CN104777315A - Chemiluminescence immunoassay method for detecting S100 based on gold magnetic particles - Google Patents
Chemiluminescence immunoassay method for detecting S100 based on gold magnetic particles Download PDFInfo
- Publication number
- CN104777315A CN104777315A CN201510184976.3A CN201510184976A CN104777315A CN 104777315 A CN104777315 A CN 104777315A CN 201510184976 A CN201510184976 A CN 201510184976A CN 104777315 A CN104777315 A CN 104777315A
- Authority
- CN
- China
- Prior art keywords
- damping fluid
- concentration
- magnetic particles
- gold
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a chemiluminescence immunoassay method for detecting S100 based on gold magnetic particles. The method mainly comprises the following steps: (1) coating, namely taking gold magnetic particles as a carrier of immunoreaction and solid-phase separation, and coupling an S100 antibody onto the surface of the gold magnetic particles; (2) confining, namely confining the blank site at which the surface of the gold magnetic particles and the S100 antibody are bound by using confining liquid, performing magnetic separation, and removing the supernatant; (3) adding a to-be-detected sample and an enzyme-labeled secondary antibody which can be subjected to specific binding with an antigen into the gold magnetic particles which are confined in the step (2) and are bound with the S100 antibody for reacting, and forming a double-antibody sandwich complex; (4) cleaning; and (5) detecting. The method disclosed by the invention is high in detection sensitivity, high in specificity, wide in linearity range, high in precision, high in stability, safe to operate, simple and rapid, and radioactive contamination is avoided.
Description
Technical field
The present invention relates to a kind of method that chemiluminescence immunoassay detects S100.
Background technology
In the market enzyme-linked immunosorbent assay, Electrochemiluminescince, radio immunoassay, immunoradiometry and fluorescence immunoassay etc. are mainly comprised to the immunological detection method of S100 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.There is radiocontamination and harm in radio immunoassay and immunoradiometry, the nucleic half life period is short, and the stabilization of kit phase is short, and complex operation etc. are not enough and apply limited.Fluorescence immunoassay needs special fluorescence equipment, therefore not easily popularizes clinically.
Traditional magnetic particle be mostly by after surface-functionalized modification with carboxyl (-COOH), hydroxyl (-OH), amino (-NH2), aldehyde radical (-CHO) isoreactivity group and antigen or antibody covalent effect these antigen-antibodies are coupled at magnetic bead surfaces.Coupling process is complicated, and easily causes antigen or antibody inactivation when coupling, and not only coupling efficiency is low, and cost of idleness, due to these reasons, the chemiluminescent popularization and application based on magnetic particle is subject to certain restrictions.
Magnetic composite particle, also known as magnetic microsphere or magnetic bead, grow up late 1970s and be widely used in the superparamagnetism nano-and micro-composite particle of biomedical sector, (comprise magnetic metal as Fe by macromolecule or inorganic material and magnetic superfine powder, Co, Ni or its metal oxide etc.) colloidal compound that forms.Under additional magnetic fields, the superparamagnetism of magnetic particle can ensure to be separated rapidly after interaction with the target molecules, and itself again can not be permanently magnetized.Nano-micro level magnetic composite particle has higher specific surface area, shows stronger adsorptive power.By physisorption, or utilize the reactive group being modified at magnetic composite particle surface the bioactivators such as enzyme, antibody, the acid of few core former times and nucleic acid can be coupled at its surface.Thus Magnetic antibody immunological technique (Magnetic Antibody Immunoassay, the advantages such as MAIA) technology tool detection speed is fast, specificity is high, highly sensitive, reproducible, no radioactivity pollute, there is good application prospect in the fields such as the method detects in medical science, environmental monitoring and food safety detection.
Collaurum is used to mark and the detection of biomolecule very early, the separability in magnetic nano-particle outside magnetic field and collaurum characteristic is combined, and preparation consists of Fe/Au, Fe
3o
4the magnetic particles such as/Au, Co/Au are called as " gold-magnetic particles ".
