CN104897901A - Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4 - Google Patents
Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4 Download PDFInfo
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Abstract
The invention provides a goldmag particle-based acridinium ester chemiluminescence immunological detection method of human epididymis secretory protein (HE4). The goldmag particle-based acridinium ester chemiluminescence immunological detection method mainly comprises following steps: (1) goldmag particle is taken as an immunoreaction and solid-phase separation carrier, and HE4 coated antibody is connected with the surface of the goldmag particle via coupling; (2) blank sites on the surface of the goldmag particle, which are not combined with the HE4 coated antibody, are blocked with a blocking solution; (3) acridinium ester (AE) is used for marking HE4 labelled antibody; (4) a sample to be detected, and the acridinium ester marked HE4 antibody are added into the blocked HE4 antibody coated goldmag particle for reaction so as to obtain a double-antibody sandwich compound, wherein the acridinium ester marked HE4 antibody is capable of realizing specific binding with HE4 antigen; (5) washing is carried out; and (6) chemiluminescence detection is carried out. The goldmag particle-based acridinium ester chemiluminescence immunological detection method is high in detection sensitivity, specificity, accuracy, and stability, and is simple and rapid; linearity range is wide; no radioactive contamination is caused; and operation is safe.
Description
Technical field
The present invention relates to a kind of method detecting people's epididymal secretory protein 4 (HE4).
Background technology
In the market enzyme-linked immunosorbent assay, Electrochemiluminescince and chemiluminescent enzyme immunoassay method etc. are mainly comprised to the immunological detection method of HE4 albumen.Enzyme-linked immunosorbent assay can be used for quantitative detection, but complex operation step, be affected by human factors large, in sensitivity, specificity and range of application, difference is very large; The accuracy that electrochemiluminescence standard measure detects is higher, and level has quantitative and stable advantage, but is not suitable for modern active immunity instrument, needs the use of special necessary instrument, and cost of investment is higher, be therefore difficult to apply; The sensitivity of chemiluminescent enzyme immunoassay method and accuracy want general higher than euzymelinked immunosorbent assay (ELISA), simultaneously to the requirement of detecting instrument equipment again far below Electrochemiluminescince, so be subject to extensive concern always, but because such chemiluminescent substance needs corresponding enzyme to carry out catalytic luminescence, cause background high sensitivity lower slightly, in addition enzymatic chemiluminescence reaches plateau needs certain hour, limits detection efficiency.
Chemiluminescence immunoassay technology is an immunology detection technology having concentrated panimmunity mark detection technique advantage, is founded in 1977 by Halmann etc. on the basis of classical radioimmunoassay technology.This detection technique comprises two Major Systems, wherein on the basis inheriting antigen-antibody high specific immune response system, combine chemiluminescence system, using chemiluminescent substance as the direct labelled antibody of label or antigen, or using the enzyme of energy cataluminescence material as label, after immune response, under the environment that chemiluminescent substance exists at corresponding oxygenant or catalyzing enzyme, form unstable intermediate excited state, the photon signal that can be detected by Chemiluminescence Apparatus is discharged rapidly when getting back to ground state, realize accurately qualitatively or quantitatively determining antigen antibody complex to be measured.Wherein using chemiluminescent substance acridinium ester directly as label, there is the signal value higher than enzymatic chemiluminescence and detection sensitivity.
Traditional chemiluminescence immune assay connects antigen or antibody mainly with microwell plate as solid phase carrier greatly, and the characteristics of luminescence that acridinium ester is concentrated fast makes immunoassay process more easily realize robotization, just has further requirement to solid phase carrier.Magnetic particle is as antigen, antibody mediated immunity react solid phase carrier more efficiently, magnetic metal or the metal oxide under a kind of outside magnetic field existent condition with response, large is core mainly with iron oxide, can at the fixing large number of biological molecule in its surface through molecular modification.In addition the superparamagnetism of magnetic particle makes it deposit at externally-applied magnetic field can to assemble rapidly in case, and can be scattered in solution by Eddy diffusion after removing external magnetic field, greatly shortens the scavenging period in immune detection operating process.
Collaurum is that one has a certain size, the gold grain of the stable colloid state formed by electrostatic interaction, is used to mark and the detection of biomolecule very early.
In conjunction with superparamagnetism and the modifiable dual speciality of gold colloid surface biomolecule of magnetic nano-particle, inventor place seminar have developed Fe
3o
4/ Au magnetic composite particle, i.e. gold-magnetic particles, and achieved commercialization.Gold-magnetic particles is a kind of novel magnetic inorganics composite particles, this material have concurrently separability in magnetic particle outside magnetic field and magnetic particle surface modify after to features such as the quick immobilizations of biomolecule, with medical domain, there is important application prospect biological.
