CN103852579B - A kind of human serum A β quantitative detecting method - Google Patents

A kind of human serum A β quantitative detecting method Download PDF

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CN103852579B
CN103852579B CN201210516697.9A CN201210516697A CN103852579B CN 103852579 B CN103852579 B CN 103852579B CN 201210516697 A CN201210516697 A CN 201210516697A CN 103852579 B CN103852579 B CN 103852579B
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姚钧
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

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Abstract

The invention discloses a kind of human serum A β quantitative detecting method, in turn include the following steps：A) use A β in the plasma sample that kit is pre-configured with the special mouse monoclonal antibody for being covered with anti-A β peptide structure N ends selectivity amino acid sequence and temperature control and constant speed centrifugal treating is crossed to carry out the immune response of capture property, obtain reaction compound；B) the reaction compound and the A β antibody of the rabbit-anti people of anti-A β specific amino acid sequences carry out secondary antigen-antibody reaction, obtain reaction bonded thing；c）The reaction bonded thing is quantitative determined after the polyclonal antibody of the rabbit-anti mouse with zymolyte carries out chromogenic reaction by spectrophotometer.The inventive method improves A β detection sensitivities, reduced sample processing and operation sequence, it is available for the laboratory diagnosis of hospital clinical senile dementia disease, to carrying dull-witted Genetic history or dull-witted dangerous gene crowd, and examination and clinical efficacy of new drug observation/judgement and the universities and colleges' experimental study of suspicious concealment patient progress disease.

Description

A kind of human serum A β quantitative detecting method
Technical field
The present invention relates to a kind of detection method, more particularly to a kind of human serum A β quantitative detecting method.
Background technology
Aβ（Amyloid peptide abbreviation A β）It is amyloid precusor protein caused metabolism production through protease hydrolytic Thing.There is less amount of background level in normal brain cells.But include hereditary amyloid precusor protein and water under pathological conditions The mutation of solution enzyme gene and environmental pollution or the effect of chemical carcinogen can cause A β products to increase and cause increasing for A β.A β increase Height forms senile plaque expelling with intracerebral is gathered in, and the final memory function that destroys causes mistake intelligence or senile dementia.Therefore A β are as old The biomarker that year learns dementia is the unique detection method for being directed to exploitation at present.
Because A β are the key pathogenetic factor of current senile dementia disease and can reflect the important symbol of pathological development process, It is directed to studying effective A β detection methods and exploitation detection kit always for many years both at home and abroad, domestic market is at present substantially Be introduce the existing A β detection kits in international market detected, in recent years the U.S. sell A β detection kits sensitivity compared with Low, the critical field of detection sensitivity is not suitable with asian population, and can not meet that A β levels are slightly elevated in disease early stage It is required that；The detection kit sensitivity that Japanese market is sold at present is higher, but its price is not only sufficiently expensive but also requires complicated Sample process and special detection technical ability；In addition the A β detection kits that European market is sold are deposited between the U.S. and Japan In specificity deficiency, the disadvantage larger with detection error is not sufficiently stable.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of human serum A β quantitative detecting method, can combine Asia The biological nature of continent crowd, improve A β detection sensitivities, reduced sample processing and operation sequence.
The present invention is to provide a kind of human serum A β quantitative inspection to solve the technical scheme that above-mentioned technical problem uses Survey method, in turn includes the following steps：A) it is pre-configured with using kit and is covered with anti-A β peptide structure N- ends selection acidic amino acid sequence It is immune anti-to carry out capture property by A β in the plasma sample that the special mouse monoclonal antibody of row is crossed with temperature control and constant speed centrifugal treating Should, obtain reaction compound；B) the reaction compound and the A β antibody of the rabbit-anti people of anti-A β specific amino acid sequences are carried out secondary Antigen-antibody reaction, obtain reaction bonded thing；c）The reaction bonded thing passes through the polyclonal antibody of the rabbit-anti mouse with zymolyte After carrying out chromogenic reaction, quantitative determined by spectrophotometer.