Gold-magnetic particles is a kind of novel magnetic inorganics composite particles, be compounded to form by colloid gold particle and magnetic particle, this material has separability in magnetic particle outside magnetic field and collaurum concurrently to features such as the quick immobilizations of biomolecule, has important application prospect biological with medical domain.In conjunction with superparamagnetism and the modifiable dual speciality of gold surface biomolecule of magnetic nano-particle, inventor have developed Fe
3o
4/ Au magnetic composite particle, and achieved commercialization.
Chemiluminescence immunoassay technology is born in 1977.High-sensitive chemiluminescence, according to the ultimate principle of radiommunoassay, combines with the immune response of high specific, establishes chemiluminescence immunoassay by Halmann etc.This technology comprises two systems, i.e. immunoassay system and chemiluminescence analysis system.Therefore, it has immunoassay high specific and chemiluminescence high sensitivity concurrently in the feature of one.The principle that immunoassay system is application antigen, react between antibody, by by chemiluminescent substance or enzyme labeling on antigen or antibody, through immune response, form antigen-antibody complex.Chemiluminescence analysis system is then after immune response terminates, add chemical luminous substrate or the oxygenant of enzyme, chemiluminescent substance forms the intermediate of excited state under oxygenant effect, unstable intermediate is when getting back to stable ground state, to photon be launched, now by chemiluminescence detector, chemiluminescence intensity be detected.The method can carry out qualitative analysis and quantitative test, is a kind of integrated technology.
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.
In actual testing process, for different detecting factor, because of its sensing range, sensitivity requirement difference, condition optimizing and the mutual coordination of immune response, Magneto separate, each system of chemiluminescence just seem extremely important.Especially the mode of new and innovative magnetic-particle coupled antigen or antibody, immunoreactive antibody consumption and reaction time, magnetic divides duration and number of times, chemiluminescence agent and substrate to select, and is all the Focal point and difficult point of the method research.
Summary of the invention
The invention provides a kind of method that chemiluminescence immunoassay based on gold-magnetic particles detects S100, to solve the problem such as limitation, specificity, sensitivity, danger that above-mentioned background technology for detection S100 exists.
For achieving the above object, the present invention provides following basic solution:
Chemiluminescence immunoassay based on gold-magnetic particles detects a method of S100, comprises the following steps:
(1) bag quilt
The carrier be separated with solid phase using gold-magnetic particles as immune response, by the surface of S100 antibody coupling at gold-magnetic particles;
(2) close
The surperficial blank site be not combined with antibody of gold-magnetic particles is closed with confining liquid; Then magnetic resolution, abandons supernatant, saves backup in preservation damping fluid with cleaning buffer solution cleaning rear overhang;
(3) be combined with determinand
By measuring samples with being combined with in the gold-magnetic particles of S100 antibody and reacting after step (2) is closed can be added with the ELIAS secondary antibody of antigen generation specific binding, S100 albumen free in testing sample is caught by specific antibody magnetic particle wrapping quilt, be combined with ELIAS secondary antibody simultaneously, formed " double-antibody sandwich compound ";
(4) clean
With cleaning buffer solution cleaning double-antibody sandwich compound, magnetic resolution, abandons supernatant;
(5) detect
In the double-antibody sandwich compound formed after step (4) process, add Chemoluminescent substrate, in 25 ~ 35min after Chemoluminescent substrate adds, measure the luminous intensity in each hole; The concentration of luminous intensity and sample to be tested inversely.
For realizing better Detection results, step (1) specifically can comprise the following steps:
(1.1) pre-service: get gold-magnetic particles, with equilibration buffer 1 ~ 3 time;
(1.2) coupling: mixed with coupling buffer by S100 antibody, adds gained mixed liquor in pretreated gold-magnetic particles, is placed in shaking table, at 35-38 DEG C, fully react 30 ~ 40min under 170 ~ 210rpm condition, after having reacted, be placed on magnetic separator, magnetic resolution, abandon supernatant;
(1.3) clean: with cleaning buffer solution cleaning 3-4 time, magnetic resolution, abandons supernatant;
(2) close
In the product of step (1), add confining liquid, be placed in shaking table, under 35-38 DEG C of condition, with the tachyphylaxis 1.3 ~ 1.7 hours of 170 ~ 210rpm, close the blank site that gold-magnetic particles surface is not combined with S100 antibody; Then magnetic resolution, abandons supernatant, saves backup in preservation damping fluid with cleaning buffer solution cleaning rear overhang.