Chemiluminescence immunoassay based on magnetic particle is novel analysis and detection technology magnetic separation technique, immuno analytical method and chemiluminescence detection technology three effectively combined, thus there is the feature of chemiluminescent high sensitivity, the strong specificity of immunoassay, the quick separating of magnetic separation system, in addition, also have that detection time is short, the range of linearity is wide and easily realize the advantages such as robotization, therefore, numerous aspects such as clinical diagnosis, biomedicine, food security and illicit drugs inspection are widely used in recent years.
For specific detected object, the foundation based on (acridinium ester chemiluminescent) immunoassay of magnetic particle (comprising gold-magnetic particles) needs to carry out a large amount of experiments and analyze and research just likely obtaining significant achievement.This is mainly because the molecular structure of different detected object, molecular weight, biologically active and stable Conservation environment etc. are not identical.Difficult point and the complicated part of setting up the method are, different detected object has different requirements to each composition proportion in detection method, immune response condition, even detection system, luminescent solution composition etc., determines that optimum result just can reach optimum detection scope and sensitivity by great many of experiments; In magnetic resolution process, different magnetic field intensity or Magneto separate time all directly affect separating effect; In this external antibody labeling process, different antibodies is synantibody consumption not even, all directly affect the quality (being mainly reflected in labeling effciency) of marked body tying fruit, the condition, preservation condition etc. of labeled reactant condition, purified pool all need repeatedly contrived experiment to determine.
Summary of the invention
The invention provides a kind of method of acridinium ester chemiluminescent immunology detection people epididymal secretory protein 4 (HE4) based on gold-magnetic particles, to solve the problem such as limitation, specificity, sensitivity, danger that above-mentioned background technology for detection HE4 exists.
For achieving the above object, the present invention provides following solution:
Based on the method for acridinium ester chemiluminescent immunology detection people epididymal secretory protein 4 (HE4) of gold-magnetic particles, comprise the following steps:
(1) bag quilt
The carrier be separated with solid phase using gold-magnetic particles as immune response, is coupled at the surface of gold-magnetic particles by HE4 coated antibody; Detailed process is as follows:
(1.1) pre-service: get gold-magnetic particles, cleans balance 1 ~ 3 time with level pad;
(1.2) coupling: mixed with coupling buffer by HE4 coated antibody, adds gained mixed liquor in pretreated gold-magnetic particles, is placed in shaking table, at 25-40 DEG C, fully react under 150 ~ 250rpm condition, after having reacted, be placed on magnetic separator, magnetic resolution, abandon supernatant;
(1.3) clean: with cleaning buffer solution cleaning 3-5 time, magnetic resolution, abandons supernatant;
(2) close
In the product of step (1), add confining liquid, be placed in shaking table, under 25-40 DEG C of condition, with the tachyphylaxis 1 ~ 2 hour of 150 ~ 250rpm, close the blank site that gold-magnetic particles surface is not combined with HE4 coated antibody; Then magnetic resolution, abandons supernatant, saves backup in preservation damping fluid with cleaning buffer solution cleaning rear overhang;
(3) mark of antibody
(3.1) pre-service: desalting column is balanced with elution buffer;
(3.2) antibody labeling: get acridinium ester solution and HE4 labelled antibody solution, carries out labeled reactant, finally adds lysine salt solution and again react in mark buffered environment;
(3.3) purified pool: the antibody-solutions loading desalting column that step (3.2) is obtained by reacting, wash-out collection is carried out with elution buffer, by luminous value and the antibody concentration screening antibodies purified product of gleanings, be namely marked with the HE4 antibody of acridinium ester;
(4) be combined with determinand
By measuring samples and can with the HE4 antibody being marked with acridinium ester of HE4 antigen generation specific binding, add being combined with in the gold-magnetic particles of HE4 coated antibody after step (2) is closed to react, HE4 antigen contained in testing sample is caught by the HE4 coated antibody on gold-magnetic particles, be combined with acridinium ester label HE4 antibody simultaneously, formed " double-antibody sandwich compound ";
(5) clean
With cleaning buffer solution cleaning double-antibody sandwich compound, magnetic resolution, abandons supernatant;
(6) chemiluminescence detection
In the double-antibody sandwich compound formed after step (5) process, add Chemoluminescent substrate, after Chemoluminescent substrate adds, measure the luminous intensity in each hole to be measured immediately; The concentration of luminous intensity and sample to be tested is proportional.
