CN105929151A - Chemiluminiscence immune detection kit for homologous isomer of prostate-specific antigen and preparation method of chemiluminiscence immune detection kit - Google Patents

Chemiluminiscence immune detection kit for homologous isomer of prostate-specific antigen and preparation method of chemiluminiscence immune detection kit Download PDF

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CN105929151A
CN105929151A CN201610505968.9A CN201610505968A CN105929151A CN 105929151 A CN105929151 A CN 105929151A CN 201610505968 A CN201610505968 A CN 201610505968A CN 105929151 A CN105929151 A CN 105929151A
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specific antigen
prostate specific
antigen isoform
monoclonal antibody
prostate
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段桂开
钱纯亘
夏福臻
王刚
祝亮
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Shenzhen Yhlo Biotech Co Ltd
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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Abstract

The invention discloses a chemiluminiscence immune detection kit for a homologous isomer of a prostate-specific antigen and a preparation method of the chemiluminiscence immune detection kit. The chemiluminiscence immune detection kit comprises carboxylated magnetic micro-particles and a chemiluminiscence marker, wherein the carboxylated magnetic micro-particles are coated with a monoclonal antibody of the homologous isomer of the prostate-specific antigen, and the chemiluminiscence marker is labeled by statin monoclonal antibody. According to the chemiluminiscence immune detection kit, a full-automatic chemiluminiscence immune analysis meter is used as a detection tool for finishing the detection of the homologous isomer of the prostate-specific antigen. Experiments show that the detection sensitivity reaches 1pg/mL and is increased by at least 10 times compared with that in a traditional detection method for the homologous isomer of the prostate-specific antigen. The chemiluminiscence immune detection kit for the homologous isomer of the prostate-specific antigen is relatively high in detection precision.

Description

Prostate specific antigen isoform chemiluminescence immune detection reagent kit and preparation method thereof
Technical field
The present invention relates to vitro detection field, particularly relate to a kind of prostate specific antigen isoform Learn electrochemiluminescent immunoassay detection kit and preparation method thereof.
Background technology
Carcinoma of prostate has become as one of the highest tumor of the Western countries sickness rate, it is estimated that, the annual whole world is new Ill patients with prostate cancer has about 914,000 people because carcinoma of prostate and the patient of death is about 258, 000 people, the carcinoma of prostate morbidity example of China was riseing year by year in recent years.Early discovery carcinoma of prostate is to prostate The treatment of cancer is particularly significant, and at present, the goldstandard of prostate cancer diagnosis is still aspiration biopsy of prostatic gland.
The end of the eighties in last century, the mankind be found that prostate specific antigen (Prostate-specific Antigen, PSA) be carcinoma of prostate prediction important symbol thing.But, in utilization for many years, the limitation of PSA is also Day by day highlight, the relatively low defect of specificity result in the most excessively puncture, the generation of over-treatment, for disease People causes psychological burden, causes financial burden for society.Therefore, people are devoted to find new specificity Tumor markers is used for predicting carcinoma of prostate.In recent years, along with developing rapidly of molecular biology, Ren Menfa Having showed the tumour-specific mark of multiple carcinoma of prostate, part has been applied to clinic, simultaneously along with mankind's base The development learned because of group, people are in succession found that prostate cancer risk gene, and expect gene test as just The instrument of step examination carcinoma of prostate high-risk group.
Recently, one is referred to as the PSA precursor quilt of prostate specific antigen isoform (p2PSA) Finding, research confirms that p2PSA and derivative index thereof have with carcinoma of prostate in the crowd of Europe and significantly associates, For predicting that carcinoma of prostate is significant clinically.
The common methods of Clinical detection p2PSA has enzyme linked immunosorbent assay, radio immunoassay, enzyme at present Promote chemoluminescence method, but in place of these methods all also exist some shortcomings.
One, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but the method there is also following weak point:
(1) use 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate as antigen bag By apparatus and reaction vessel, 12 batches, 6 batches, 8 batches or imposite can only be divided in use and once make With, it is impossible to carry out independent, the detection of single part；
(2) reagent type used by quantitative determination is more, and each detectable will contain with reagent bottle, And it is required for changing imbibition nozzle when often using a kind of reagent to be filled into respectively in the micropore of microwell plate, not only Reagent bottle kind is many, and the operation of filling reagent is the most loaded down with trivial details；
(3) the corresponding mark to detection information is lacked, can only be by checking the mark ability of test kit external packing box Understand or know product batch number and the effect duration information of detectable, and the information known is in detection process In uncontrolled, there is the biggest randomness；
(4) detectable is in open space during detection, easily causes the intersection between various reagent Pollute and affect the accuracy of testing result；
(5) detection process uses manual operations, and the dosage of reagent or sample is not bery accurate, and operating process is extremely Loaded down with trivial details and complicated, it is susceptible to bust, accuracy and the precision of testing result are poor；
(6) in the quantity configuration and use of detection project reagent set, it is item number × 48/96 person-portion, if Need to detect 10 projects, then configuration and the use number of reagent must be 10 × 48/96 person-portions, if only one Part sample needs to detect 10 different projects, it is also desirable to the reagent of configuration 10 × 48/96 person-portions, also exists The shortcoming of economical rationality not.
