CN102721813A - Homogeneous luminous immunoassay assay kit for prostate specific antigen and detection method therefor - Google Patents
Homogeneous luminous immunoassay assay kit for prostate specific antigen and detection method therefor Download PDFInfo
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Abstract
The invention discloses a homogeneous luminous immunoassay assay kit for prostate specific antigen (PSA) and a detection method therefor. The assay kit comprises a homogeneous luminous 96-pore detection board, a PSA standard product and a streptavidin labeling donor microsphere solution, and further comprises a biotin labeling PSA antibody and a PSA antibody coating receptor microsphere solution. During detection, a to-be-detected sample, a biotin labeling antibody and specific antibody coating receptor microspheres are sequentially added into the detection board and subjected to incubation after being mixed, then streptavidin labeling donor microspheres are added into the mixture for continuous incubation; a homogeneous luminousimmunoassay instrument is used for detecting the luminous intensities of all pores; a four-parameter fitting method is adopted to draw a standard curve so as to calculate the concentration of PSA in the to-be-detected sample; a 'detection after mixing' mode is formed and washing is not required in the detection process; the advantages of both enzyme immunoassay and electrochemical luminescence immunoassay are integrated; the cost is lower; and therefore, the assay kit is suitable for being popularized and applied in basic hospitals and applicable to high-throughput screening of large samples.
Description
Technical field
The present invention relates to optical excitation coupling immunoassay technology, specifically is a kind of prostate specific antigen homogeneous luminescent immunoassay detection kit and detection method thereof.
Background technology
1971, people such as Hara found that at first (prostate specific antigen, PSA), it to seminal fluid, is one of principal ingredient of refining by the synthetic justacrine of prostate epithelial cell to prostate specific antigen.Prostate specific antigen has organ specificity, exists only in the kytoplasm and mucus of prostate epithelial cell, ductal epithelial cell, has chymotrypsin appearance and tryptic activity.Under the normal condition, exist the barrier that constitutes by endodermis, basal-cell layer and basilar memebrane to be separated by between prostate acinus and the lymphatic system, prostate specific antigen can not occur in the peripheral blood.When tumour or other pathology take place when, destroy barrier between prostate and lymphatic system tissue, the acinus content that the is rich in prostate specific antigen lymphatic system that bleeds, and get into blood circulation in succession, make prostate specific antigen rising in the peripheral blood.Prostate specific antigen is that generally acknowledge at present unique has organ specific blood serum tumor markers.The serum prostate specific antigen raises generally, and there is pathology (prostatitis, benign prostatic hyperplasis, prostate cancer) in the prompting prostate.Detect serum or blood plasma prostate specific antigen content, not only can be in the people at highest risk early detection prostate cancer, also can be used for the aspects such as monitoring, surgical result assessment after the patients with prostate cancer treatment simultaneously.
Prostate specific antigen has good immunogenicity, can prepare specific antibody and measures through the immunochemistry principle.The labelled immune analysis is divided into radiommunoassay, EIA enzyme immunoassay and luminescence immunoassay according to the label difference, and the serum prostate specific antigen mainly adopts EIA enzyme immunoassay and electrochemiluminescence immunoassay.No matter adopt which kind of analysis mode, it is basic identical that it detects principle, promptly adopts double antibody sandwich method to measure unknown prostate specific antigen: wherein a kind of antibody is connected with solid phase carrier as capture antibody, is used for catching sample prostate specific antigen to be measured; With another kind of antibody and probe material (horseradish peroxidase or tris (bipyridine) ruthenium) (detection) antibody that combines to serve as a mark; When labelled antibody with after prostate specific antigen combines, form capture antibody-determined antigen-labelled antibody compound at surface of solid phase carriers.With the solid phase carrier washing, remove superfluous label or participate in the composition that reacts; EIA enzyme immunoassay through enzymatic reaction intensity, carries out quantitative test to unknown antigen through adding substrate.And electrochemiluminescence adds tripropyl amine (TPA) and electrode surface generation redox reaction again, through light signal unknown antigen is carried out quantitative test.