The present invention also establishes following optimization link and parameter:
Any one being chosen as in following damping fluid better of step (1.1) level pad:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration be 0.005M ~ 1M Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid,
Step (1.2) coupling buffer is selected from any one in following damping fluid:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
The cleaning buffer solution adopted in step (1.3) is selected from any one in following damping fluid or contains this kind of damping fluid of 0.05-0.2% (volume fraction, lower same) tween:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
The cleaning buffer solution adopted in step (2) is selected from any one in following damping fluid or contains this kind of damping fluid of 0.05-0.2% tween:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
Step (2) described confining liquid is the potpourri of any one or wherein 2-3 kind among bovine serum albumin(BSA), skimmed milk power, casein, gelatin or animal blood serum, and the mass body volume concentrations (g/mL) of this confining liquid is 1-5%; Described animal blood serum is hyclone or horse serum, and sealer configuration buffer solution is coupling buffer;
Described sealer configuration damping fluid is selected from any one in following damping fluid:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
The preservation damping fluid adopted in step (2) is selected from any one in following damping fluid:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
The elution buffer adopted in step (3) is selected from following damping fluid containing 0.10-0.15M NaCl:
PH5.0 ~ 7.0, concentration is phosphate (PBS) damping fluid of 0.05M ~ 0.15M;
The mark damping fluid adopted in step (3.2) is selected from following damping fluid containing 0.10-0.15M NaCl:
PH7.0 ~ 10.0, concentration is phosphate (PBS) damping fluid of 0.05M ~ 0.15M;
Step (5) described cleaning buffer solution is selected from any one in following damping fluid or also contains this kind of damping fluid of 0.05-0.2% tween:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M.
The cleaning buffer solution then best results containing 0.05% tween is adopted in described step (1.3).
The cleaning buffer solution then best results containing 0.1% tween is adopted in described step (2).
The cleaning buffer solution then best results containing 0.05% tween is adopted in described step (4).
The HE4 monoclonal antibody adopted in step (1) is Meridian, Hytest or Abcam Products.
In step (3.2), acridinium ester solution concentration is the input amount of 0.05-1mM, HE4 labelled antibody is 50-500 μ g.The condition of reaction is specially: get acridinium ester solution and HE4 labelled antibody solution, with the mixing of mark damping fluid, is placed in shaking table, under 20-25 DEG C of condition, with the tachyphylaxis 10 ~ 30 minutes of 150 ~ 250rpm; Add lysine salt solution, be again placed in shaking table, under 20-25 DEG C of condition, with the tachyphylaxis 20 ~ 50 minutes of 150 ~ 250rpm; The labelled antibody adopted is the mouse monoclonal HE4 antibody of acridinium ester or acridine sulfonamides chemiluminescent substance mark.
The condition that step (4) is reacted is: add in gold-magnetic particles by the HE4 antibody of measuring samples and acridinium ester label, be placed in shaking table, under 25-40 DEG C of condition, with the tachyphylaxis 30 ~ 90 minutes of 150 ~ 250rpm.
Above-mentioned mark acridinium ester class chemiluminescent substance is 4-(2-succinimide base carboxyl) phenyl-10-methylacridine-9-carboxylate fluoro sulfonate, acridinium ester-NSP-DMAE-NHS, or adopts acridine sulfonamide NSP-SA-NHS.
The chemical luminous substrate adopted in step (6) is the solution containing hydrogen peroxide and NaOH, or contains in the solution of hydrogen peroxide and NaOH also containing Qu Latong-100 and nitric acid described.The chemical luminous substrate adopted in specific operation process can be divided into the front self-existent luminous exciting liquid A and luminous exciting liquid B of use, wherein contain 0.1% (volume fraction) hydrogen peroxide, 0.05-0.5M nitric acid in luminous exciting liquid A, the Qu Latong-100 containing 0.25M NaOH, 0.5%-5% (volume fraction) in luminous exciting liquid B.
The present invention has the following advantages:
The present invention is carrier with gold-magnetic particles, in conjunction with chemiluminescence immune detection system, establishes the method for the chemiluminescence immunoassay detection HE4 based on gold-magnetic particles.The antibody used is monoclonal antibody, and atopic is good between antigen, effectively avoid cross reaction, and the high absorption capacity had macromolecular substances such as protein due to gold-magnetic particles, in addition the high specific of antibody, antigen immune reaction, the high sensitivity and the label that also have acridinium ester chemiluminescent method concurrently are stablized simultaneously, free of contamination feature.In preparation process, different step uses suitable buffer solution and pH value, makes effectively to be combined between antibody with solid phase carrier, and effectively carry out site and close, the generation of nonspecific reaction avoided.