Further, the kit in the step a) includes box body, lid and fixed plate, and ten are placed with built in the box body Individual reagent bottle and a plate for detecting immunity, described ten reagent bottles and plate for detecting immunity are separated by agent plate to be fixed, described each Care label is attached on reagent bottle, each reagent bottle inner sealing is packaged with reagent.
Further, the box body of the kit and lid are made up of cardboard or plastic plate, the agent plate by cardboard or Plastic foam is made.
Further, it is respectively provided with described ten reagent bottles：Reagent I：Human body A beta-amyloyd peptides；Reagent II： NaH2PO4H2O·Na2HPO4Cushioning liquid；Reagent III：NaN3Block AcePBS solution；Reagent IV：NaH2PO4H2O· Na2HPO4Solution；Reagent V：BSA and Block Ace powder；Reagent VI：The A β detection antibody of rabbit-anti people；Reagent VII：Cleaning is exempted from The equilibrium liquid of epidemic disease reaction；Reagent VIII：The secondary antibody of mouse anti-rabbit；Reagent Ⅸ：TMB chromogenic reaction solution；Reagent X： Peroxidase solution.
Further, the reagent bottle equipped with reagent V, reagent Ⅸ and reagent X is brown, and other reagent bottles are white.
Further, NaH in the reagent II2PO4H2O·Na2HPO4Concentration is 20mM, and EDTA concentration is 2mM, and NaCl is dense Spend and be followed successively by 0.2%, 0.05%, 0.4% and 0.05% for 400mM, BSA, CHAPS, Block Ace and NaN3 concentration, solution ph For 7.0；NaN3 and Block Ace weight percent concentrations are respectively the solution 10% and 1% in PBS solution in the reagent III PH value is 7.4；NaH in reagent IV2PO4H2O·Na2HPO4Concentration is 20mM, and EDTA concentration is 2mM, NaCl concentration 400mM, BSA concentration is 1%, solution ph 7.0；The reagent VII is NaCl, KCl, Na2HPO4·7H2O and KH2PO4Mixed liquor, pH It is worth for 7.4.
Further, two kinds of powder of BSA and Block Ace are divided in respective two tubules in the reagent V, Block Ace powder is dissolved in reagent II and III solution respectively using before, and the BSA powder is dissolved in respectively using preceding In reagent II and IV solution；It is dissolved in before the use of reagent VI in reagent IV solution, and is diluted to pattern detection concentration；Institute The capacity for stating reagent VII is 250ml, dilutes 1 times with distilled water using preceding；The reagent VIII is preceding in the molten of reagent IV using using Pattern detection concentration is diluted in liquid.
Further, following solution is first configured before A β quantitative detection is carried out using the kit：1）Prepare work Make the sample and standard dilutions of concentration：The reagent II of refrigeration is taken out, 10 times of 25ml reagents II is diluted to 250ml；Take Go out the reagent V of refrigeration, be warmed to room temperature naturally, a wherein tubule 0.5g BSA and a tubule 1g Block Ace are dissolved in into dilution In reagent II afterwards；2）Prepare the buffering base fluid of working concentration：The 10xPBS for taking out 100ml adds distilled water to check pH value to 1L； 3）Prepare lock solution：Another tubule 1g Block Ace powder is taken out from reagent V, and to dissolve in reagent II molten to 900ml's 1xPBS adds 100ml NaN3 to be configured to 1X Block Ace lock solutions again；4）Prepare the antibody dilute solution of working concentration： 10X 25ml reagent IV is taken out, the antibody dilute solution that 2475ml distilled waters are made into 1X is added, then adds another tubule 0.5g BSA powder is added in the antibody dilute solution of reagent working concentration.