Two, radio immunoassay
Radioimmunoassay method radioactive pollution is big, load procedure is loaded down with trivial details, operation is complicated, operating time length, Measurement result is unstable, the reagent holding time is short, test kit operating automation degree is low, necessary instrument price The weak points such as costliness.
Two, chemoluminescence method
Chemoluminescence method can be divided into direct chemiluminescence and enzyme-catalyzed chemical luminescence by principle of luminosity.
Enzyme-catalyzed chemical luminescence mainly has horseradish peroxidase (HRP) and alkali phosphatase two kinds, but has Certain limitation, horseradish peroxidase major defect is: luminol is deposited not having horseradish peroxidase In the case of, also can be aoxidized self luminescence by H2O2, background is of a relatively high, affects signal to noise ratio, and reaction is dynamic Mechanics is complicated, and influence factor is many, and result is not sufficiently stable, and will obtain the substrate of highly sensitive and plateau length not Easily.Alkali phosphatase major defect is: the time that substrate reaches plateau is long, and substrate cost is high, causes Testing cost is high, patient burden's weight.
Acridinium ester is compared enzyme-catalyzed chemical luminescence as the direct chemiluminescence of label and is had detailed advantage, mainly Show: reaction need not catalyst, as long as alkaline environment can be carried out, is swift in response, and background luminescence is low, Signal to noise ratio is high, and interference factor is few, and reagent stability is good, can be with two-point calibration, and system is simple, and exciting liquid becomes This is the lowest, acridinium ester easily and after protein bind, and connection photon productivity do not reduce.
Summary of the invention
Based on this, it is necessary to provide the prostate specific antigen isoform that a kind of detection sensitivity is higher Chemiluminescence immune detection reagent kit and preparation method thereof.
A kind of prostate specific antigen isoform chemiluminescence immune detection reagent kit, including: prostatitis The coated carboxylated magnetic particle of gland specific antigen isoform monoclonal antibody and inhibin monoclonal anti The chemiluminescent labels of body tag.
In the described coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody, Described prostate specific antigen isoform monoclonal antibody with the ratio of described carboxylated magnetic particle is 1:25～35.
In the chemiluminescent labels of described inhibin labeling of monoclonal antibody, described prostate specific antigen Isoform monoclonal antibody is 50:1～10 with the ratio of described chemiluminescent labels.
The particle diameter of described carboxylated magnetic particle is 0.05 μm～1 μm.
Described chemiluminescent labels is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
Described prostate specific antigen isoform chemiluminescence immune detection reagent kit, also including Learning luminous substrate liquid, described Chemoluminescent substrate includes A liquid and B liquid.
Described A liquid is H2O2Solution, described B liquid is NaOH solution.
Described prostate specific antigen isoform chemiluminescence immune detection reagent kit, before also including Row gland specific antigen isoform calibration product.
Described prostate specific antigen isoform calibration product be concentration be respectively 0pg/mL, 50pg/mL, The prostate specific antigen homology isomery of 200pg/mL, 500pg/mL, 1000pg/mL and 1600pg/mL The solution of body.
A kind of preparation of above-mentioned prostate specific antigen isoform chemiluminescence immune detection reagent kit Method, comprises the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, then adds Enter EDC aqueous solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into prostate specific antigen Isoform monoclonal antibody, suspendible 2h～10h under room temperature, Magneto separate uses Tris buffer after removing supernatant Resuspended, obtain the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody； And
Take prostate specific antigen isoform monoclonal antibody, mix after adding carbonate buffer solution, Mixing after being subsequently adding chemiluminescent labels, remove impurity after lucifuge reaction 1h～2h under room temperature, be inhibited element The chemiluminescent labels of labeling of monoclonal antibody.