The method detection sensitivity is high, specificity good, the range of linearity is wide, precision is high, good stability, no radioactivity pollute, and handling safety, method is fast easy.Especially the immune detection all participated in than enzyme in detection sensitivity, specificity, accuracy and precision is significantly increased.
Accompanying drawing explanation
Fig. 1 is the principle schematic of acridinium ester label antibody of the present invention;
Fig. 2 is acridinium ester luminous value and the antibody concentration testing result of antibody labeling elution fraction of the present invention in embodiment one;
Fig. 3 is the typical curve that in embodiment one, the present invention quantitatively detects HE4.
Embodiment
The form example that following examples operate with specific experiment the solution of the present invention, experiment condition wherein and setup parameter should not be considered as the limitation to basic technical scheme of the present invention; Those skilled in the art should determine experiment condition and setup parameter based on principle of the present invention in rational scope, still can realize goal of the invention of the present invention.
Embodiment one: a kind of method of acridinium ester chemiluminescent immunology detection people epididymal secretory protein 4 (HE4) based on gold-magnetic particles
(1) bag quilt
(l.1) pre-service: the gold-magnetic particles 100 μ l getting 10mg/ml, cleans 2 times with pH7.4,0.02M Tris-HCl level pad 200 μ l, the pH of balance magnetic grain.
(l.2) coupling: the HE4 coated antibody of 50 μ g is dissolved in 200 μ l pH7.4, in 0.02M Tris-HCl coupling buffer, add in pretreated gold-magnetic particles after mixing, in 37 DEG C, 180rpm, reacts 30min in shaking table.Take out after completion of the reaction, magnetic resolution, abandon supernatant.
(l.3) clean: add 300 μ l pH7.4, containing the 0.01M PBS cleaning buffer solution of 0.05% Tween-20, magnetic resolution, abandons supernatant, cleans 3 times.
(2) close: in above-mentioned reacted gold-magnetic particles, add the pH7.4 that 1ml contains 5% skimmed milk power and 2% hyclone, in 0.01M PBS damping fluid, in 37 DEG C, 180rpm, reacts 2h in shaking table, and magnetic resolution abandons supernatant.Use 1ml pH7.4, containing the 0.01M PBS buffer solution for cleaning 3 times of 0.1% Tween-20, magnetic grain is suspended from the preservation damping fluid of the 0.01M PBS containing 1% bovine serum albumin(BSA) of 1ml pH7.4, and 4 DEG C for subsequent use.
(3) mark of antibody:
(3.1) pre-service: G-25 sephadex desalting column 25mL elution buffer is balanced.
(3.2) mark of acridinium ester: the HE4 antibody getting 250 μ g, add acridinium ester (4-(2-succinimide base carboxyl) phenyl-10-methylacridine-9-carboxylate fluoro sulfonate) the solution 50 μ l of 0.5mM, the 0.1M PBS containing 0.15M NaCL adding pH8.0 marks damping fluid makes total reaction volume be 1000 μ l, lucifuge is in 25 DEG C, 180rpm, reacts 20min in shaking table.Take out after completion of the reaction, add the lysine salt solution 100 μ l of 10%, continue lucifuge in 25 DEG C, 180rpm, reacts 30min in shaking table.
(3.3) post purifying is crossed: by above-mentioned reactant loading in the G-25 desalting column balanced, carry out wash-out with the 0.1M PBS elution buffer containing 0.15M NaCL of pH 6.3 and collect, elution speed is 120drops/min, eluent is collected with 1.5mL test tube, often pipe is collected 500 μ l and is collected 24 pipes altogether, and the luminous value of elution fraction in collection tube and antibody concentration are measured, result as shown in Figure 2, collect the 4th, 5,6 pipes mixing as the HE4 antibody being marked with acridinium ester, add 1% bovine serum albumin(BSA) and 50% glycerine saves backup in-20 DEG C.
(4) be combined with determinand: take out the gold-magnetic particles being coated with HE4 antibody, in equilibrate at room temperature 15min, take out luminous test tubes, add the HE4 antibody 100 μ l being marked with acridinium ester of above-mentioned gold-magnetic particles 30 μ l, each 100 μ l of sample to be tested (0ng/ml, 0.3ng/ml, 0.6ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 25ng/ml), 50 times of dilutions successively, to put in shaking table 37 DEG C, 180rpm, reaction 40min, forms double-antibody sandwich compound.