The main advantage that EIA enzyme immunoassay detects prostate specific antigen shows: detectable has realized production domesticization, ELIASA popularity height; Therefore, EIA enzyme immunoassay has the low cost that detects, and can carry out the work at basic hospital.But owing to adopt horseradish peroxidase as probe material, less stable and enzymatic activity disturbing factor are more, cause the precision of EIA enzyme immunoassay not ideal enough.In addition, EIA enzyme immunoassay adopts micro reaction plate as solid phase material, since the restriction of coated antibody, the sensing range of can not realizing ideal.The electrochemiluminescence immunoassay is to detect the higher labelled immune analytical technology of performance at present, has higher automatization level, higher detection performance (aspects such as susceptibility, precision, stability).But the electrochemiluminescence detector is expensive, the detectable dependence on import, thus make the detection cost very high, and basic medical treatment unit is difficult to carry out.For these reasons, enzyme immunoassay (EIA) is mainly used in prostate cancer people at highest risk's examination, and patients with prostate cancer is made a definite diagnosis and aspects such as surgical result assessment and monitoring tumor recurrence and the electrochemiluminescence immunoassay is mainly used in.
In labelled immune is analyzed,, after reaction reaches balance, still there is remaining, the labelled antibody of conjugated antigen not in the system in reaction system because labelled antibody (or antigen) is excessive.Wherein, heterogeneous immunoassay adopts solid phase adsorption method separating and combining label and free label.The solid phase adsorption partition method is that antigen (antibody) is adsorbed on surface of solid phase carriers; Immune response is carried out on surface of solid phase carriers; Test substance (antibody or antigen), labelled antibody (antigen) all can be combined in the solid phase material surface through immune response, and bound substances (containing free label) is not present in the liquid phase.At this moment, discard liquid phase and, just can remove free label through washing.ELISA and electrochemiluminescence immunoassay detect prostate specific antigen in the serum; Two kinds of detection methods all belong to heterogeneous immunoassay from detecting on the principle; EIA enzyme immunoassay adopts the enzyme linked immunoassay plate as solid phase material, and the electrochemiluminescence immunoassay adopts magnetic microsphere as solid phase material.
The solid phase adsorption separation method has two important steps: encapsulate and wash.The design of solid phase adsorption separation method is ingenious, simple to operate, so use very extensive.But this kind heterogeneous reaction pattern is because of encapsulating and wash the existence of link, and analysis causes very big defective to labelled immune, mainly shows following two aspects:
1. in the process of encapsulating, no matter physisorption is still chemical connects, and its conformation of antibody molecule that encapsulates solid phase material surface, back is different from the antibody molecule that is in the liquid phase, will receive certain limitation with the antigen molecule binding ability because of space steric effect; No matter adopt microballoon still be microwell plate as solid phase material because it is limited to encapsulate area, the capture antibody molecule can not satisfy the needs of a large amount of determined antigens to greatest extent, will influence sensing range thus.
2. " wash plate " or " washing ball " is the important step in the non-homogeneous immunoassay and repeatedly occurs.Washing process increases the complicacy of trace routine, increases detection time and brings obstacle for the realization robotization.In addition, because washing process EIA enzyme immunoassay particularly is difficult to realize standardization, clean result is different between each instrument connection, the precision that detects of influence to a certain extent, between occurring batch, batch in difference.
In a word, with regard to serum prostate specific antigen test item, no matter be to adopt EIA enzyme immunoassay or adopt the electrochemiluminescence immunoassay, all there is the defective on the certain methods in the two.The operation that has is loaded down with trivial details, and the requirement for experiment condition that has is too high; The proving time that has is oversize, and the consumptive material expense that has is too high.These weak points not only increase the cost of seeking medical advice to the patient, also limit the popularization and application of this test item simultaneously, and the diagnosis and treatment work that influences different medical unit is carried out.
Summary of the invention
The present invention is the problems referred to above that exist for the prior art that solves, and a kind of prostate specific antigen homogeneous luminescent immunoassay detection kit and the detection method thereof based on optical excitation coupling immunoassay principle that can carry out easily and fast and accurately detect serum prostate specific antigen content is provided.
The present invention realizes by following technical scheme.
A kind of prostate specific antigen homogeneous luminescent immunoassay detection kit; Mainly comprise homogeneous luminescent 96 hole assay plate; The prostate specific antigen standard items; Marked by streptavidin donor microspheres solution, and this kit also includes biotin labeling anti-prostate-specific-antigen antibody, anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution.