(3) be combined with determinand
Measuring samples is reacted with adding in the gold-magnetic particles in conjunction with S100 antibody after step (2) is closed with the ELIAS secondary antibody of antigen generation specific binding, S100 albumen free in testing sample is caught by specific antibody magnetic particle wrapping quilt, be combined with ELIAS secondary antibody simultaneously, formed " double-antibody sandwich compound ";
(4) clean
With cleaning buffer solution cleaning double-antibody sandwich compound, magnetic resolution, abandons supernatant;
(5) detect
In the double-antibody sandwich compound formed after step (4) process, add Chemoluminescent substrate, in 25 ~ 35min after Chemoluminescent substrate adds, measure the luminous intensity in each hole; The concentration of luminous intensity and sample to be tested inversely.
Step (1.1) level pad is selected from any one in following damping fluid:
PH6.5 ~ 7.8, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.2 ~ 7.8, concentration be 0.01M ~ 0.05M Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid,
PH7.4 ~ 8.2, concentration is the BS damping fluid of 0.01M ~ 0.04M;
Step (1.2) coupling buffer is selected from any one in following damping fluid:
PH3.6 ~ 5.8, concentration be 0.01M ~ 0.02M acetate buffer,
PH6.5 ~ 7.8, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.2 ~ 7.8, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
The cleaning buffer solution adopted in step (l.3) is selected from any one in following damping fluid or contains this kind of damping fluid of 0.04-0.07% tween:
PH3.6 ~ 5.8, concentration be 0.01M ~ 0.02M acetate buffer,
PH6.5 ~ 7.8, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.2 ~ 7.8, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
Step (2) described confining liquid is the potpourri of any one or wherein 2-3 kind among bovine serum albumin(BSA), skimmed milk power, lysine or animal blood serum, and the mass body volume concentrations (g/ml) of this confining liquid is 2-5%; 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:
PH7.7 ~ 8.0, concentration be 0.01M ~ 0.04M phosphate (PB) damping fluid,
PH7.6 ~ 8.2, concentration be 0.01M ~ 0.05M BS damping fluid,
PH7.9 ~ 8.3, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.05M;
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 0.08-0.15% tween:
PH7.7 ~ 8.0, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.6 ~ 8.2, concentration be 0.01M ~ 0.05M BS damping fluid,
PH7.9 ~ 8.3, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
The preservation damping fluid adopted in step (2) is selected from any one in following damping fluid:
PH7.7 ~ 8.0, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.6 ~ 8.2, concentration be 0.01M ~ 0.05M BS damping fluid,
PH7.9 ~ 8.3, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
Step (4) described cleaning buffer solution is selected from any one in following damping fluid or contains this kind of damping fluid of 0.02-0.1% tween:
PH7.7 ~ 8.0, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.6 ~ 8.2, concentration be 0.01M ~ 0.05M BS damping fluid,
PH7.9 ~ 8.2, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
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 (4) is this kind of damping fluid containing 0.05% tween.
The ELIAS secondary antibody that step (3) adopts is goat against murine or the goat antirabbit of alkali phosphatase enzyme mark or horseradish peroxidase-labeled.
The chemical luminous substrate adopted in step (5) is luminol, different luminol or 1,2 one dichloroethane analog derivatives.
Above-mentioned 1,2 one dichloroethane analog derivatives are AMPPD, CSPD or CDP-STAR.
The present invention has the following advantages:
The present invention is carrier with gold-magnetic particles, in conjunction with chemiluminescence detection system, establishes the method for the chemiluminescence immunoassay detection S100 based on gold-magnetic particles.Antibody used in the method 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 it has both the high specific of enzyme-linked immuno assay, free of contamination feature, also retains the high sensitivity of chemoluminescence method, compensate for its deficiency in specificity.
In preparation process, different step establishes 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 be all significantly increased than the inspection-free survey of development process enzyme in detection sensitivity, specificity, accuracy and precision.