Further, after the solution of configuration good berth concentration, quantitative detection, including following step are carried out to human serum A β Suddenly：1）The plate for detecting immunity that refrigeration is covered with A β antibody and vinyl cover membrane closure is taken out, is warmed to room temperature naturally；2）Prepare quantitative detection A β standard items：Reagent I standard items A β are dissolved in the sample and standard dilutions containing BSA and Block Ace；And will Lysate is diluted to the gradient concentration of standard curve successively；3）Prepare sample to be tested：By blood sample to be measured in temperature control and constant speed Supernatant is taken out after centrifugation, carries out protein quantification；4）Test sample is treated into collection with the sample and standard dilutions of working concentration Protein concentration in this is uniformly diluted to consistent concentration aequum；5）The close membrane of plate for detecting immunity is opened, by working concentration Sample and standard dilutions are added in the 96 holes reaction sulculus on plate for detecting immunity；By the standard items of A β difference diluted concentrations according to After the secondary corresponding aperture of addition, then the sample to be tested of consistent concentration protein concentration is added into detection and reacted in sulculus；6）Closing 96 Hole plate for detecting immunity, by plate for detecting immunity in constant speed concussion instrument incubation at room temperature 1-3 hours or 4 degree without concussion 12-36 hours；7）With The buffering base fluid cleaning reaction sulculus of working concentration is three times；8）Lock solution is added to be incubated at room temperature 1-3 hours or 4 in constant speed concussion instrument Degree is without concussion 12-36 hours；9）The reagent VI of refrigeration is taken out, is warmed to room temperature naturally；The antibody dilute solution of working concentration by its It is diluted to 10ml；10）Add the A β of dilution to detect antibody to enter to react sulculus, 1-3 hours or 4 degree of nothings are incubated at room temperature in constant speed concussion instrument Shake 12-36 hours；11）Reaction sulculus is cleaned with the buffering base fluid of working concentration four times；12）Take out the mouse of the reagent VIII of refrigeration The secondary antibody of anti-rabbit, is warmed to room temperature naturally；Diluted with the antibody dilute solution of working concentration；13）The A β of dilution are added to examine Survey antibody and enter to react sulculus in constant speed concussion instrument incubation at room temperature 0.5-1.5 hours；14）Cleaned instead with the buffering base fluid of working concentration Answer sulculus three times；15）The reagent Ⅸ and X of refrigeration are taken out, is heated up to 37 degree；Reagent Ⅸ and reagent X is taken uniformly to mix；Add mixing In liquid to each reaction sulculus；According to colour developing from it is shallow to navy blue when, add reagent Ⅸ, stop chromogenic reaction；Detection plate is put Enter OD value of the spectrophotometer in 450nm detection reaction solutions；Containing for A β in sample is determined according to the standard curve of OD values and A β Amount.
Further, the step 1）It is middle quantitative determination standard curve gradient concentration for 500pg/ml, 250pg/ml, 125pg/ml、62.5pg/ml、31.25pg/ml、15.63pg/ml、7.86pg/ml、0pg/ml。
Present invention contrast prior art has following beneficial effect：Human serum A β provided by the invention quantitative detection side Method, the special mouse monoclonal for being covered with anti-A β peptide structure N- ends selectivity amino acid sequence is pre-configured with by using kit A β carry out the immune response of capture property in antibody and plasma sample, then with the A β antibody of the rabbit-anti people of anti-A β specific amino acid sequences Secondary antigen-antibody reaction is carried out, after the polyclonal antibody of the rabbit-anti mouse with zymolyte carries out chromogenic reaction, by being divided Photometer is quantitative determined；The biological nature of this method combination asian population, improves A β detection sensitivities, at reduced sample Reason and operation sequence provide outsourcing service to make up the shortcomings that external existing kit is detected and deficiency, are available for hospital The laboratory diagnosis of clinical senile dementia disease, the dull-witted Genetic history of carrying or dull-witted dangerous gene crowd, and suspicious concealment are suffered from Person carry out disease examination and clinical efficacy of new drug observation/judgement and universities and colleges' experimental study, as long as operator according to Kit specification completes the reagent configuration of final step when in use, implements to detect to include blood by the operating procedure of design Liquid, cerebrospinal fluid, cell culture fluid and brain tissue extract.The tedious steps in kinds of experiments detection are simplified, therefore are shortened Detection cycle is tested, the time of financial resource and material resource and test operation is saved, meets the simplification of testing process and the standard of testing result True property.