This prostate specific antigen isoform chemiluminescence immune detection reagent kit can be with automatically Chemical illumination immunity analysis instrument is detection instrument, complete prostate specific antigen isoform detection this Plant prostate specific antigen isoform chemiluminescence immune detection reagent kit, through experiment, its detection Sensitivity reaches 1pg/mL, relative to the detection method spirit of traditional prostate specific antigen isoform Sensitivity at least improves 10 times, the detection examination of this prostate specific antigen isoform chemiluminescence immunoassay The accuracy of detection of agent box is higher.
Accompanying drawing explanation
Fig. 1 is the prostate specific antigen isoform chemiluminescence immunoassay detectable of an embodiment The flow chart of the preparation method of box；
Fig. 2 is the prostate specific antigen isoform canonical plotting that embodiment 3 obtains.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, below in conjunction with the accompanying drawings and The detailed description of the invention of the present invention is described in detail by specific embodiment.Elaborate in the following description very Many details are so that fully understanding the present invention.But the present invention can be described here to be much different from Alternate manner is implemented, and those skilled in the art can do similar changing in the case of intension of the present invention Entering, therefore the present invention is not limited by following public being embodied as.
The prostate specific antigen isoform chemiluminescence immune detection reagent kit of one embodiment, bag Include: the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody and inhibin The chemiluminescent labels of labeling of monoclonal antibody.
Preferably, the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody In, prostate specific antigen isoform monoclonal antibody is 1 with the ratio of carboxylated magnetic particle: 25～35.
Preferably, in the chemiluminescent labels of inhibin labeling of monoclonal antibody, prostate specific antigen Isoform monoclonal antibody is 50:1～10 with the ratio of chemiluminescent labels.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm～1 μm.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, Chemiluminescent labels is preferably acridinium ester.
In other examples, above-mentioned prostate specific antigen isoform chemiluminescence immunoassay detection Test kit also includes Chemoluminescent substrate.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH Solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be 0.25mol/L NaOH solution.
In other examples, above-mentioned prostate specific antigen isoform chemiluminescence immunoassay detection Test kit also includes that prostate specific antigen isoform calibrates product.
Prostate specific antigen isoform calibration product be concentration be respectively 0pg/mL, 50pg/mL, The prostate specific antigen homology isomery of 200pg/mL, 500pg/mL, 1000pg/mL and 1600pg/mL The solution of body.
Concrete, prostate specific antigen isoform calibration product can use standard substance buffer by front Row gland specific antigen isoform be configured to concentration be respectively 0pg/mL, 50pg/mL, 200pg/mL, The solution of the prostate specific antigen isoform of 500pg/mL, 1000pg/mL and 1600pg/mL.
This prostate specific antigen isoform chemiluminescence immune detection reagent kit is special for prostate During the detection of Specific Antigen isoform, utilize Full-automatic chemiluminescence immunoassay analysis meter to prostate specific Antigen isoform calibration product detect, and draw standard curve, are built in computer software；Then test Actual sample, calculates concentration of specimens according to sample luminous value；Finally to prostate specific antigen homology isomery Body automatic chemiluminescence immunoassay system carries out performance (sensitivity, linear, precision, interference) Evaluation.
This prostate specific antigen isoform chemiluminescence immune detection reagent kit can be with automatically Chemical illumination immunity analysis instrument is detection instrument, complete prostate specific antigen isoform detection this Plant prostate specific antigen isoform chemiluminescence immune detection reagent kit, through experiment, its detection Sensitivity reaches 1pg/mL, relative to the detection method spirit of traditional prostate specific antigen isoform Sensitivity at least improves 10 times, the detection examination of this prostate specific antigen isoform chemiluminescence immunoassay The accuracy of detection of agent box is higher.
Additionally, this prostate specific antigen isoform chemiluminescence immune detection reagent kit also has Advantages below:
1, selection acridinium ester is as marker material, and is applied to chemiluminescence immunoassay system, this luminous body System is direct chemiluminescence, and compared with traditional enzyme-catalyzed chemical luminescence, this reaction need not the participation of enzyme, more Add cost-effective；
2, select acridinium ester chemiluminescence immunoassay system range of linearity width, can reach 1pg/mL～ 1000pg/mL, and the inspection range of linearity of the detection method of traditional prostate specific antigen isoform is 10pg/mL～1000pg/mL；
3, acridinium ester chemiluminescent immunoassay system repeatability is high, in batch and difference between batch is all within 5%, This is that other chemiluminescence immunoassay system is unapproachable；
4, chemiluminescence immunoassay system has realized the quantitative of sample, soft to test by built-in standard curve Part, only needs test sample just can directly obtain the concentration value of sample；
5, chemiluminescence immunoassay system can realize the interpolation of full-automation, reagent and sample instrument entirely Complete, operate easier, decrease artificial error.