Accompanying drawing explanation
Fig. 1 is the structural representation of the present invention's solid phase carrier-gold-magnetic particles used;
Fig. 2 be in method of the present invention with gold-magnetic particles bag by the operation chart of S100;
Fig. 3 is of the present invention based on gold-magnetic particles double antibody sandwich method principle schematic;
Fig. 4 is the quantitative reaction curve that the embodiment of the present invention 1 detects S100.
Fig. 5 is the quantitative reaction curve that the embodiment of the present invention 2 detects S100.
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 recognize, determine concrete experiment condition and parameter, still can obtain enough satisfied effect based in the scope that claim scheme limits.
Embodiment 1: the chemiluminescence immunoassay based on gold-magnetic particles quantitatively detects the method for S100 in human serum
(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 100 μ l, the pH of balance magnetic grain.
(l.2) coupling: 160 μ g S100 antibody are dissolved in 200 μ l pH7.4, and in 0.02M Tris-HCl coupling buffer, add in pretreated gold-magnetic particles after mixing, in 37 DEG C, 180rpm, reacts 35min in shaking table.Take out after completion of the reaction, magnetic resolution 2min, abandon supernatant.
(l.3) clean: add 200 μ l pH7.4, containing the 0.02M Tris-HCl cleaning buffer solution of 0.05% Tween-20, magnetic resolution 2min, abandons supernatant.
(2) close: in gold-magnetic particles, add 1ml containing 5% bovine serum albumin(BSA), in the pH8.0 of 2.5% skimmed milk power and 3% hyclone, 0.01M PB damping fluid, in 37 DEG C, 180rpm, reacts 1.5h in shaking table, and magnetic resolution 2min abandons supernatant.With 1ml pH 8.0, containing the 0.01M PB buffer solution for cleaning 3 times of 0.1% Tween-20, magnetic grain is suspended from the preservation damping fluid of the 0.02M Tris-HCl of 1ml pH8.0, and 4 DEG C for subsequent use.
(3) be combined with determinand: take out bag by after gold-magnetic particles, in equilibrate at room temperature 20min, add gold-magnetic particles 20 μ l, each 50 μ l, the ALP of sample to be tested (0ng/ml, 0.05ng/ml, 0.1ng/ml, 0.3ng/ml, 0.6ng/ml, 1ng/ml, 3ng/ml, 6ng/ml, 12ng/ml, 35ng/ml) of being coated with S100 antibody successively and mark sheep anti mouse 50 μ l, to put in shaking table 37 DEG C, 180rpm, reaction 2h, forms double-antibody sandwich compound.
(4) clean: take out centrifuge tube and be placed on magnetic separator, abandon supernatant, clean 3 times, magnetic resolution 2min with the 0.02M Tris-HCl cleaning buffer solution that pH 8.0 contains 0.05% Tween-20, abandon supernatant.
(5) detect: the gold-magnetic particles of the double-antibody sandwich compound formed after step (4) process is proceeded to 96 orifice plates, and add luminous substrate AMPPD solution 100 μ l to it, the luminous intensity (RLU) in each hole is detected in 30min after adding, its luminous intensity and sample to be tested concentration relationship are as shown in Figure IV, better in 0.05ng/ml ~ 35ng/ml scope internal linear, minimum detectability can reach 0.02ng/ml.
In implementation process, we have chosen and have Suitable pH ranges and the buffer solution that can adapt to gold-magnetic particles carrier, and in the OK range of reaction conditions, have chosen the value with optimum efficiency.During coupling, the optimal proportion of immunoreactive S100 antibody consumption and gold-magnetic particles is 1: 0.15.
Embodiment 2: the chemiluminescence immunoassay based on gold-magnetic particles quantitatively detects the method for S100 in human serum
(1) bag quilt
(l.1) pre-service: the gold-magnetic particles 100 μ l getting 10mg/ml, cleans 2 times with pH7.6,0.02M Tris-HCl level pad 100 μ l, the pH of balance magnetic grain.
(l.2) coupling: 150 μ g S100 antibody are dissolved in 200 μ l pH7.6, and in 0.02M Tris-HCl coupling buffer, add in pretreated gold-magnetic particles after mixing, in 35 DEG C, 200rpm, reacts 38min in shaking table.Take out after completion of the reaction, magnetic resolution 2min, abandon supernatant.