Said prostate specific antigen homogeneous luminescent immunoassay detection kit, its anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution prepares by following proportion raw material,
A. the anti-prostate-specific-antigen antibody that will desire mark places the dialysis of 0.1M pH 8.0 phosphate buffers, and adjustment concentration is subsequent use to the 1-2 mg/ml;
B. the acceptor microballoon joins in 0.1M pH 8.0 phosphate buffered salt solutions, and centrifugal 16000 rev/mins of washing microballoons are abandoned supernatant and with above-mentioned solution microballoon concentration are adjusted to the 10-20 mg/ml;
C. by the acceptor microballoon: dialysis back anti-prostate-specific-antigen antibody is that the mass ratio of 1:10 is mixed in 0.1M pH 8.0 phosphate buffered salt solutions; Adding percent by volume successively is the freshly prepared 400 mM sodium cyanoborohydride solution of 10% polysorbas20 solution, 4-5% of 0.6-0.625%; Abundant mixing, 37 ℃ of reactions are more than 48 hours;
D. using 800mM sodium hydroxide solution preparation solubility is the carboxymethyl methylamino amine aqueous solution of 65 mg/ml, adds percent by volume and is in 5% the centrifuge tube of carboxymethyl methylamino amine aqueous solution behind mark, 37 ℃ of cappings 1 hour;
E. draw supernatant solution, add the 100mM Tris-HCl solution of pH 8.0 again, the microballoon that suspends again, the centrifugal ball of washing, it is subsequent use to the 5-10 mcg/ml finally to adjust concentration.
Said prostate specific antigen homogeneous luminescent immunoassay detection kit, its biotin labeling anti-prostate-specific-antigen antibody prepares by following proportion raw material,
A. the anti-prostate-specific-antigen antibody with purifying is adjusted into 5.0 mg/ml, uses 0.1M, and the dialysis of the carbonate buffer solution of pH9.5 is spent the night and in A
280nmThe calculating antibody total amount is 5.35 milligrams;
B. the biotin with activation is dissolved in the dimethyl formamide, is that the ratio of 20:1 is mixed the two according to the biotin and the mol ratio of the anti-prostate-specific-antigen antibody of purifying, reacts 1 hour;
C. with reacted liquid with the 0.01M phosphate buffered saline (PBS) in 4 ℃ the dialysis 24 hours, promptly process biotin labeling anti-prostate-specific-antigen antibody.
A kind of prostate specific antigen homogeneous luminescent immunoassay detection method, it carries out according to the following steps,
A. will distinguish bottled biotin labeling anti-prostate-specific-antigen antibody, prostate specific antigen standard items, anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution, marked by streptavidin donor microspheres solution and take out, to equilibrium at room temperature 20 minutes; Homogeneous luminescent 96 hole assay plate are marked as required;
B. add 25 microlitre samples to be tested (or prostate specific antigen standard items), 25 microlitre biotin labeling anti-prostate-specific-antigen antibody, 25 microlitre anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution in the assay plate micropore successively; Rearmounted 37 ℃ of incubators of mixing or water-bath, incubation 30 minutes;
C. each hole adds 100 microlitre marked by streptavidin donor microspheres solution again, and rearmounted 37 ℃ of incubators of mixing or water-bath continued incubation 15 minutes;
D. use the homogeneous luminescent immunity analysis instrument and measure each hole luminous intensity, excitation wavelength adopts 680 nanometers, detects wavelength and adopts 615 nanometers;
E. standard items are measured the result, adopt four parameter fitting mode drawing standard curves or obtain mathematical function, calculate testing sample prostate specific antigen concentration through typical curve or mathematical function;
The present invention integrates the advantage of EIA enzyme immunoassay and electrochemiluminescence immunoassay, and overcomes the shortcoming of two kinds of methods, accurately measures prostate specific antigen level in the serum.Whole mensuration process reduces the deviation that causes owing to " washing "; Have very strong anti-interference ability, low background signal makes testing result have degree of precision, particularly determinand content is in the sample of " gray area (critical point) ", and the homogeneous luminescent immunoassay has very high resolution.Can a shared typical curve with batch kit, each mensuration does not need independent calibration.The antigen and antibody specific association reaction takes place and have only, acceptor microballoon and donor microballoon could near, and then cause follow-up cascade and amplify reaction, be a kind of " after the mixing i.e. mensuration " pattern.Be applicable to prostate cancer people at highest risk examination, auxiliary diagnosis and result of treatment assessment, and the antidiastole of benign prostatic hyperplasia and malignant prostate cancer.
Description of drawings
Fig. 1 is a detection principle schematic of the present invention;
Fig. 2 is an operating process synoptic diagram of the present invention.