Above-mentioned prostate specific antigen isoform chemiluminescence immune detection reagent kit as shown in Figure 1 Preparation method, comprise the steps:
The suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC aqueous solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into prostate specific antigen same Source isomer monoclonal antibody, suspendible 2h～10h under room temperature, Magneto separate uses Tris buffer weight after removing supernatant Outstanding, obtain the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody.
The concentration of MES (2-(N-morpholine) ethyl sulfonic acid) buffer be 0.02M, pH be 5.5.
The concentration of Tris buffer is 0.1M and is 8.0 containing 2%BSA, pH.
The concentration of EDC (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides) aqueous solution is 10mg/mL～20mg/mL, EDC are 0.05:0.1～1 with the ratio of carboxylated magnetic particle.
Preferably, the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody In, prostate specific antigen isoform monoclonal antibody is 1 with the ratio of carboxylated magnetic particle: 25～35.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm～1 μm.
Take prostate specific antigen isoform monoclonal antibody, mix after adding carbonate buffer solution, Mixing after being subsequently adding chemiluminescent labels, remove impurity after lucifuge reaction 1h～2h under room temperature, be inhibited element The chemiluminescent labels of labeling of monoclonal antibody.
Carbonate buffer solution concentration be 0.1M, pH be 9.0～9.5,
The operation of remove impurity is centrifugal desalting column desalination, and concrete operations are: the most respectively by pure water and TBS buffering Liquid (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0) processes centrifugal desalting column, finally adds Enter the molten of the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody that obtains Liquid, finally collects the liquid in centrifuge tube.
Preferably, in the chemiluminescent labels of inhibin labeling of monoclonal antibody, prostate specific antigen Isoform monoclonal antibody is 50:1～10 with the ratio of chemiluminescent labels.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, Chemiluminescent labels is preferably acridinium ester.
The coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody obtained and The chemiluminescent labels combination of inhibin labeling of monoclonal antibody i.e. can get above-mentioned prostate specific antigen Isoform chemiluminescence immune detection reagent kit.
This prostate specific antigen isoform chemiluminescence immune detection reagent kit in use, is gone back Need Chemoluminescent substrate and prostate specific antigen isoform calibration product.
Chemoluminescent substrate and prostate specific antigen isoform calibration product can be prepared voluntarily Arrive.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH Solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be 0.25mol/L NaOH solution.
Concrete, prostate specific antigen isoform calibration product can use standard substance buffer by front Row gland specific antigen isoform be configured to concentration be respectively 0pg/mL, 50pg/mL, 200pg/mL, The solution of the prostate specific antigen isoform of 500pg/mL, 1000pg/mL and 1600pg/mL.
The preparation method letter of this prostate specific antigen isoform chemiluminescence immune detection reagent kit Folk prescription just, the detection of prostate specific antigen isoform chemiluminescence immune detection reagent kit prepared Sensitivity is higher, has a good application prospect.
It it is below specific embodiment.
Embodiment 1: the preparation of prostate specific antigen isoform chemiluminescence immune detection reagent kit
(1) system of the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody Standby:
Take containing the carboxylated magnetic particle (MagnaBind that 50mg particle diameter is 0.05 μm～1 μmTM, article No. 21353) suspension, Magneto separate removes supernatant, is that 5.5MES buffer is resuspended with 0.02M, pH, adds The EDC aqueous solution of the 10mg/mL that 1mL is newly configured, activated magnetic beads surface carboxyl groups, add 4mg prostate Specific antigen isoform monoclonal antibody (biorbyt, article No. orb48780), suspendible 6h under room temperature, Magneto separate, removes supernatant, is resuspended to 1mg/mL with the Tris buffer that the 0.1M containing 2%BSA, pH are 8.0, Obtain the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody, every bottle 5mL subpackage be stored in 4 DEG C standby.
(2) preparation of the acridinium ester of inhibin labeling of monoclonal antibody:
Take the prostate specific antigen isoform monoclonal antibody that 50 μ L concentration are 25mg/mL, add Enter 150 μ L concentration be 0.1M, pH be the carbonate buffer solution of 9.0～9.5, mixing, be subsequently adding 1.5 μ L Concentration is the acridinium ester solution mixing of 5mg/mL, and lucifuge reaction under room temperature is taken out after 1.5h, with 2mL's Zeba is centrifuged desalting column desalting processing, the most respectively with at pure water and TBS buffer in desalination processes Reason, is eventually adding the acridinium ester solution of the inhibin labeling of monoclonal antibody obtained, and collects the liquid in centrifuge tube Body is in control the acridinium ester of inhibin labeling of monoclonal antibody to preserving, every bottle of 5mL subpackage be stored in 4 DEG C standby With.