(l.3) clean: add 200 μ l pH7.6, containing the 0.02M Tris-HCl cleaning buffer solution of 0.07% Tween-20, magnetic resolution 2min, abandons supernatant.
(2) close: in gold-magnetic particles, add 1ml containing 5% bovine serum albumin(BSA), in the pH8.0 of 2.5% skimmed milk power and 3% hyclone, 0.01M PB damping fluid, in 38 DEG C, 200rpm, reacts 1.7h in shaking table, and magnetic resolution 2min abandons supernatant.With 1ml pH 7.8, containing the 0.01M PB buffer solution for cleaning 3 times of 0.1% Tween-20, magnetic grain is suspended from the preservation damping fluid of the 0.02M Tris-HCl of 1ml pH7.8, and 4 DEG C for subsequent use.
(3) be combined with determinand: take out bag by after gold-magnetic particles, in equilibrate at room temperature 20min, add gold-magnetic particles 20 μ l, each 50 μ l, the ALP of sample to be tested (0ng/ml, 0.05ng/ml, 0.1ng/ml, 0.3ng/ml, 0.6ng/ml, 1ng/ml, 3ng/ml, 6ng/ml, 12ng/ml, 35ng/ml) of being coated with S100 antibody successively and mark sheep anti mouse 50 μ l, to put in shaking table 35 DEG C, 190rpm, reaction 2.2h, forms double-antibody sandwich compound.
(4) clean: take out centrifuge tube and be placed on magnetic separator, abandon supernatant, clean 3 times, magnetic resolution 2min with the 0.02M Tris-HCl cleaning buffer solution that pH 8.2 contains 0.05% Tween-20, abandon supernatant.
(5) detect: the gold-magnetic particles of the double-antibody sandwich compound formed after step (4) process is proceeded to 96 orifice plates, and add luminous substrate AMPPD solution 100 μ l to it, the luminous intensity (RLU) in each hole is detected in 35min after adding, its luminous intensity and sample to be tested concentration relationship are as shown in Figure IV, better in 0.05ng/ml ~ 35ng/ml scope internal linear, minimum detectability can reach 0.02ng/ml.
Conclusion: Fig. 4 is the dose-effect curve of the chemiluminescence detection S100 based on golden magnetic, and linearly good at 0.05ng/ml ~ 35ng/ml, lowest detectable limit reaches 0.02ng/ml.
Fig. 5 is the dose-effect curve of the chemiluminescence detection S100 based on golden magnetic, and linearly good at 0.05ng/ml ~ 35ng/ml, lowest detectable limit reaches 0.02ng/ml.
Claims (7)
1., based on a method of the chemiluminescence immunoassay detection S100 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, by the surface of S100 antibody coupling at gold-magnetic particles; Detailed process is as follows:
(1.1) pre-service: get gold-magnetic particles, with equilibration buffer 1 ~ 3 time;
(1.2) coupling: mixed with coupling buffer by S100 antibody, adds gained mixed liquor in pretreated gold-magnetic particles, is placed in shaking table, at 35-38 DEG C, fully react 30 ~ 40min under 170 ~ 210rpm condition, after having reacted, be placed on magnetic separator, magnetic resolution, abandon supernatant;
(1.3) clean: with cleaning buffer solution cleaning 3-4 time, magnetic resolution, abandons supernatant;
(2) close
In the product of step (1), add confining liquid, be placed in shaking table, under 35-38 DEG C of condition, with the tachyphylaxis 1.3 ~ 1.7 hours of 170 ~ 210rpm, close the blank site that gold-magnetic particles surface is not combined with S100 antibody; Then magnetic resolution, abandons supernatant, saves backup in preservation damping fluid with cleaning buffer solution cleaning rear overhang;
(3) be combined with determinand
Measuring samples is reacted with adding in the gold-magnetic particles in conjunction with S100 antibody after step (2) is closed with the ELIAS secondary antibody of antigen generation specific binding, S100 albumen free in testing sample is caught by specific antibody magnetic particle wrapping quilt, be combined with ELIAS secondary antibody simultaneously, formed " double-antibody sandwich compound ";
(4) clean
With cleaning buffer solution cleaning double-antibody sandwich compound, magnetic resolution, abandons supernatant;
(5) detect
In the double-antibody sandwich compound formed after step (4) process, add Chemoluminescent substrate, in 25 ~ 35min after Chemoluminescent substrate adds, measure the luminous intensity in each hole; The concentration of luminous intensity and sample to be tested inversely.
