFT and SP is recognized as the pathological diagnosis of AD and depends on According to.Amyloid beta (β-Amyloid) is to be formed 40 to 43 by precursor substance amyloid beta-protein precursor (A β) through zymolysis Individual amino acid whose polypeptide.In body, APP is prevalent in the organs such as the heart of human body, brain, kidney, lung, intestinal.In brain, maincenter Nervous system neuronal, spider cell, microglia, oligodendrocyte, endotheliocyte all can express APP.Normal condition Under, amyloid beta only has very small amount and expresses, and the amyloid beta of low concentration is nutritious to neuron undifferentiated, jejune Effect, the amyloid beta of high concentration is to the differentiated the most toxic effect of mature neuron.The precipitation of amyloid beta Overexpression and abnormal processing with APP are relevant, APP through β, g secretase degrade, produce amyloid beta generate solubility and Insolubility two states, insoluble based on β-pleated sheet, above-mentioned conformation is conducive to the gathering of amyloid beta.Beta-amyloyd egg Neurotoxicity can be caused after forming high density, fibrous polymer in vain, thus hinder normal growth and the biography of neurocyte Lead.

At present, the clinical treatment for senile dementia, first it is considered that symptomatic treatment, e.g., ganglioside can have Effect repairs impaired or dying cranial nerve cell, promotes the reparative regeneration of nerve and reinventing of neutral net；Supplement choline medicine Thing can improve its memory and ability of thinking.

Summary of the invention

It is an object of the invention to provide a kind of highly stable biomacromolecule being combined with amyloid beta, should Biomacromolecule comprises SEQ ID NO.2 aminoacid sequence.

The purposes also providing for above-mentioned biomacromolecule of the present invention, i.e. this biomacromolecule are for qualitative or/and quantitatively examine Surveying the purposes of people's amyloid beta content in the body fluid of people, this biomacromolecule comprises SEQ ID NO.2 aminoacid sequence.

Also provide for the encoding DNA of above-mentioned biomacromolecule, this DNA of the present invention contain such as the sequence of SEQ ID NO.1.

The present invention also provides for amyloid beta detection kit, comprising:

-solid phase, above-mentioned solid phase secures seizure antibody, and above-mentioned seizure antibody includes that anti-human amyloid beta strand resists Body,

-cause the solution of degeneration, and

-liquid phase containing detection antibody, above-mentioned detection antibody includes anti-human amyloid beta polyclonal antibody.Above-mentioned anti- People's amyloid beta single-chain antibody or anti-human amyloid beta polyclonal antibody have such as the sequence of SEQ ID NO.2.

The further Optimized Measures taked includes:

Above-mentioned detection antibody can sandwich detection amyloid beta with above-mentioned seizure antibody.

Above-mentioned anti-human amyloid beta polyclonal antibody is through biotin labeling；Above-mentioned liquid phase also includes for quantitative test The standard substance of reference.

Above-mentioned liquid phase also includes developer；Above-mentioned developer is tetramethyl biphenyl diamidogen；Above-mentioned cause degeneration solution be Reaction terminating liquid；Above-mentioned reaction terminating liquid is HCl.

Above-mentioned liquid phase also includes cleaning mixture；Above-mentioned cleaning mixture be pH value be the phosphate buffer containing polysorbas20 of 7.2；

Above-mentioned liquid phase also includes concentrating enzyme conjugates, and above-mentioned concentration enzyme conjugates includes but not limited to horseradish peroxidase-labeled Avidin.

The invention has the advantages that:

1, the antibody of present invention screening is highly stable, uses the reaction pattern of double-antibody method during detection, and the method has operation Easy, react advantage efficiently, the highest to laboratory condition and equipment requirements, it is easy to grasp and promote；

2, the test kit detection range of the present invention is relatively wide, and its detection range is 0 ~ 1000pg/ml, the linear correlation coefficient of this test kit Higher；

3, the test kit sensitivity of the present invention is higher, and its detection sensitivity reaches as high as 3.0pg/ml, can be used for senile dementia Early prevention.

Accompanying drawing explanation

Fig. 1 is the plasmid vector collection of illustrative plates of the present invention；

Fig. 2 is that after the present invention uses amyloid beta immune mouse, blood drawing ELISA method detects titer；

Fig. 3 is PCR sample agarose gel electrophoresis figure of the present invention.