Embodiment
Human serum A β provided by the invention quantitative detecting method, in turn includes the following steps：
Step S1：It is pre-configured with using kit and is covered with the special small of anti-A β peptide structure N- ends selectivity amino acid sequence A β carry out the immune response of capture property in the plasma sample that mouse monoclonal antibody is crossed with temperature control and constant speed centrifugal treating, must react compound Thing；
Step S2：The reaction compound and the A β antibody of the rabbit-anti people of anti-A β specific amino acid sequences carry out secondary anti- Antigen-antibody reaction, obtain reaction bonded thing；
Step S3：After the polyclonal antibody of rabbit-anti mouse of the reaction bonded thing through carrying zymolyte carries out chromogenic reaction, Quantitative determined by spectrophotometer.
Kit in step S1 includes box body, lid and fixed plate, and ten reagent bottles and one are placed with built in the box body Individual plate for detecting immunity, fixed wherein ten reagent bottles and 96 hole plate for detecting immunity are separated by agent plate, on each reagent bottle Care label is attached to, each reagent bottle inner sealing is packaged with reagent.It is preferred that the box body and lid of the kit by Cardboard or plastic plate are made, and the agent plate is made up of cardboard or plastic foam.
Ten reagent bottles specifically form as follows：
Reagent bottle 1：White plastic bottle, bottle inner sealing encase people's A beta-amyloyd peptides of accurate weighing（A β 40 and 42）Standard Product, its net weight are managed for 1ug/；Drafting for standard curve.This is reagent I.
Reagent bottle 2：White plastic bottle, bottle is built with the NaH by fixed weight and proportioning2PO4H2O·Na2HPO4Buffer molten Liquid, for the dissolving and dilution of sample and standard items, wherein NaH2PO4H2O·Na2HPO4For 20mM, EDTA concentration is 2mM, NaCl concentration is 400mM, BSA, CHAPS, Block Ace and NaN3Concentration is followed successively by 0.2%, 0.05%, 0.4% and 0.05%, molten Liquid pH value is 7.0, capacity 25ml（10X）；This is reagent II.
Reagent bottle 3：White plastic bottle, bottle is built with the NaN by fixed weight and proportioning3Block AcePBS are molten Liquid, wherein NaN3It is respectively the solution ph 7.4 10% and 1% in PBS solution with Block Ace weight percent concentrations, uses In the non-specific A β detection signals of reduction；This is reagent III.
Reagent bottle 4：White plastic bottle, bottle is built with the NaH by fixed weight and proportioning2PO4H2O·Na2HPO4Solution. Wherein NaH2PO4H2O·Na2HPO4For 20mM, EDTA concentration is 2mM, and NaCl concentration 400mM, BSA concentration is 1%, pH value of solution For 7.0,25ml (10X), the configuration for A β detection antibody concentrations；This is reagent IV.
Reagent bottle 5：Brown plastic bottle, bottle inner sealing encase BSA the and Block Ace powder of accurate weight, two kinds of powder End is divided in respective two tubules, and often pipe net weight is 1g to Block Ace, be dissolved in respectively before use reagent II and III it is molten In liquid, often pipe net weight is 0.5g to BSA, for improving detection signal resolution ratio, using it is preceding be dissolved in respectively reagent II and IV it is molten In liquid, this is reagent V.