2. the chemiluminescence immunoassay based on gold-magnetic particles according to claim 1 detects the method for S100, it is characterized in that:
Step (1.1) level pad is selected from any one in following damping fluid:
PH6.5 ~ 7.8, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.2 ~ 7.7, concentration be 0.01M ~ 0.05M Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid,
PH7.4 ~ 8.2, concentration is the BS damping fluid of 0.01M ~ 0.04M;
Step (1.2) coupling buffer is selected from any one in following damping fluid:
PH3.6 ~ 5.8, concentration be 0.01M ~ 0.02M acetate buffer,
PH6.5 ~ 7.8, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.2 ~ 7.7, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
The cleaning buffer solution adopted in step (l.3) is selected from any one in following damping fluid or contains this kind of damping fluid of 0.04-0.07% tween:
PH3.6 ~ 5.8, concentration be 0.01M ~ 0.02M acetate buffer,
PH6.5 ~ 7.8, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.2 ~ 7.7, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
Step (2) described confining liquid is the potpourri of any one or wherein 2-3 kind among bovine serum albumin(BSA), skimmed milk power, lysine or animal blood serum, and the mass body volume concentrations (g/ml) of this confining liquid is 2-5%; 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:
PH7.7 ~ 8.0, concentration be 0.01M ~ 0.04M phosphate (PB) damping fluid,
PH7.6 ~ 8.2, concentration be 0.01M ~ 0.05M BS damping fluid,
PH7.9 ~ 8.3, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.05M;
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 0.08-0.15% tween:
PH7.7 ~ 8.0, concentration be 0.005M ~ 0.02M phosphate (PB) damping fluid,
PH7.6 ~ 8.2, concentration be 0.01M ~ 0.05M BS damping fluid,
PH7.9 ~ 8.3, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
The preservation damping fluid adopted in step (2) is selected from any one in following damping fluid:
PH7.7 ~ 8.0, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.6 ~ 8.2, concentration be 0.01M ~ 0.05M BS damping fluid,
PH7.9 ~ 8.3, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M;
Step (4) described cleaning buffer solution is selected from any one in following damping fluid or contains this kind of damping fluid of 0.02-0.1% tween:
PH7.7 ~ 8.0, concentration be 0.01M ~ 0.05M phosphate (PB) damping fluid,
PH7.6 ~ 8.2, concentration be 0.01M ~ 0.05M BS damping fluid,
PH7.9 ~ 8.2, concentration is Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) damping fluid of 0.01M ~ 0.04M.
3. the chemiluminescence immunoassay based on gold-magnetic particles according to claim 2 detects the method for S100, it 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 (4) is this kind of damping fluid containing 0.05% tween.
4. the chemiluminescence immunoassay based on gold-magnetic particles according to claim 1 detects the method for S100, it is characterized in that:
The S100 monoclonal antibody adopted in step (1) is Meridian or Hytest Products.
5. the chemiluminescence immunoassay based on gold-magnetic particles according to claim 1 detects the method for S100, it is characterized in that:
The condition that step (3) is reacted is: add in gold-magnetic particles by measuring samples and ELIAS secondary antibody, be placed in shaking table, under 35-38 DEG C of condition, with the tachyphylaxis 1.8 ~ 2.2 hours of 170 ~ 210rpm; The ELIAS secondary antibody adopted is goat against murine or the goat antirabbit of alkali phosphatase enzyme mark or horseradish peroxidase-labeled.
6. the chemiluminescence immunoassay based on gold-magnetic particles according to claim 1 detects the method for S100, it is characterized in that: the chemical luminous substrate adopted in step (5) is luminol, different luminol or 1,2 one dichloroethane analog derivatives.