Detailed description of the invention

Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.

Embodiment: the technology path of the present invention is, first, uses antigen 43 peptide amyloid beta immune mouse, determines and exempt from After epidemic disease success, isolating mouse spleen and lymph node, extract total RNA, reverse transcription becomes cDNA, amplifies antibody by PCR Gently, heavy chain gene.Then assemble and expand scFv DNA, build pCANTAB5E-scFv recombiant plasmid.Convert plasmid again to obtain Single chain antibody phage storehouse, obtains target antibody by screening.Finally assemble adult's amyloid beta detection kit.

Built by display technique of bacteriophage and screen single-chain antibody

Cell and cell strain use:E.coliTG1, for the conversion of pCANTAB-5E；E.coliHB2151, for scFv's Express.

Trizol total serum IgE extraction agent box, M-MLV reverse transcription, RNase inhibitor are purchased from Invitrogen company.Taq Archaeal dna polymerase, T4 DNA ligase and restriction endonuclease are purchased from NEB company.DNA glue reclaims test kit purchased from Tian Gen company. M13KO7 helper phage is purchased from GE company.The primer sequence of gene amplification such as following table:

Experimental technique

Animal immune:

After the Freund's complete adjuvant mixing of amyloid beta antigen and equivalent, use abdominal part and dorsal sc multi-point injection mode Immunity Balb/C mice.Every immunity in 14 days once, after 4 immunity, blood drawing ELISA method detects titer, method particularly includes:

1), after being diluted by a certain percentage by serum with phosphate buffer (dilution ratio is shown in figure), every hole 50 l is added in and is coated plate On, stand overnight in 4 DEG C；

2) with 200 l containing bovine serum albumin be 2%(W/V), sucrose be 3%(W/V) phosphate buffer confining liquid close bag By plate；

3) being simultaneously introduced biotinylated anti-human amyloid beta (A β) antibody and standard substance, sample, every hole is 50 l；Sample In people's amyloid beta can be with the biotinylated anti-human amyloid beta antibody being incorporated in hole and be coated in ELISA Plate On many anti-bindings, formed immune complex；

4) being coated plate with phosphate buffer washing, free composition is washed away；

5) adding the affinity element of horseradish peroxidase-labeled, affinity element is specific binding with biotin, and free composition is washed Go；

6) having people's amyloid beta in reacting hole, horseradish peroxidase can make colourless developer become indigo plant, adds stop buffer and becomes Yellow.OD value is surveyed at 490nm.

Isolated and purified lymphocyte:

Take out mouse spleen and lymph node.Wash with PBS and remove superabundant fats tissue, spleen and lymph node are placed in 20 mesh not In rust steel mesh film, it is lightly ground tissue, makes lymphocyte pass through nethike embrane and flow in plate.Rustless steel nethike embrane is rinsed gently with PBS After plate, after piping and druming uniformly, cell suspension is placed in 15ml centrifuge tube gently, centrifugal collecting cell.

Extraction lymphocyte total serum IgE:

Lymphocyte total serum IgE is extracted according to the Trizol total serum IgE extraction agent box description of Invitrogen company.

Reverse transcription synthesis cDNA:

Response procedures is: 94 DEG C of 5min, and 42 DEG C of 60min, product is cDNA.

Reverse transcription system is as follows:

2.5 amplifications are light, heavy chain gene

Gently, heavy chain gene amplification system is as follows:

Amplification program is: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 circulations, 72 DEG C extend 10min.Running gel recovery purification is light, heavy chain gene segment.The amplification of chain variable region gene fragment: with cDNA as mould Plate, carries out PCR amplification with the upstream and downstream primer V λ back and MJ λ for-Mix of VL.By VL downstream primer MJ λ 1for, MJ λ 2for, MJ λ 3for and MJ λ 4for is mixed into MJ λ for-Mix.The amplification of heavy chain variable region gene fragment: with cDNA as template, PCR amplification is carried out with the upstream and downstream primer VH back and VH for of VH.