Wherein:
1: marked by streptavidin donor microballoon 2: biotin labeling anti-prostate-specific-antigen antibody
3: anti-prostate-specific-antigen antibody sandwich acceptor microballoon 4: prostate specific antigen to be measured
5: excitation source 6: fluorescence detecting system.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is carried out detailed explanation.
Present embodiment is based on optical excitation coupling immunoassay, to the double antibody detection of prostate specific antigen content in the serum.
One. detect principle
Optical excitation coupling immune analysis method system is made up of the microballoon of two kind of 100 nanometer, utilizes the interaction that comes the detection of biological molecule as the microballoon of donor and acceptor, and microsphere surface has covered one deck hydrogel, as the functional group of biology connection.Can donor and acceptor microballoon be furthered when biomolecule exists when interacting, thereby the chemical reaction that excites cascade to amplify produces the several times enhancing signal.Specifically, under the irradiation of laser (wavelength 680 nanometers), the photosensitizer on the donor microballoon is more active free oxygen with the oxygen conversion in the surrounding environment.Free oxygen diffuses to the acceptor microballoon, and the chemiluminescence agent reaction surperficial with it further activated same fluorophor on acceptor, makes it to send fluorescence, and wavelength is the 520-620 nanometer.
As shown in Figure 1 based on optical excitation coupling immunoassay to the measuring principle of the homogeneous luminescent immune reagent kit of prostate specific antigen content in the serum.
Utilize optical excitation coupling immune analysis method; At acceptor microballoon (luminous microballoon) pan coating anti-prostate-specific-antigen antibody (polyclonal antibody); With anti-prostate-specific-antigen antibody (monoclonal antibody) mark biotin molecule, preparation biotin labeling anti-prostate-specific-antigen antibody; Simultaneously, encapsulate Streptavidin in advance on donor microballoon (sensitization microballoon) surface.As there being prostate specific antigen (standard items or testing sample) in the reaction system; Specificity takes place with the anti-prostate-specific-antigen antibody of biotin labeling anti-prostate-specific-antigen antibody and acceptor microsphere surface simultaneously and combines in prostate specific antigen simultaneously, forms the double-antibody sandwich compound in the acceptor microsphere surface; At this moment; As add the donor microballoon that Streptavidin encapsulates; Biotin combine with Streptavidin and make two microballoons each other near, under the exciting of LASER Light Source (680 nanometer), the photosensitizer of donor microsphere surface converts the normality oxygen molecule in the solution to an electronics singlet oxygen.The oxygen molecule of singlet spreads in solution, produces chemiluminescence after in solution, running into the acceptor microballoon, thus further excite fluorophor on the same microballoon to produce cascade to amplify reaction and produce fluorescence (615 nanometer).At this moment, prostate specific antigen to be measured is many more, each other near the D-A microballoon many more, then fluorescence intensity is strong more.Concrete quantivative approach is to use the prostate specific antigen of known variable concentrations to be standard items, by obtaining a dose-response curve (or mathematical function relationship) after above-mentioned pattern reaction, the mensuration; As unknown concentration sample to be measured is operated equally, above-mentioned dose-effect curve then capable of using obtains the concentration of prostate specific antigen to be measured in the sample.
Two. reagent constituents
Prostate specific antigen homogeneous luminescent immunoassay detection kit mainly comprises following component:
1. biotin labeling anti-prostate-specific-antigen antibody,
2. prostate specific antigen standard items,
3. anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution,
4. marked by streptavidin donor microspheres solution,
5. homogeneous luminescent 96 hole assay plate.
Three. the preparation method
1. critical materials
1. 96 hole luminescent immunoassay plates available from platinum-Elmer Co., Ltd, are different from the EIA enzyme immunoassay reaction plate, and it is a reaction vessel, not envelope antigen or antibody.
2. acceptor microballoon 50 μ l 20 mg/ml are available from platinum-Elmer Co., Ltd.
3. marked by streptavidin donor microballoon 200 μ l 5 mg/ml are available from platinum-Elmer Co., Ltd.
4. biotin labeling anti-prostate-specific-antigen antibody, preparation voluntarily.
5. anti-prostate-specific-antigen antibody is available from Britain Abcam company.
6. pure article of prostate specific antigen are available from U.S. Sigma company.
7. the biotin of activation (available from U.S. Sigma company).
2. reagent constituents preparation
1. prostate specific antigen standard items
Take by weighing the pure article of prostate specific antigen, use 0.1M pH 7.4 phosphate buffered salt solutions that contain 20% deactivation calf serum to be mixed with the series standard article solution of 0,2,10,25,50,100 nanograms/milliliter, standard items must be through international quality controlled serum calibration.
2. anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution
A. pre-service antibody and assay: the anti-prostate-specific-antigen antibody that will desire mark is packed in the bag filter, places the dialysis of 0.1M pH 8.0 phosphate buffers, removes Tris, glycocoll etc. and contains amino composition.Antibody-solutions after the dialysis is measured antibody content with ultraviolet spectrophotometer, adjustment concentration to 2 mg/ml.
B. wash microballoon: get 50 microlitres (20 mg/ml) acceptor microballoon and place 1.5 milliliters of plastic centrifuge tubes, add 0.1M pH 8.0 phosphate buffered salt solutions, centrifuge washing 2 times, each 16000 * G 15 minutes, it is for use to blot supernatant.
C. mark: add anti-prostate-specific-antigen antibody after 0.1 milligram of dialysis in the above-mentioned centrifuge tube; And be settled to 200 microlitres with 0.1M pH 8.0 phosphate buffered salt solutions, add 1.25 microlitres, 10% polysorbas20s (Tween-20) solution, the freshly prepared 400 mM sodium cyanoborohydride (NaBH of 10 microlitres more successively
3CN) solution, fully mixing was put 37 ℃ of incubator internal reactions more than 48 hours.
D. sealing: using 800mM sodium hydroxide solution preparation solubility is the carboxymethyl methylamino amine aqueous solution of 65 mg/ml.Add in the centrifuge tube behind 10 microlitre carboxymethyl methylamino amine aqueous solutions and the mark, put 37 ℃ of incubator internal reactions 1 hour.
E. wash ball: centrifuge tube is placed in the hydro-extractor centrifugal 15 minutes of 16000 * G; With pipette, extract supernatant solution, add 200 microlitre 100mM Tris-HCl (pH 8.0) damping fluids again, microballoon again suspends; Repeated centrifugation, it is subsequent use to be suspended into 50 microlitres once more.
3. the preparation method for antibody of biotin labeling anti-prostate-specific-antigen:
A. the anti-prostate-specific-antigen antibody with purifying is adjusted into 5.0 mg/ml, uses 0.1M, and the dialysis of the carbonate buffer solution of pH9.5 is spent the night and in A
280nmThe calculating antibody total amount is 5.35 milligrams;
B. the biotin with activation is dissolved in the dimethyl formamide (DMF), is that the ratio of 20:1 is mixed the two according to the biotin and the mol ratio of anti-prostate-specific-antigen antibody, reacts 1 hour.
C. with reacted liquid with 0.01M phosphate buffered saline (PBS) (PBS) in 4 ℃ the dialysis 24 hours, promptly process biotin labeling anti-prostate-specific-antigen antibody.
4. prepare the acceptor microspheres solution with pH 8.0 0.1 M Tris-HCl damping fluids with mark after the acceptor microballoon be diluted to 20 mcg/ml (25 microlitre/tests, 0.5 microgram).
5. 10 * Tris-HCl measures the damping fluid preparation: 1M Tris, 0.1% Tween-20,0.05% Proclin-300, during use with 10 times of distilled water dilutings.
6. prepare the donor microspheres solution
With pH 8.0 0.1 M Tris-HCl damping fluids with mark after the donor microballoon be diluted to 5 mcg/ml (100 microlitre/tests, 0.5 microgram).
Four. method of application
As shown in Figure 2 to the measuring principle of the homogeneous luminescent immune reagent kit of prostate specific antigen content in the serum based on optical excitation coupling immunoassay, it is following to detect step.
1. will distinguish bottled biotin labeling anti-prostate-specific-antigen antibody, prostate specific antigen standard items, anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution, marked by streptavidin donor microspheres solution and take out, to equilibrium at room temperature 20 minutes; Homogeneous luminescent 96 hole assay plate are marked as required;
2. add 25 microlitre samples to be tested (or prostate specific antigen standard items), 25 microlitre biotin labeling anti-prostate-specific-antigen antibody, 25 microlitre anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution in the assay plate micropore successively; Rearmounted 37 ℃ of incubators of mixing or water-bath, incubation 30 minutes;
3. each hole adds 100 microlitre marked by streptavidin donor microspheres solution again, and rearmounted 37 ℃ of incubators of mixing or water-bath continued incubation 15 minutes.