(5) clean: take out centrifuge tube and be placed on magnetic separator, abandon supernatant, the 0.01M PBS cleaning buffer solution containing 0.05% Tween-20 with pH7.4 cleans 3 times, magnetic resolution, abandons supernatant.
(6) chemiluminescence detection: to the gold-magnetic particles of the double-antibody sandwich compound formed after step (5) process, add 0.1M HNO
3, 0.1%H
2o
2luminous exciting liquid A 100 μ l, put into chemiluminescence immunoassay detector immediately, adjustment instrument adds 0.25M NaOH automatically, 2%Triton-100 luminous exciting liquid B100 μ l, detect cumulative time 15s, detect the luminous intensity (RLU) in each hole, its luminous intensity and sample to be tested concentration relationship are as shown in Figure 3, better in the scope internal linear of 0.3ng/ml ~ 25ng/ml, minimum detectability can reach 0.069ng/ml.
Conclusion: in the acridinium ester label of antibody, accounted for 69.77% of total antibody dropped into by the HE4 antibody on acridinium ester label, and labeling effciency is up to 1.92, namely on per molecule antibody, average marker has 1.92 acridinium ester molecules;
Based on the concentration of the chemiluminescence detection HE4 method of gold-magnetic particles-luminous value typical curve display, good linear should be had in the scope of 0.05ng/ml ~ 25ng/ml, and can 0.069ng/ml be reached by calculating rear lowest detectable limit.
Embodiment two: a kind of method of acridinium ester chemiluminescent immunology detection people epididymal secretory protein 4 (HE4) based on gold-magnetic particles
(1) bag quilt
(l.1) pre-service: the gold-magnetic particles 100 μ l getting 10mg/ml, cleans 2 times with 0.01M phosphate (PBS) the level pad 200 μ l of pH7.4, the pH of balance magnetic grain.
(l.2) coupling: be dissolved in by the HE4 coated antibody of 50 μ g in 0.01M phosphate (PBS) coupling buffer of 200 μ l pH7.4, add in pretreated gold-magnetic particles after mixing, in 37 DEG C, 180rpm, reacts 30min in shaking table.Take out after completion of the reaction, magnetic resolution, abandon supernatant.
(l.3) clean: add 300 μ l pH7.4, containing the 0.01M PBS cleaning buffer solution of 0.05% Tween-20, magnetic resolution, abandons supernatant, cleans 3 times.
(2) close: in above-mentioned reacted gold-magnetic particles, add the pH7.4 that 1ml contains 5% skimmed milk power and 2% bovine serum albumin(BSA), in 0.01M PBS damping fluid, in 37 DEG C, 180rpm, reacts 2h in shaking table, and magnetic resolution abandons supernatant.Use 1ml pH7.4, containing the 0.01M PBS buffer solution for cleaning 3 times of 0.1% Tween-20, magnetic grain is suspended from the preservation damping fluid of the 0.01M PBS containing 1% bovine serum albumin(BSA) of 1ml pH7.4, and 4 DEG C for subsequent use.
(3) mark of antibody:
(3.1) pre-service: G-25 sephadex desalting column 25mL elution buffer is balanced.
(3.2) mark of acridinium ester: the HE4 antibody getting 500 μ g, add acridinium ester (4-(2-succinimide base carboxyl) phenyl-10-methylacridine-9-carboxylate fluoro sulfonate) the solution 50 μ l of 0.1mM, the 0.1M PBS containing 0.15M NaCL adding pH8.0 marks damping fluid makes total reaction volume be 1000 μ l, lucifuge is in 25 DEG C, 180rpm, reacts 30min in shaking table.Take out after completion of the reaction, add the lysine salt solution 100 μ l of 10%, continue lucifuge in 25 DEG C, 180rpm, reacts 40min in shaking table.
(3.3) post purifying is crossed: by above-mentioned reactant loading in the G-25 desalting column balanced, carry out wash-out with the 0.1M PBS elution buffer containing 0.15M NaCL of pH6.3 and collect, elution speed is 120drops/min, eluent is collected with 1.5mL test tube, often pipe is collected 500 μ l and is collected 24 pipes altogether, and the luminous value of elution fraction in collection tube and antibody concentration are measured, collect the 4th, 5,6 pipes mixing as the HE4 antibody being marked with acridinium ester, add 1% bovine serum albumin(BSA) and 50% glycerine saves backup in-20 DEG C.