The assembling of 2.6 scFv DNA and amplification

Linker primer and light, heavy chain gene amplified fragments, by Overlapping PCR PCR(SOE-PCR), make antibody V_H、V_L Gene and Linker are assembled into V_H-Linker-V_LSingle-chain antibody.Then by scFv single-chain antibody primer amplification scFv DNA.Wherein forward primer is SfiI back, and reverse primer is J λ 1-NotI, J λ 2-NotI, J λ 3-NotI and J λ 4-NotI Mixture.

Overlapping PCR PCR amplification condition is: 94 DEG C of 1min, 63 DEG C of 4min, acts on 7 circulations；Add scFv mono- Expanding scFv DNA after chain antibody primer again, program is 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, acts on 30 circulations, Last 72 DEG C extend 10min.

Overlapping PCR PCR reaction system is:

The structure of single-chain antibody library and qualification

Single-chain antibody scFv fragment and phagemid vector pCANTAB-5E use S respectivelyfiI and NotI carries out enzyme action, connect afterwards, Electricity is transformed into E.coliTG1 competent cell.The cell converted adds 37 DEG C of shaken cultivation of culture medium, when OD600 is 0.8, Add M13KO7 helper phage, continue to cultivate to final concentration 10¹⁰pfu/ml.Plus ampicillin to final concentration 100 g/ml, Continue overnight incubation, i.e. can get single chain antibody phage Antibodies Primary storehouse.Optimal single-chain antibody is screened by ELISA, And it is purified into plasmid, proceed toE.coliHB2151 is for the expression of scFv.

Experimental result

ELISA method detection serum titer

After using amyloid beta immune mouse, blood drawing ELISA method detects titer, and result is shown in that Fig. 2, result show dilution When multiple increases to 1:50000, the antibody in serum still can substantially detect, shows that immune mouse is successful.

Heavy chain, light chain gene amplification

Taking 7 l PCR sample agarose gel electrophoresiies, agarose gel concentration is 1.3%, 6V/cm electrophoresis 20min.Result such as Fig. 3 Shown in, V_HAnd V_LIt is respectively positioned between 250bp and 500bp.

V_HAnd V_LScFv gene after connection

Taking 7 l PCR sample agarose gel electrophoresiies, agarose gel concentration is 1.2%, 6V/cm electrophoresis 20min.Result such as Fig. 3 Shown in, V_HAnd V_LAfter splicing, the ScFv gene obtained is about 750bp, its sequence as shown in SEQ ID NO.1, coded amino Acid sequence is as shown in SEQ ID NO.2, specific as follows:

ScFv gene, SEQ ID NO.1:

ATGGCCCAGGTCAAACTGCAGGAGTCAGGCGGCGACCTGAAGAAGCCCGCCGCCACCGTGAGAATCAGCTGCA AGGCCACCGCCTACAGCTGGAGCACCTACGGCATGCACTTCGTGAAGAACGCCCCCGGCCAGCACCTGGAGTTCATG GGCTTCATCAACGCCGGCCAGGGCGCCAGCCACTACAGCCAGAGATTCAACGGCAGAGTGAGCATCACCAAGGAGAG CACCGCCAGCACCGGCTACATGGACGTGACCACCATCAGAAGCGACGAGACCGCCGTGTTCTACTGCGCCCACAGCA GAAAGAAGAACTTCGGCCAGGGCAGCCTGGTGACCGTGAGCAGAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCAGCGACATCACCAACGACCCCGGCGTGACCCTGGCCCTGGGCAACACCGTGAGACTGACCTGCCA GGGCGAGAGCATCAAGAGCTACTACGGCAGCTGGTACAACAACAAGCCCGCCCAGGCCCCCCTGGTGCTGATCTACG GCAAGCAGAACAAGCCCAGCGGCCTGCCCGACAGAAAGTTCAGCGGCACCACCAGCGGCCAGACCGGCAGCGTGACC ATCACCGCCGCCCAGGCCGACGAGGAGGCCGAGTTCTACTGCAACACCAGAGAGACCAGCGCCAACCACCTGGTGTT CGGCGCCGCCACCAGAATCAGCCTGGTGGGCGCCGGCGCCGAGCAGGGCACCAAGCTGGAAATCAAACGGGCGGCCG CAGAATGACTC

The aminoacid sequence of ScFv gene, SEQ ID NO.2:

MAQVKLQESGGDLKKPAATVRISCKATAYSWSTYGMHFVKNAPGQHLEFMGFINAGQGASHYSQRFNGRVSIT KESTASTGYMDVTTIRSDETAVFYCAHSRKKNFGQGSLVTVSRGGGGSGGGGSGGGGSSDITNDPGVTLALGNTVRL TCQGESIKSYYGSWYNNKPAQAPLVLIYGKQNKPSGLPDRKFSGTTSGQTGSVTITAAQADEEAEFYCNTRETSANH LVFGAATRISLVGAGAEQGTKLEIKRAAAE*

SEQ ID NO.1 with NO.2 is corresponding as follows:

The qualification of single-chain antibody

After screening obtains the single-chain antibody of sequence described in step 3.3, it is purified into plasmid, proceeds toE.coliHB2151 is used for scFv Expression, gained single-chain antibody molecules amount is about 25KD.

Assemble people's amyloid beta-protein precursor test kit

After ELISA filters out suitable single-chain antibody, assemble adult's beta-amyloid kit.This test kit includes that antibody is coated Lath, standard substance, concentrated biological element antibody (single-chain antibody labelling biotin), concentration enzyme conjugates, reagent dilutions, concentration Cleaning mixture, developer and stop buffer.Particular make-up is as shown in table 1.

The composition of table 1 the present inventor's beta-amyloid kit

Title	96 Tests	48 Tests	Preservation condition
				Antibody coating plate bar	8×12	8×6	4℃
Standard substance	2 (lyophilizing)	1 (lyophilizing)	4℃
				Concentrated biological element antibody	2	1	4℃
Concentrate enzyme conjugates	2	1	4 DEG C (lucifuge)
				Reagent dilutions (25ml/ bottle)	2 bottles	1 bottle	4℃
Concentrated cleaning solution 20 × (30ml/ bottle)	1 bottle	1 bottle	4℃
				Developer	1 bottle (12ml)	1 bottle (6ml)	4 DEG C (lucifuge)
Stop buffer	1 bottle	1 bottle	4℃
				Shrouding gummed paper	3	2
Description	1 part	1 part

The preparation of antibody coating plate bar comprises the following steps: the Mus present invention prepared with the phosphate buffer that pH value is 7.2 Anti-human amyloid beta single-chain antibody is diluted to 1 g/ml, is added in and is coated on plate, with shrouding paper shrouding, places 24h mistake at 4 DEG C After night, plate will be coated and be placed in and wash on trigger, be 0.1%(V/V with the polysorbas20 content that pH value is 7.2) phosphate buffer wash Plate once, is subsequently adding pH value and is 7.2, is 2%(W/V containing bovine serum albumin), sucrose be 3%(W/V) phosphate buffer, At room temperature place 2h, dry confining liquid, air-dry.The plate that is coated air-dried loads in aluminium foil bag, adds desiccant, seal i.e. obtain anti- Body coated slab.

The preparation of standard substance comprises the following steps: with pH value for 7.2, is 5%(W/V containing bovine serum albumin) phosphate Buffer is solvent, and people's amyloid beta recombiant protein standard substance are configured to the solution that 2000pg/50 l/ props up, by this solution It is placed in lyophilizing 2h in-70 DEG C of low temperature environments, is then placed on lyophilizing 8h in freezer dryer, seal and i.e. obtain standard substance.

Concentrated biological element antibody is biotin labeled anti-human amyloid beta single-chain antibody, specifically uses Thermo Fisher:Sulfo-NHS-LC-Biotin Kit carrys out the anti-human amyloid beta single-chain antibody of the labelling present invention.Concentration enzyme is tied Compound is the streptavidin of horseradish peroxidase-labeled.Reagent dilutions be pH value be 7.2, bovine serum albumin content be 1% (W/V) phosphate buffer.Concentrated cleaning solution be pH value be 7.2, polysorbas20 content be 0.1%(V/V) phosphate-buffered Liquid.Developer is tetramethyl biphenyl diamidogen.Stop buffer is 1N HCL(1N hydrochloric acid).