7. the chemiluminescence immunoassay based on gold-magnetic particles according to claim 6 detects the method for S100, and it is characterized in that: described 1,2 one dichloroethane analog derivatives are AMPPD, CSPD or CDP-STAR.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510184976.3A CN104777315A (en) | 2015-04-17 | 2015-04-17 | Chemiluminescence immunoassay method for detecting S100 based on gold magnetic particles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510184976.3A CN104777315A (en) | 2015-04-17 | 2015-04-17 | Chemiluminescence immunoassay method for detecting S100 based on gold magnetic particles |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104777315A true CN104777315A (en) | 2015-07-15 |
Family
ID=53618907
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510184976.3A Pending CN104777315A (en) | 2015-04-17 | 2015-04-17 | Chemiluminescence immunoassay method for detecting S100 based on gold magnetic particles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104777315A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061186A (en) * | 2018-08-14 | 2018-12-21 | 深圳天辰医疗科技有限公司 | A kind of Procalcitonin detection kit and Procalcitonin detection method |
| CN109596835A (en) * | 2018-11-23 | 2019-04-09 | 深圳天辰医疗科技有限公司 | A kind of detection method of detection kit and preparation method thereof and troponin T |
| CN111487409A (en) * | 2019-01-28 | 2020-08-04 | 艾维可生物科技有限公司 | Chemiluminescence detection kit for S100B protein and use method thereof |
| CN112834742A (en) * | 2021-01-11 | 2021-05-25 | 中国科学院苏州生物医学工程技术研究所 | Non-cleaning magnetic particle chemiluminescence immunoassay method |
| CN112986555A (en) * | 2021-02-07 | 2021-06-18 | 武汉生之源生物科技股份有限公司 | GPC-3 chemiluminescence kit |
| CN114152742A (en) * | 2021-11-30 | 2022-03-08 | 深圳市易瑞生物技术股份有限公司 | Kit for light-activated chemiluminescence immunoassay containing magnetic luminescent microspheres and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101165487A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for biological molecule detection by nanometer gold magnetic particle |
| CN101713780A (en) * | 2009-12-07 | 2010-05-26 | 陕西北美基因股份有限公司 | Method for magnetic antibody immunoassay chemiluminescence detection of treponema pallidum antibodies |
| CN102331502A (en) * | 2011-08-31 | 2012-01-25 | 内蒙古科慧生物科技有限责任公司 | Quantitative measurement kit and detection method for human S100 protein (S-100) |
| CN102721804A (en) * | 2012-05-29 | 2012-10-10 | 西安金磁纳米生物技术有限公司 | Electrical chemiluminescence immunoassay based on gold magnetic particles |
| CN102735842A (en) * | 2012-05-29 | 2012-10-17 | 西安金磁纳米生物技术有限公司 | Method of detecting small molecule substances based on chemiluminescence immunology of golden-magnetic particles |
| CN103604931A (en) * | 2013-11-15 | 2014-02-26 | 陆上苏 | Human S100 protein detection reagent and preparation method thereof |
-
2015
- 2015-04-17 CN CN201510184976.3A patent/CN104777315A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101165487A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for biological molecule detection by nanometer gold magnetic particle |
| CN101713780A (en) * | 2009-12-07 | 2010-05-26 | 陕西北美基因股份有限公司 | Method for magnetic antibody immunoassay chemiluminescence detection of treponema pallidum antibodies |
| CN102331502A (en) * | 2011-08-31 | 2012-01-25 | 内蒙古科慧生物科技有限责任公司 | Quantitative measurement kit and detection method for human S100 protein (S-100) |
| CN102721804A (en) * | 2012-05-29 | 2012-10-10 | 西安金磁纳米生物技术有限公司 | Electrical chemiluminescence immunoassay based on gold magnetic particles |
| CN102735842A (en) * | 2012-05-29 | 2012-10-17 | 西安金磁纳米生物技术有限公司 | Method of detecting small molecule substances based on chemiluminescence immunology of golden-magnetic particles |
| CN103604931A (en) * | 2013-11-15 | 2014-02-26 | 陆上苏 | Human S100 protein detection reagent and preparation method thereof |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061186A (en) * | 2018-08-14 | 2018-12-21 | 深圳天辰医疗科技有限公司 | A kind of Procalcitonin detection kit and Procalcitonin detection method |