(4) be combined with determinand: take out the gold-magnetic particles being coated with HE4 antibody, in equilibrate at room temperature 15min, take out luminous test tubes, add the HE4 antibody 100 μ l being marked with acridinium ester of above-mentioned gold-magnetic particles 30 μ l, each 100 μ l of sample to be tested (0ng/ml, 0.3ng/ml, 0.6ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 25ng/ml), 50 times of dilutions successively, to put in shaking table 37 DEG C, 180rpm, reaction 50min, forms double-antibody sandwich compound.
(5) clean: take out centrifuge tube and be placed on magnetic separator, abandon supernatant, the 0.01M PBS cleaning buffer solution containing 0.05% Tween-20 with pH7.4 cleans 3 times, magnetic resolution, abandons supernatant.
(6) chemiluminescence detection: to the gold-magnetic particles of the double-antibody sandwich compound formed after step (5) process, add 0.3M HNO
3, 0.1%H
2o
2luminous exciting liquid A 100 μ l, put into chemiluminescence immunoassay detector immediately, adjustment instrument adds 0.25M NaOH automatically, 0.5%Triton-100 luminous exciting liquid B 100 μ l, detect cumulative time 15s, detect the luminous intensity (RLU) in each hole, its luminous intensity is directly proportional in certain limit to sample to be tested concentration relationship, wherein better in the scope internal linear of 0.3ng/ml ~ 25ng/ml, minimum detectability can reach 0.086ng/ml.
Conclusion: in the acridinium ester label of antibody, accounted for 63.51% of total antibody dropped into by the HE4 antibody on acridinium ester label, and labeling effciency is up to 1.83, namely on per molecule antibody, average marker has 1.83 acridinium ester molecules;
Based on the concentration of the chemiluminescence detection HE4 method of gold-magnetic particles-luminous value typical curve display, good linear should be had in the scope of 0.05ng/ml ~ 25ng/ml, and can 0.086ng/ml be reached by calculating rear lowest detectable limit.
Embodiment three: a kind of method of acridinium ester chemiluminescent immunology detection people epididymal secretory protein 4 (HE4) based on gold-magnetic particles
(1) bag quilt
(l.1) pre-service: the gold-magnetic particles 100 μ l getting 10mg/ml, cleans 2 times with pH8.0,0.1M Tris-HCl level pad 200 μ l, the pH of balance magnetic grain.
(l.2) coupling: the HE4 coated antibody of 50 μ g is dissolved in 200 μ l pH8.0, in 0.1M Tris-HCl coupling buffer, add in pretreated gold-magnetic particles after mixing, in 37 DEG C, 180rpm, reacts 30min in shaking table.Take out after completion of the reaction, magnetic resolution, abandon supernatant.
(l.3) clean: add 300 μ l pH7.4, containing the 0.1M Tris-HCl cleaning buffer solution of 0.05% Tween-20, magnetic resolution, abandons supernatant, cleans 3 times.
(2) close: in above-mentioned reacted gold-magnetic particles, add the pH8.0 that 1ml contains 5% skimmed milk power and 2% hyclone, in 0.1M Tris-HCl damping fluid, in 37 DEG C, 180rpm, reacts 2h in shaking table, and magnetic resolution abandons supernatant.Use 1ml pH7.4, containing the 0.01M PBS buffer solution for cleaning 3 times of 0.1% Tween-20, magnetic grain is suspended from the preservation damping fluid of the 0.01M PBS containing 1% bovine serum albumin(BSA) of 1ml pH7.4, and 4 DEG C for subsequent use.
(3) mark of antibody:
(3.1) pre-service: G-25 sephadex desalting column 25mL elution buffer is balanced.
(3.2) mark of acridinium ester: the HE4 antibody getting 250 μ g, add acridinium ester (acridinium ester-NSP-DMAE-NHS) the solution 50 μ l of 0.5mM, the 0.15M PBS containing 0.10M NaCL adding pH9.0 marks damping fluid makes total reaction volume be 1000 μ l, lucifuge is in 25 DEG C, 180rpm, reacts 20min in shaking table.Take out after completion of the reaction, add the lysine salt solution 100 μ l of 10%, continue lucifuge in 25 DEG C, 180rpm, reacts 30min in shaking table.
(3.3) post purifying is crossed: by above-mentioned reactant loading in the G-25 desalting column balanced, carry out wash-out with the 0.15M PBS elution buffer containing 0.10M NaCL of pH5.8 and collect, elution speed is 120drops/min, eluent is collected with 1.5mL test tube, often pipe is collected 500 μ l and is collected 24 pipes altogether, and the luminous value of elution fraction in collection tube and antibody concentration are measured, collect the 4th, 5,6 pipes mixing as the HE4 antibody being marked with acridinium ester, add 1% bovine serum albumin(BSA) and 50% glycerine saves backup in-20 DEG C.