Reagent bottle 6：White plastic bottle, bottle inner sealing encase the rabbit-anti people A beta-amyloyd peptide antibody 25ul of Precise levels, For the secondary antigen-antibody reaction of sample to increase the sensitivity of detection, it is dissolved in the solution of reagent IV, dilutes before use To suitable pattern detection concentration；This is reagent VI.
Reagent bottle 7：White plastic bottle, bottle is built with the equilibrium liquid that immune response is cleaned by fixed weight and proportioning （10xPBS）That is NaCl, KCl, Na2HPO4·7H2O、KH2PO4, pH value 7.4,250ml, will be diluted to before use with distilled water 1X（1 times）, this is reagent Ⅸ.
Reagent bottle 8：White plastic bottle, bottle inner sealing encase the secondary antibody 25ul of the mouse anti-rabbit of Precise levels, are used for The A signal betas of amplification detection, suitable pattern detection concentration is diluted in the solution of reagent IV using preceding；This is reagent VIII.
Reagent bottle 9：Brown plastic bottle, bottle inner sealing pack the TMB solution of redox reaction, can make secondary antibody Integrated enzyme reaction, TMB solution are 25ml；This is reagent Ⅸ.
Reagent bottle 10:Brown plastic bottle, bottle inner sealing pack the peroxidase of redox reaction（Oxidizing ferment）Solution, The measure for the colour developing degree quantized samples antigenic content that can be presented with TMB solution reactions according to reactant, this is reagent X.
Following working strength solution is first configured before human serum A β quantitative detection is carried out using kit：
1. prepare the sample and standard dilutions of working concentration：The reagent II of refrigeration is taken out, by 10 times of 25ml reagents II is diluted to 250ml (1X)；The reagent V of refrigeration is taken out, is warmed to room temperature naturally, will an a wherein tubule 0.5g BSA and tubule 1g Block Ace dissolve in dilution after reagent II in.
2. prepare the buffering base fluid of working concentration；The 10xPBS for taking out 100ml adds distilled water to check pH value to 1L.
3. prepare lock solution：Taken out from reagent V another tubule 1g Block Ace powder dissolve in reagent II it is molten to 900ml 1xPBS adds 100ml NaN3 to be configured to 1X Block Ace lock solutions again.
4. prepare the antibody dilute solution of working concentration：10X 25ml reagent IV is taken out, 2475ml distilled waters is added and matches somebody with somebody Into 1X antibody dilute solution, then add another tubule 0.5g BSA powder be added to reagent working concentration antibody it is dilute Release in solution.
After the solution of configuration good berth concentration, quantitative detection is carried out to human serum A β, is specifically comprised the following steps：
1. taking out the plate for detecting immunity that refrigeration is covered with A β antibody and vinyl cover membrane closure, it is warmed to room temperature naturally.
2. prepare quantitatively to detect A β standard items：The gradient concentration for quantitative determining standard curve is 500pg/ml, 250pg/ ml、125pg/ml、62.5pg/ml、31.25pg/ml、15.63pg/ml、7.86pg/ml、0pg/ml.By reagent I standard items A β is dissolved in the sample and standard dilutions that 200ul contains BSA and Block Ace, and lysate addition 1.8ml is made into 1000pg/ml mother liquors, take out 1ml mother liquors and add 1ml dilutions that 250ul 500pg/ml then is added into equivalent to 500pg/ml Dilution is diluted to the gradient concentration of standard curve successively.