The test kit utilizing the present invention carries out people's amyloid beta detection, uses double antibody sandwich ELISA.Anti-human β forms sediment Powder sample albumen single-chain antibody is coated in ELISA Plate, is simultaneously introduced specimen, standard substance and biotinylated anti-human amyloid beta Single-chain antibody, the people's amyloid beta in specimen, standard substance can be with the biotinylated anti-human beta-amyloyd egg being incorporated in hole Bai Kangti and the monoclonal antibody being coated in ELISA Plate combine, and form immune complex, and free composition is washed away.Add Radix Cochleariae officinalis peroxide The affinity element of compound enzyme labelling, affinity element is specific binding with biotin, and free composition is washed away.Add chromogenic substrate (aobvious Toner), if there being people's amyloid beta in reacting hole, horseradish peroxidase can make colourless developer become indigo plant, adds stop buffer and becomes Yellow.OD value, people's amyloid beta concentration and OD is surveyed at 490nm₄₉₀It is proportionate between value, can ask by drawing standard curve Go out people's amyloid beta concentration in specimen.Specifically include following steps:

Test equipment is provided for oneself needed for test:

1. microplate reader (490nm detects wavelength filter, 570nm or 630nm tuning wavelength optical filter)；

2. high-precision liquid adding device and disposable tip: 0.5-10 l, 2-20 l, 20-200 l, 200-1000 l；

3. microwell plate agitator, distilled water or deionized water, graph paper.

Specimen collection:

1. the test tube collecting blood should be disposable apyrogeneity, without endotoxin test tube；

2. plasma anticoagulant agent recommends EDTA, it is to avoid use haemolysis, hyperlipidemia specimen；

3. specimen should be as clear as crystal, and float answers centrifugal segregation；

If 4. detecting not in time after specimen collection, need to be frozen in-20 DEG C or-70 DEG C of refrigerators by first use amount subpackage, keep away Exempt from multigelation；

5. can do suitable multiple dilution according to the practical situation of specimen；

Note: the albumen of serum or the frozen post polymerization of plasma sample can hide the epi-position of antigen, it is proposed that with reagent dilutions by serum Or plasma sample does detection after 1:2 dilution.Extension rate should be multiplied by when calculating sample content.

Points for attention:

1. test kit please be saved in 2-8 DEG C before using, and except the standard substance after redissolving, other composition can not freeze；

2. concentrated biological element antibody, concentrate enzyme conjugates volume little, transport moderately gusty air and possible inversion liquid can be made to be stained with Tube wall or bottle cap.Therefore please get rid of several times with hands before using or 1000rpm is centrifuged 1 minute, so that attachment tube wall or the liquid of bottle cap Deposit at the bottom of pipe；

3. the concentrated cleaning solution taken out from refrigerator may have crystallization, belongs to normal phenomenon, micro-be heated to 40 DEG C and make crystallization complete Cleaning mixture is prepared again after dissolving；

Use standard substance the most by several times ,-20 or-70 should be placed it in by consumption subpackage each time after standard substance redissolve DEG C storage.Avoid multigelation；

5. the test kit component of different lot numbers can not use (except cleaning mixture and reaction terminating liquid) with；

6. the slightest mixing is particularly important to reaction result, it is preferred to use micro oscillator (use low-limit frequency), as without micro- Amount agitator, can rock ELISA Plate the most gently, makes the reactant liquor mixing adding in hand-hole；

7. when enzyme is excused from an examination to test Plays product and pattern detection, multiple hole is made in suggestion.

Preparation before detection:

1. within 20 minutes in advance, from refrigerator, take out test kit, with balance to room temperature；

2. concentrated cleaning solution distilled water is diluted (1:20), unspent put back to 4 DEG C of refrigerators；

3. standard substance: in addition reagent dilutions 1.0ml to lyophilizing standard substance, stand 15 minutes, after it fully dissolves, gently Light mixing (concentration is 2000pg/ml).It is diluted the most as required.Recommended standard curve uses following concentration: 1000, 500、250、125、62.5、31.25、15.625、0 pg/ml；

4. biotinylated antibody working solution: by when time test institute expense, dilute concentrated biological element antibody with reagent dilutions (1:100) biotinylated antibody working solution it is configured to.Use preparation in first 30 minutes.It is intended for using the same day；

5. enzyme conjugates working solution: by when time required consumption of test, with reagent dilutions dilution concentrate enzyme conjugates (1: 100) enzyme conjugates working solution it is configured to.Use preparation in first 30 minutes.It is intended for using the same day.