4. use the homogeneous luminescent immunity analysis instrument and measure each hole luminous intensity, excitation wavelength adopts 680 nanometers, detects wavelength and adopts 615 nanometers.
5. standard items are measured the result, adopt four parameter fitting mode drawing standard curves or obtain mathematical function, calculate testing sample prostate specific antigen concentration through typical curve or mathematical function.
Claims (4)
1. prostate specific antigen homogeneous luminescent immunoassay detection kit; Mainly comprise homogeneous luminescent 96 hole assay plate; The prostate specific antigen standard items; Marked by streptavidin donor microspheres solution is characterized in that: this kit also includes biotin labeling anti-prostate-specific-antigen antibody and anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution.
2. according to the said prostate specific antigen homogeneous luminescent of claim 1 immunoassay detection kit, it is characterized in that:
Anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution prepares by following proportion raw material,
A. the anti-prostate-specific-antigen antibody that will desire mark places the dialysis of 0.1M pH 8.0 phosphate buffers, and adjustment concentration is subsequent use to the 1-2 mg/ml;
B. the acceptor microballoon joins in 0.1M pH 8.0 phosphate buffered salt solutions, and centrifugal 16000 rev/mins of washing microballoons are abandoned supernatant, with above-mentioned solution microballoon concentration are adjusted to the 10-20 mg/ml;
C. by the acceptor microballoon: dialysis back anti-prostate-specific-antigen antibody is that the mass ratio of 1:10 mixes; In 0.1M pH 8.0 phosphate buffered salt solutions; Adding percent by volume successively is the freshly prepared 400 mM sodium cyanoborohydride solution of 10% polysorbas20 solution, 4-5% of 0.6-0.625%; Abundant mixing, 37 ℃ of reactions are more than 48 hours;
D. using 800mM sodium hydroxide solution preparation solubility is the carboxymethyl methylamino amine aqueous solution of 65 mg/ml, and adding percent by volume is in the centrifuge tube of carboxymethyl methylamino amine aqueous solution behind mark of 4-5%, 37 ℃ of cappings 1 hour;
E. draw supernatant solution, add the 100mM Tris-HCl solution of pH 8.0 again, the microballoon that suspends again, the centrifugal ball of washing, it is subsequent use to the 5-10 mcg/ml finally to adjust concentration.
3. according to the said prostate specific antigen homogeneous luminescent of claim 1 immunoassay detection kit, it is characterized in that: biotin labeling anti-prostate-specific-antigen antibody prepares by following proportion raw material,
A. the anti-prostate-specific-antigen antibody with purifying is adjusted into 5.0 mg/ml, uses 0.1M, and the dialysis of the carbonate buffer solution of pH9.5 is spent the night and in A
280nmThe calculating antibody total amount is 5.35 milligrams;
B. the biotin with activation is dissolved in the dimethyl formamide, is that the ratio of 20:1 is mixed the two according to the biotin and the mol ratio of the anti-prostate-specific-antigen antibody of purifying, reacts 1 hour;
C. with reacted liquid with the 0.01M phosphate buffered saline (PBS) in 4 ℃ the dialysis 24 hours, promptly process biotin labeling anti-prostate-specific-antigen antibody.
4. prostate specific antigen homogeneous luminescent immunoassay detection method, it carries out according to the following steps,
A. will distinguish bottled biotin labeling anti-prostate-specific-antigen antibody, prostate specific antigen standard items, anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution, marked by streptavidin donor microspheres solution and take out, to equilibrium at room temperature 20 minutes; Homogeneous luminescent 96 hole assay plate are marked as required;
B. add 25 microlitre samples to be tested, 25 microlitre biotin labeling anti-prostate-specific-antigen antibody, 25 microlitre anti-prostate-specific-antigen antibody sandwich acceptor microspheres solution in the assay plate micropore successively, rearmounted 37 ℃ of incubators of mixing or water-bath, incubation 30 minutes;
C. each hole adds 100 microlitre marked by streptavidin donor microspheres solution again, and rearmounted 37 ℃ of incubators of mixing or water-bath continued incubation 15 minutes;
D. use the homogeneous luminescent immunity analysis instrument and measure each hole luminous intensity, excitation wavelength adopts 680 nanometers, detects wavelength and adopts 615 nanometers;
E. standard items are measured the result, adopt four parameter fitting mode drawing standard curves or obtain mathematical function, calculate testing sample prostate specific antigen concentration through typical curve or mathematical function.
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