3. prepare sample to be tested：Blood sample to be measured is taken out into supernatant after temperature control and constant speed centrifugation, albumen is carried out and determines Amount；
4. the protein concentration unification in the sample to be tested of collection is adjusted with the sample and standard dilutions of working concentration （Dilution）To consistent concentration aequum；
5. opening the close membrane of plate for detecting immunity, the sample of 25ul working concentrations and standard dilutions are added into immune inspection In 96 hole reactive tanks of drafting board.After the standard items 100ul of A β difference diluted concentrations is sequentially added into corresponding reactive tank, then will The sample to be tested of the consistent concentration protein concentration of 100ul is added in detection reaction sulculus；
6. closing 96 hole plate for detecting immunity, plate for detecting immunity is incubated at room temperature 2 hours in constant speed concussion instrument or at 4 degree without shake Swing overnight；
7. clean reaction sulculus three times with the buffering base fluid of working concentration；
8. 100ul lock solutions are added to be incubated at room temperature 2 hours in constant speed concussion instrument or stayed overnight at 4 degree without concussion；
9. take out the reagent VI of refrigeration（A β detect antibody）, it is warmed to room temperature naturally.The antibody dilute solution of working concentration will It is diluted to 10ml；
10. adding the A β detection antibody of 100ul dilutions to enter to react sulculus, it is incubated at room temperature 2 hours in constant speed concussion instrument or at 4 degree Without concussion overnight；
11. clean reaction sulculus four times with the buffering base fluid of working concentration；
12. take out VIII antibody of refrigeration（The secondary antibody of mouse anti-rabbit）, it is warmed to room temperature naturally, the antibody dilution of working concentration Solution is diluted to 10ml；
13. add the A β detection antibody of 100ul dilutions to enter to react sulculus to be incubated at room temperature 1 hour in constant speed concussion instrument；
14. clean reaction sulculus three times with the buffering base fluid of working concentration；
15. taking out the reagent Ⅸ and X of refrigeration, water-bath is heated up to 37 degree, takes 5.5ml reagents Ⅸ and 5.5ml reagents X uniform Mixing, 100ul is added to be reacted to each in aperture；According to colour developing from it is shallow to navy blue when, add reagent Ⅸ, stop chromogenic reaction； Detection plate is inserted into OD value of the spectrophotometer in 450nm detection reaction solutions；According to the quantitative sample of OD values and A β standard curve The level of A β in this.
To sum up, human serum A β provided by the invention quantitative detecting method, is pre-configured with by using kit and is covered with A β carry out capture in the special mouse monoclonal antibody and plasma sample of anti-A β peptide structure N- ends selectivity amino acid sequence Immune response, secondary antigen-antibody reaction then is carried out with the A β antibody of the rabbit-anti people of anti-A β specific amino acid sequences, through band After the polyclonal antibody for having the rabbit-anti mouse of zymolyte carries out chromogenic reaction, quantitative determined by spectrophotometer；This method knot The biological nature of asian population is closed, A β detection sensitivities, reduced sample processing and operation sequence is improved or outsourcing clothes is provided It is engaged in, to make up the shortcomings that external existing kit is detected and deficiency, being available for the experiment of hospital clinical senile dementia disease to examine Examination and clinic disconnected, to the dull-witted Genetic history of carrying or dull-witted dangerous gene crowd, and suspicious concealment patient progress disease Efficacy of new drug observation/judgement and universities and colleges' experimental study, as long as operator completes when in use according to kit specification The reagent configuration of final step, is implemented to detect to include blood, cerebrospinal fluid by the operating procedure of design, cell culture fluid and brain Tissue extract.The tedious steps in kinds of experiments detection are simplified, therefore shorten experiment detection cycle, save financial resource and material resource With the time of test operation, meet the simplification of testing process and the accuracy of testing result.
Although the present invention is disclosed as above with preferred embodiment, so it is not limited to the present invention, any this area skill Art personnel, without departing from the spirit and scope of the present invention, when a little modification and perfect, therefore the protection model of the present invention can be made Enclose to work as and be defined by what claims were defined.