Washing methods:

1. automatic washer: require that the cleaning mixture injected is 350 l, injects and the sucking-off interval 20-30 second.Wash plate 4 times；

2. manual plate of washing: every hole adds cleaning mixture 350 l, gets rid of liquid in most hole, pat dry in thickness repeatedly absorbent paper after standing 30 seconds. Wash plate 5 times.

Operating procedure:

1., from balancing to the sealing bag of room temperature the required lath of taking-up test, unused lath and desiccant please put back to aluminium foil 4 DEG C it are sealed in Dai；

2. blank hole (if using dual wavelength to read plate, blank well can not set)；

Get out sample, standard substance and biotinylated antibody working solution the most in advance；

The most first variable concentrations standard substance (0mIU/ml hole reagent adding diluent) are separately added in respective aperture (100 l/ hole)；? (now sample has been 1:2 times and has diluted, result of calculation up-to-date style to add 50 l Sample dilution and 50 l samples in each sample aperture This content to be multiplied by 2)；In sample and standard sample wells, add 50 l biotinylated antibody working solutions subsequently, seal with shrouding gummed paper Live reacting hole；

5. room temperature (20 ~ 25 DEG C) hatches 120 minutes.It is preferably used micro oscillator (low-limit frequency, 100rpm)；

Within 15 minutes the most in advance, prepare enzyme conjugates working solution.Room temperature lucifuge is placed；

7. wash plate 5 times；

8., in addition to blank well, add enzyme conjugates working solution (100 l/ hole).Reacting hole is sealed with shrouding gummed paper；

9. room temperature (20 ~ 25 DEG C) lucifuge hatches 60 minutes.It is preferably used micro oscillator (low-limit frequency, 100rpm)；

10. wash plate 5 times；

11. add chromogenic substrate (including blank well) 100 l/ hole, and room temperature (22 ~ 25 DEG C), lucifuge hatches 15 minutes；

12. add stop buffer (including blank well) 100 l/ hole, at once measure OD490 value (in 10 minutes) after mixing.

Result judges:

The OD value of the most each standard substance and specimen should deduct the OD value in zero hole, and (if the hole value that do not subtracts zero, zero hole of standard curve should phase Meet at Y-axis)；

2. manual drawing standard curve.Making abscissa with standard concentration, OD value makees vertical coordinate, connects each standard with sweep The coordinate points of product.Its concentration can be found on standard curve by the OD value of specimen；

3. if specimen OD value is higher than the standard curve upper limit, should resurvey after suitably diluting, when calculating concentration, extension rate should be multiplied by.

The stability of test kit of the present invention and sensitivity test

Relatively the present inventor's beta-amyloid kit and the stability of human beta-amyloid kit in the market And sensitivity.

1 experimental technique

(1) with phosphate buffer, mouse-anti people's amyloid beta antibody is diluted to 1 g/ml, every hole 50 l be added in and be coated plate On, stand overnight in 4 DEG C；

(2) with 200 l containing bovine serum albumin be 2%(W/V), sucrose be 3%(W/V) phosphate buffer close be coated plate；

(3) being simultaneously introduced standard substance and biotinylated anti-human amyloid beta antibody, every hole is 50 l, each Concentraton gradient In triplicate；People's amyloid beta in standard substance can be with the biotinylated anti-human amyloid beta antibody being incorporated in hole And the monoclonal antibody being coated in ELISA Plate combines, form immune complex；

(4) being coated plate with phosphate buffer washing, free composition is washed away；

(5) adding the affinity element of horseradish peroxidase-labeled, affinity element is specific binding with biotin, and free composition is washed Go；

(6) having people's amyloid beta in reacting hole, horseradish peroxidase can make colourless developer become indigo plant, adds stop buffer and becomes Yellow.OD value is surveyed at 490nm.Such as following table.

Although having combined preferred embodiment to describe the present invention, so it is not limited to the present invention, any art technology Personnel, without departing from the spirit and scope of the present invention, it is possible to the theme various changes of enforcement here listed, together Deng displacement and the amendment of thing, therefore protection scope of the present invention is when the claim restriction depending on being proposed is in the range of standard.