(4) be combined with determinand: take out the gold-magnetic particles being coated with HE4 antibody, in equilibrate at room temperature 15min, take out luminous test tubes, add the HE4 antibody 100 μ l being marked with acridinium ester of above-mentioned gold-magnetic particles 30 μ l, each 100 μ l of sample to be tested (0ng/ml, 0.3ng/ml, 0.6ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 25ng/ml), 50 times of dilutions successively, to put in shaking table 37 DEG C, 180rpm, reaction 40min, forms double-antibody sandwich compound.
(5) clean: take out centrifuge tube and be placed on magnetic separator, abandon supernatant, the 0.1M Tris-HCl cleaning buffer solution containing 0.05% Tween-20 with pH8.0 cleans 3 times, magnetic resolution, abandons supernatant.
(6) chemiluminescence detection: to the gold-magnetic particles of the double-antibody sandwich compound formed after step (5) process, add 0.1M HNO
3, 0.1%H
2o
2luminous exciting liquid A 100 μ l, put into chemiluminescence immunoassay detector immediately, adjustment instrument adds 0.25M NaOH automatically, 2%Triton-100 luminous exciting liquid B100 μ l, detect cumulative time 15s, detect the luminous intensity (RLU) in each hole, its luminous intensity is directly proportional within the specific limits to sample to be tested concentration relationship, better in the scope internal linear of 0.3ng/ml ~ 25ng/ml, minimum detectability can reach 0.12ng/ml.
Conclusion: in the acridinium ester label of antibody, accounted for 72.53% of total antibody dropped into by the HE4 antibody on acridinium ester label, and labeling effciency is up to 1.73, namely on per molecule antibody, average marker has 1.73 acridinium ester molecules;
Based on the concentration of the chemiluminescence detection HE4 method of gold-magnetic particles-luminous value typical curve display, good linear should be had in the scope of 0.05ng/ml ~ 25ng/ml, and can 0.12ng/ml be reached by calculating rear lowest detectable limit.
Claims (9)
1., based on a method of the acridinium ester chemiluminescent immunology detection HE4 of gold-magnetic particles, comprise the following steps:
(1) bag quilt
The carrier be separated with solid phase using gold-magnetic particles as immune response, is coupled at the surface of gold-magnetic particles by HE4 coated antibody; Detailed process is as follows:
(1.1) pre-service: get gold-magnetic particles, cleans balance 1 ~ 3 time with level pad;
(1.2) coupling: mixed with coupling buffer by HE4 coated antibody, adds gained mixed liquor in pretreated gold-magnetic particles, is placed in shaking table, at 25-40 DEG C, fully react under 150 ~ 250rpm condition, after having reacted, be placed on magnetic separator, magnetic resolution, abandon supernatant;
(1.3) clean: with cleaning buffer solution cleaning 3-5 time, magnetic resolution, abandons supernatant;
(2) close
In the product of step (1), add confining liquid, be placed in shaking table, under 25-40 DEG C of condition, with the tachyphylaxis 1 ~ 2 hour of 150 ~ 250rpm, close the blank site that gold-magnetic particles surface is not combined with HE4 coated antibody; Then magnetic resolution, abandons supernatant, saves backup in preservation damping fluid with cleaning buffer solution cleaning rear overhang;
(3) mark of antibody
(3.1) pre-service: desalting column is balanced with elution buffer;
(3.2) antibody labeling: get acridinium ester solution and HE4 labelled antibody solution, carries out labeled reactant, finally adds lysine salt solution and again react in mark buffered environment;
(3.3) purified pool: the antibody-solutions loading desalting column that step (3.2) is obtained by reacting, wash-out collection is carried out with elution buffer, by luminous value and the antibody concentration screening antibodies purified product of gleanings, be namely marked with the HE4 antibody of acridinium ester;
(4) be combined with determinand
By measuring samples and can with the HE4 antibody being marked with acridinium ester of HE4 antigen generation specific binding, add being combined with in the gold-magnetic particles of HE4 coated antibody after step (2) is closed to react, HE4 antigen contained in testing sample is caught by the HE4 coated antibody on gold-magnetic particles, be combined with acridinium ester label HE4 antibody simultaneously, formed " double-antibody sandwich compound ";
(5) clean
With cleaning buffer solution cleaning double-antibody sandwich compound, magnetic resolution, abandons supernatant;
(6) chemiluminescence detection
In the double-antibody sandwich compound formed after step (5) process, add Chemoluminescent substrate, after Chemoluminescent substrate adds, measure the luminous intensity in each hole to be measured immediately; The concentration of luminous intensity and sample to be tested is proportional.