Claims (10)

1. the special mouse monoclonal antibody of anti-A β peptide structure N- ends selectivity amino acid sequence, anti-A β specific amino acid sequences The A β antibody of rabbit-anti people and the polyclonal antibody of rabbit-anti mouse with zymolyte be used to quantitatively examine by the following method preparing The purposes surveyed in human serum A β kit, it is characterised in that methods described in turn includes the following steps：
A) the special mouse monoclonal for being covered with anti-A β peptide structure N- ends selectivity amino acid sequence is pre-configured with using kit A β carry out the immune response of capture property in the plasma sample that antibody is crossed with temperature control and constant speed centrifugal treating, obtain reaction compound；
B) it is anti-to carry out secondary Ag-Ab for the reaction compound and the A β antibody of the rabbit-anti people of anti-A β specific amino acid sequences Should, obtain reaction bonded thing；
C) the reaction bonded thing is after the polyclonal antibody of the rabbit-anti mouse with zymolyte carries out chromogenic reaction, by spectrophotometric Instrument is quantitative determined.
2. purposes as claimed in claim 1, it is characterised in that the kit in the step a) includes box body, lid and consolidated Fixed board, is placed with ten reagent bottles and a plate for detecting immunity built in the box body, described ten reagent bottles and plate for detecting immunity by Agent plate, which separates, is fixed, and care label is attached on each reagent bottle, and each reagent bottle inner sealing is packaged with reagent.
3. purposes as claimed in claim 2, it is characterised in that the box body and lid of the kit are by cardboard or plastic plate system Into the agent plate is made up of cardboard or plastic foam.
4. purposes as claimed in claim 2, it is characterised in that be respectively provided with described ten reagent bottles：Reagent I：Human body A β 4 amyloid；Reagent II：NaH2PO4H2O·Na2HPO4Cushioning liquid；Reagent III：NaN3Block AcePBS solution；Examination Agent IV：NaH2PO4H2O·Na2HPO4Solution；Reagent V：BSA and Block Ace powder；Reagent VI：The A β detections of rabbit-anti people are anti- Body；Reagent VII：Clean the equilibrium liquid of immune response；Reagent VIII：The secondary antibody of mouse anti-rabbit；Reagent Ⅸ：TMB chromogenic reactions are molten Liquid；Reagent X：Peroxidase solution.
5. purposes as claimed in claim 4, it is characterised in that the reagent bottle equipped with reagent V, reagent Ⅸ and reagent X is Brown, other reagent bottles are white.
6. purposes as claimed in claim 4, it is characterised in that NaH in the reagent II2PO4H2O·Na2HPO4Molar concentration For 20mM, EDTA molar concentrations are 2mM, and NaCl molar concentrations are 400mM, BSA, CHAPS, Block Ace and NaN3Weight hundred Specific concentration is divided to be followed successively by 0.2%, 0.05%, 0.4% and 0.05%, solution ph 7.0；NaN in the reagent III3With Block Ace weight percent concentrations are respectively the solution ph 7.4 10% and 1% in PBS solution；In reagent IV NaH2PO4H2O·Na2HPO4Molar concentration is 20mM, and EDTA molar concentrations are 2mM, and NaCl molar concentrations are 400mM, BSA weight Percent concentration is 1%, solution ph 7.0；The reagent VII is NaCl, KCl, Na2HPO4·7H2O and KH2PO4Mixing Liquid, pH value 7.4.
7. purposes as claimed in claim 6, it is characterised in that two kinds of powder packing of BSA and Block Ace in the reagent V In respective two tubules, Block Ace powder is dissolved in reagent II and III solution respectively using preceding, the BSA powder End is dissolved in reagent II and IV solution respectively before using；It is dissolved in before the use of reagent VI in reagent IV solution, and it is dilute Release to pattern detection concentration；The capacity of the reagent VII is 250ml, dilutes 1 times with distilled water using preceding；The reagent VIII makes Pattern detection concentration is diluted in the solution of reagent IV with preceding.