SEQUENCE LISTING

<110>Zhejiang Ju Kang biological engineering company limited

<120>biomacromolecule that can be combined with amyloid beta and encode the DNA of this macromole, amyloid beta

Detection kit and application thereof

<130> YYF-201501

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 777

<212> DNA

<213>synthetic

<400> 1

atggcccagg tcaaactgca ggagtcaggc ggcgacctga agaagcccgc cgccaccgtg 60

agaatcagct gcaaggccac cgcctacagc tggagcacct acggcatgca cttcgtgaag 120

aacgcccccg gccagcacct ggagttcatg ggcttcatca acgccggcca gggcgccagc 180

cactacagcc agagattcaa cggcagagtg agcatcacca aggagagcac cgccagcacc 240

ggctacatgg acgtgaccac catcagaagc gacgagaccg ccgtgttcta ctgcgcccac 300

agcagaaaga agaacttcgg ccagggcagc ctggtgaccg tgagcagagg cggcggcggc 360

agcggcggcg gcggcagcgg cggcggcggc agcagcgaca tcaccaacga ccccggcgtg 420

accctggccc tgggcaacac cgtgagactg acctgccagg gcgagagcat caagagctac 480

tacggcagct ggtacaacaa caagcccgcc caggcccccc tggtgctgat ctacggcaag 540

cagaacaagc ccagcggcct gcccgacaga aagttcagcg gcaccaccag cggccagacc 600

ggcagcgtga ccatcaccgc cgcccaggcc gacgaggagg ccgagttcta ctgcaacacc 660

agagagacca gcgccaacca cctggtgttc ggcgccgcca ccagaatcag cctggtgggc 720

gccggcgccg agcagggcac caagctggaa atcaaacggg cggccgcaga atgactc 777

<210> 2

<211> 257

<212> PRT

<213>synthetic

<400> 2

Met Ala Gln Val Lys Leu Gln Glu Ser Gly Gly Asp Leu Lys Lys Pro

1 5 10 15

Ala Ala Thr Val Arg Ile Ser Cys Lys Ala Thr Ala Tyr Ser Trp Ser

20 25 30

Thr Tyr Gly Met His Phe Val Lys Asn Ala Pro Gly Gln His Leu Glu

35 40 45

Phe Met Gly Phe Ile Asn Ala Gly Gln Gly Ala Ser His Tyr Ser Gln

50 55 60

Arg Phe Asn Gly Arg Val Ser Ile Thr Lys Glu Ser Thr Ala Ser Thr

65 70 75 80

Gly Tyr Met Asp Val Thr Thr Ile Arg Ser Asp Glu Thr Ala Val Phe

85 90 95

Tyr Cys Ala His Ser Arg Lys Lys Asn Phe Gly Gln Gly Ser Leu Val

100 105 110

Thr Val Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Ser Asp Ile Thr Asn Asp Pro Gly Val Thr Leu Ala Leu