| CN109596835A (en) * | 2018-11-23 | 2019-04-09 | 深圳天辰医疗科技有限公司 | A kind of detection method of detection kit and preparation method thereof and troponin T |
| CN109596835B (en) * | 2018-11-23 | 2022-02-08 | 深圳天辰医疗科技有限公司 | Detection kit, preparation method thereof and troponin T detection method |
| CN111487409A (en) * | 2019-01-28 | 2020-08-04 | 艾维可生物科技有限公司 | Chemiluminescence detection kit for S100B protein and use method thereof |
| CN112834742A (en) * | 2021-01-11 | 2021-05-25 | 中国科学院苏州生物医学工程技术研究所 | Non-cleaning magnetic particle chemiluminescence immunoassay method |
| CN112834742B (en) * | 2021-01-11 | 2024-05-03 | 中国科学院苏州生物医学工程技术研究所 | Non-cleaning magnetic particle chemiluminescence immune detection method |
| CN112986555A (en) * | 2021-02-07 | 2021-06-18 | 武汉生之源生物科技股份有限公司 | GPC-3 chemiluminescence kit |
| CN112986555B (en) * | 2021-02-07 | 2024-05-07 | 武汉生之源生物科技股份有限公司 | GPC-3 chemiluminescence kit |
| CN114152742A (en) * | 2021-11-30 | 2022-03-08 | 深圳市易瑞生物技术股份有限公司 | Kit for light-activated chemiluminescence immunoassay containing magnetic luminescent microspheres and application thereof |
| CN114152742B (en) * | 2021-11-30 | 2024-05-28 | 深圳市易瑞生物技术股份有限公司 | Kit for photoexcitation chemiluminescence immunoassay containing magnetic luminescence microspheres and application of kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104897901B (en) | A kind of method of the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles | |
| CN104777315A (en) | Chemiluminescence immunoassay method for detecting S100 based on gold magnetic particles | |
| CN101713779B (en) | Method for performing immunological test on biomolecules by avidin/streptavidin magnetic composite particles | |
| US10732111B2 (en) | Automated immunoanalyzer system for performing diagnostic assays for allergies and autoimmune diseases | |
| CN105548565B (en) | A kind of kit and its preparation and application for being used to detect schizotrypanum cruzi antibody | |
| JP5200003B2 (en) | Detection of target molecules in a sample using a magnetic field | |
| CN101441210B (en) | Nano magnetic particle chromatography test paper detection method | |
| US6110660A (en) | Procedure for quantitative and qualitative determination of chemical substances, based on molecular recognition and measurement of magnetic permeability | |
| JP5628793B2 (en) | Method for detecting a substance in a biological sample | |
| CN110988331A (en) | Microfluidic chip detection method based on magnetic bead technology and reagent freeze-drying technology and microfluidic chip | |
| EP3315969B1 (en) | Method of detecting test substance by immune complex transfer method | |
| CN102735842A (en) | Method of detecting small molecule substances based on chemiluminescence immunology of golden-magnetic particles | |
| CN101313217A (en) | Sensitive magnetic catch assay by building a strong binding couple | |
| CN108508001A (en) | Chemiluminescence detection kit | |
| WO1996004558A1 (en) | Measurement system using whole blood | |
| WO2007092302A2 (en) | Test device for analyte detection | |
| US20200326338A1 (en) | Detection agent for bioassay and signal amplification method using same | |
| CN101165490A (en) | Method for immunological detection for biological molecule of body fluid by gold magnetic particle | |
| EP0660935B1 (en) | Immunological detection using two detectable labels | |
| WO1994011734A1 (en) | Two-site immunoassay for an antibody with chemiluminescent label and biotin bound ligand | |
| CN103175858A (en) | Method for quickly detecting NMR (Nuclear Magnetic Resonance) food-borne pathogenic bacteria based on indirectly enriched Fe3O4 nano particles | |
| CN106442480A (en) | OTA chemiluminescence detection method based on horseradish peroxidase marker aptasensor | |
| KR20010025027A (en) | Immunoassay reagents and immunoassay method | |
| CN103364428A (en) | Rapid detection method for NMR (nuclear magnetic resonance) nano-probe food-borne pathogenic bacteria based on gamma-Fe2O3 | |
| JP2596959B2 (en) | Ligand assays |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150715 |