2. the method for the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles according to claim 1, is characterized in that:
Step (1.1) level pad is selected from any one in following damping fluid:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration be 0.005M ~ 1M Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid,
Step (1.2) coupling buffer is selected from any one in following damping fluid:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
The cleaning buffer solution adopted in step (1.3) is selected from any one in following damping fluid or contains this kind of damping fluid of 0.05-0.2% tween:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
The cleaning buffer solution adopted in step (2) is selected from any one in following damping fluid or contains this kind of damping fluid of 0.05-0.2% tween:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
Step (2) described confining liquid is the potpourri of any one or wherein 2-3 kind among bovine serum albumin(BSA), skimmed milk power, casein, gelatin or animal blood serum, and the mass body volume concentrations (g/mL) of this confining liquid is 1-5%; Described animal blood serum is hyclone or horse serum, and sealer configuration buffer solution is coupling buffer;
Described sealer configuration damping fluid is selected from any one in following damping fluid:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
The preservation damping fluid adopted in step (2) is selected from any one in following damping fluid:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M;
The elution buffer adopted in step (3) is: pH5.0 ~ 7.0, and concentration is phosphate (PBS) damping fluid of 0.05M ~ 0.15M, wherein also containing 0.10-0.15M NaCl;
The mark damping fluid adopted in step (3.2) is: pH7.0 ~ 10.0, and concentration is phosphate (PBS) damping fluid of 0.05M ~ 0.15M, wherein also containing 0.10-0.15M NaCl;
Step (5) described cleaning buffer solution is selected from any one in following damping fluid or also contains this kind of damping fluid of 0.05-0.2% tween:
PH5.0 ~ 8.0, concentration be 0.005M ~ 0.1M phosphate (PBS) damping fluid,
PH 7.0 ~ 9.0, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.005M ~ 1M.
3. the method for the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles according to claim 2, is characterized in that:
Cleaning buffer solution in described step (1.3) is this kind of damping fluid containing 0.05% tween;
Cleaning buffer solution in described step (2) is this kind of damping fluid containing 0.1% tween;
Cleaning buffer solution in described step (5) is this kind of damping fluid containing 0.05% tween.
4. the method for the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles according to claim 1, is characterized in that:
The HE4 monoclonal antibody adopted in step (1) is Meridian, Hytest or Abcam Products.
5. the method for the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles according to claim 1, is characterized in that:
In step (3.2), acridinium ester solution concentration is the input amount of 0.05-1mM, HE4 labelled antibody is 50-500 μ g.
6. the method for the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles according to claim 1, is characterized in that:
The condition that step (3.2) is reacted is: get acridinium ester solution and HE4 labelled antibody solution, with the mixing of mark damping fluid, is placed in shaking table, under 20-25 DEG C of condition, with the tachyphylaxis 10 ~ 30 minutes of 150 ~ 250rpm; Add lysine salt solution, be again placed in shaking table, under 20-25 DEG C of condition, with the tachyphylaxis 20 ~ 50 minutes of 150 ~ 250rpm; The label that HE4 antibody labeling adopts is acridinium ester class chemiluminescent substance or acridine sulfonamides chemiluminescent substance;
The condition that step (4) is reacted is: add in gold-magnetic particles by the HE4 antibody of measuring samples and acridinium ester label, be placed in shaking table, under 25-40 DEG C of condition, with the tachyphylaxis 30 ~ 90 minutes of 150 ~ 250rpm.
7. the method for the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles according to claim 6, is characterized in that:
Described acridinium ester class chemiluminescent substance is 4-(2-succinimide base carboxyl) phenyl-10-methylacridine-9-carboxylate fluoro sulfonate, acridinium ester-NSP-DMAE-NHS, and described acridine sulfonamides chemiluminescent substance is acridine sulfonamide NSP-SA-NHS.
8. the method for the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles according to claim 1, is characterized in that:
The chemical luminous substrate adopted in step (6) is the solution containing hydrogen peroxide and NaOH, or contains in the solution of hydrogen peroxide and NaOH also containing Qu Latong-100 and nitric acid described.
9. the method for the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles according to claim 8, is characterized in that:
The chemical luminous substrate adopted in step (6) comprises the front self-existent luminous exciting liquid A and luminous exciting liquid B of use, wherein contain 0.1% hydrogen peroxide, 0.05-0.5M nitric acid in luminous exciting liquid A, the Qu Latong-100 containing 0.25M NaOH, 0.5%-5% in luminous exciting liquid B.
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