130 135 140

Gly Asn Thr Val Arg Leu Thr Cys Gln Gly Glu Ser Ile Lys Ser Tyr

145 150 155 160

Tyr Gly Ser Trp Tyr Asn Asn Lys Pro Ala Gln Ala Pro Leu Val Leu

165 170 175

Ile Tyr Gly Lys Gln Asn Lys Pro Ser Gly Leu Pro Asp Arg Lys Phe

180 185 190

Ser Gly Thr Thr Ser Gly Gln Thr Gly Ser Val Thr Ile Thr Ala Ala

195 200 205

Gln Ala Asp Glu Glu Ala Glu Phe Tyr Cys Asn Thr Arg Glu Thr Ser

210 215 220

Ala Asn His Leu Val Phe Gly Ala Ala Thr Arg Ile Ser Leu Val Gly

225 230 235 240

Ala Gly Ala Glu Gln Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala

245 250 255

Glu

<210> 3

<211> 22

<212> DNA

<213>synthetic

<400> 3

aggtsmarct gcagsagtcw gg 22

<210> 4

<211> 32

<212> DNA

<213>synthetic

<400> 4

tgaggagacg gtgaccgtgg tcccttggcc cc 32

<210> 5

<211> 24

<212> DNA

<213>synthetic

<400> 5

gacattgagc tcacccagtc tcca 24

<210> 6

<211> 24

<212> DNA

<213>synthetic

<400> 6

ccgtttgatt tccagcttgg tgcc 24

<210> 7

<211> 24

<212> DNA

<213>synthetic

<400> 7

ccgttttatt tccagcttgg tccc 24

<210> 8

<211> 24

<212> DNA

<213>synthetic

<400> 8

ccgttttatt tccaactttg tccc 24

<210> 9

<211> 24

<212> DNA

<213>synthetic

<400> 9

ccgtttcagc tccagcttgg tccc 24

<210> 10

<211> 69

<212> DNA

<213>synthetic

<400> 10

ggtggaggcg gttcaggcgg aggtgggtct ggcggtggcg gatcggacat tgagctcacc 60

cagtctcca 69

<210> 11

<211> 77

<212> DNA

<213>synthetic

<400> 11

cgatccgcca ccgccagagc cacctccgcc tgaaccgcct ccacctgagg agacggtgac 60

cgtggtccct tggcccc 77

<210> 12

<211> 56

<212> DNA

<213>synthetic

<400> 12

gtcctcgcaa ctgcggccca gccggccatg gcccaggtsm arctgcagsa gtcwgg 56

<210> 13

<211> 42

<212> DNA

<213>synthetic

<400> 13

gagtcattct gcggccgccc gtttgatttc cagcttggtg cc 42

<210> 14

<211> 42

<212> DNA

<213>synthetic

<400> 14

gagtcattct gcggccgccc gttttatttc cagcttggtc cc 42

<210> 15

<211> 42

<212> DNA

<213>synthetic

<400> 15

gagtcattct gcggccgccc gtttcagttc caactttgtc cc 42

<210> 16

<211> 42

<212> DNA

<213>synthetic

<400> 16

gagtcattct gcggccgccc gtttcagctc cagcttggtc cc 42

Claims

1. amyloid beta detection kit, is characterized in that: include

-solid phase, described solid phase secures seizure antibody, and described seizure antibody includes anti-human amyloid beta list Chain antibody,

-cause the solution of degeneration, and

-liquid phase containing detection antibody, described detection antibody includes anti-human amyloid beta polyclonal antibody.

A kind of amyloid beta detection kit the most according to claim 1, is characterized in that: described anti-human beta-amyloyd Albumen single-chain antibody or anti-human amyloid beta polyclonal antibody have such as the sequence of SEQ ID NO.2.

A kind of amyloid beta detection kit the most according to claim 2, is characterized in that: described detection antibody Detection amyloid beta can be sandwiched with described seizure antibody.

4., according to a kind of amyloid beta detection kit described in claims 1 to 3 any claim, it is characterized in that: institute The anti-human amyloid beta polyclonal antibody stated is through biotin labeling；Described liquid phase also includes for quantitative test reference Standard substance.

5., according to a kind of amyloid beta detection kit described in claims 1 to 3 any claim, it is characterized in that: institute The liquid phase stated also includes developer；Described developer is tetramethyl biphenyl diamidogen；The described solution causing degeneration is reaction Stop buffer；Described reaction terminating liquid is HCl.

6., according to a kind of amyloid beta detection kit described in claims 1 to 3 any claim, it is characterized in that: institute The liquid phase stated also includes cleaning mixture；Described cleaning mixture be pH value be the phosphate buffer containing polysorbas20 of 7.2；Described Liquid phase also includes concentrating enzyme conjugates, and the described enzyme conjugates that concentrates includes but not limited to the affine of horseradish peroxidase-labeled Element.

7. the biomacromolecule being combined with amyloid beta, is characterized in that: described biomacromolecule comprises SEQ ID NO.2 aminoacid sequence.

8. a purposes for the biomacromolecule being combined with amyloid beta, is characterized in that: this biomacromolecule is for qualitative Or/and the purposes of people's amyloid beta content in the in vitro body fluid of detection by quantitative people；Described biomacromolecule comprises SEQ ID NO.2 aminoacid sequence.

9. encode a DNA for the biomacromolecule being combined with amyloid beta, it is characterized in that: this DNA contains such as SEQ The sequence of ID NO.1.