8. purposes as claimed in claim 7, it is characterised in that before A β quantitative detection is carried out using the kit first Configure following solution：
1) sample and standard dilutions of working concentration are prepared：The reagent II of refrigeration is taken out, 10 times of 25ml reagents II is dilute Release to 250ml；The reagent V of refrigeration is taken out, is warmed to room temperature naturally, by a wherein tubule 0.5g BSA and a tubule 1g Block Ace is dissolved in the reagent II after dilution；
2) the buffering base fluid of working concentration is prepared：The 10xPBS for taking out 100ml adds distilled water to check pH value to 1L；
3) lock solution is prepared：Another tubule 1g Block Ace powder is taken out from reagent V, and to dissolve in reagent II molten to 900ml 1xPBS add 100ml NaN3 to be configured to 1X Block Ace lock solutions again；
4) the antibody dilute solution of working concentration is prepared：10X 25ml reagent IV is taken out, 2475ml distilled waters is added and is made into 1X Antibody dilute solution, then add another tubule 0.5g BSA powder be added to reagent working concentration antibody dilution it is molten In liquid.
9. purposes as claimed in claim 8, it is characterised in that after the solution of configuration good berth concentration, to human serum A β Quantitative detection is carried out, is comprised the following steps：
1) plate for detecting immunity that refrigeration is covered with A β antibody and vinyl cover membrane closure is taken out, is warmed to room temperature naturally；
2) quantitatively detection A β standard items are prepared：By reagent I standard items A β be dissolved in the sample containing BSA and Block Ace and In standard dilutions；And lysate is diluted to the gradient concentration of standard curve successively；
3) sample to be tested is prepared：Blood sample to be measured is taken out into supernatant after temperature control and constant speed centrifugation, carries out protein quantification；
4) protein concentration in the sample to be tested of collection is uniformly diluted to one with the sample and standard dilutions of working concentration Cause concentration aequum；
5) close membrane of plate for detecting immunity is opened, the sample of working concentration and standard dilutions are added on plate for detecting immunity In 96 holes reaction sulculus；After the standard items of A β difference diluted concentrations are sequentially added into corresponding aperture, then by consistent concentration protein The sample to be tested of concentration is added in detection reaction sulculus；
6) 96 hole plate for detecting immunity are closed, by plate for detecting immunity in constant speed concussion instrument incubation at room temperature 1-3 hours or 4 degree without concussion 12-36 hours；
7) reaction sulculus is cleaned three times with the buffering base fluid of working concentration；
8) add lock solution in constant speed concussion instrument incubation at room temperature 1-3 hours or 4 degree without concussion 12-36 hours；
9) the reagent VI of refrigeration is taken out, is warmed to room temperature naturally；The antibody dilute solution of working concentration is diluted to 10ml；
10) the A β detection antibody of dilution is added to enter to react sulculus, in constant speed concussion instrument incubation at room temperature 1-3 hours or 4 degree without concussion 12- 36 hours；
11) reaction sulculus is cleaned four times with the buffering base fluid of working concentration；
12) secondary antibody of the mouse anti-rabbit of the reagent VIII of refrigeration is taken out, is warmed to room temperature naturally；It is molten with the antibody dilution of working concentration Liquid is diluted；
13) add the A β detection antibody of dilution to enter to react sulculus and be incubated at room temperature 0.5-1.5 hours in constant speed concussion instrument；
14) reaction sulculus is cleaned three times with the buffering base fluid of working concentration；
15) reagent Ⅸ and X of refrigeration are taken out, is heated up to 37 degree；Reagent Ⅸ and reagent X is taken uniformly to mix；Add mixed liquor to each React in sulculus；According to colour developing from it is shallow to navy blue when, add reagent Ⅸ, stop chromogenic reaction；Detection plate is inserted into light splitting light OD value of the degree meter in 450nm detection reaction solutions；The content of A β in sample is determined according to the standard curve of OD values and A β.
10. purposes as claimed in claim 9, it is characterised in that the gradient concentration of the step 2) standard curve is 500pg/ml、250pg/ml、125pg/ml、62.5pg/ml、31.25pg/ml、15.63pg/ml、7.86pg/ml、0pg/ml。
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