(3) preparation of prostate specific antigen isoform calibration product:
With standard substance buffer (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0) by prostatitis It is 0pg/mL, 50pg/mL, 200pg/mL, 500pg that gland specific antigen isoform is configured to concentration / mL, 1000pg/mL and 1600pg/mL, every bottle of 0.5mL subpackage lyophilizing, 4 DEG C save backup.
Embodiment 2: prostate specific antigen isoform chemical luminous immune detection method
It is detection instrument with Full-automatic chemiluminescence immunoassay analysis meter (YHLO, article No. iFlash3000), side Science of law pattern is that double antibody sandwich method, i.e. instrument are sequentially added into the sample of 50 μ L, the prostate-specific of 50 μ L The property coated carboxylated magnetic particle of antigen isoform monoclonal antibody and the prostate-specific of 50 μ L Property antigen isoform treatment fluid, after reaction 20min, then add the prostate specific antigen of 50 μ L with The coated acridinium ester of source isomer, after reaction 20min, carries out Magneto separate, and reactant mixture is sent into by instrument Darkroom, is sequentially added into luminous substrate A liquid (H2O2) and B liquid (NaOH) carry out luminescence-producing reaction, finally Record luminous value.
Embodiment 3: prostate specific antigen isoform chemiluminescence immune detection reagent kit performance evaluation
Use the method in embodiment 2 that prostate specific antigen isoform calibration product are detected, Obtain drawing standard curve as shown in Figure 2.
Then to then testing actual sample, concentration of specimens is calculated according to sample luminous value.
The detection of sensitivity:
With reference to CLSI EP17-A file recommendation experimental program, calculate prostate specific antigen isoform The sensitivity of chemiluminescence immune detection reagent kit, the sensitivity tried to achieve is 1pg/mL.
Linear detection:
It it is 50pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL, 1600pg/mL mark to concentration Quasi-product do linear analysis, calculate linearly dependent coefficient, and r=0.9996, it addition, this test kit is to prostate-specific The range of linearity of property antigen isoform sample detection is 1pg/ml～1000pg/mL.
Precision measures:
Taking concentration is 10pg/mL and two prostate specific antigen isoform samples of 100pg/mL, often The each concentration of individual sample respectively do 3 parallel, detect with three batches of test kits, calculate test kit criticize interior and batch Between poor, result shows that this test kit is criticized interior and difference between batch and is respectively less than 5%.
Interference is tested:
Take pooled serum to add chaff interference respectively and include: conjugated bilirubin, unconjugated bilirubin, hemoglobin, Ascorbic acid, glyceride, adding proportion is carried out according to 1:20, measures pooled serum respectively and with the addition of each After kind chaff interference, the measured value of pooled serum, calculates deviation therebetween, with ± 10% as tolerance interval.Knot Fruit shows, interference all reaches the file standard of NCCLS, can be used for clinical laboratory's prostate specific The accurate evaluation of antigen isoform situation.
Embodiment 4, the contrast reality of prostate specific antigen isoform chemiluminescence immune detection reagent kit Test
Respectively with chemical luminescence detection method and traditional enzyme linked immunosorbent assay to concentration be 50pg/mL, The prostate specific antigen homology of 200pg/mL, 500pg/mL, 1000pg/mL, 1600pg/mL is different Structure body sample detects, and two kinds of method detection sensitivities are compared, and data are as shown in the table:
As can be seen from the above table, the sensitivity of chemical luminescence detection method relatively enzyme linked immunosorbent assay improves about 10 times.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, But therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for this area Those of ordinary skill for, without departing from the inventive concept of the premise, it is also possible to make some deformation and Improving, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended Claim is as the criterion.

Claims (10)

1. a prostate specific antigen isoform chemiluminescence immune detection reagent kit, its feature exists In, including: the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody and The chemiluminescent labels of inhibin labeling of monoclonal antibody.
Prostate specific antigen isoform chemiluminescence immunoassay the most according to claim 1 detects Test kit, it is characterised in that the described prostate specific antigen coated carboxylic of isoform monoclonal antibody In the magnetic particle of base, described prostate specific antigen isoform monoclonal antibody is carboxylated with described The ratio of magnetic particle be 1:25～35.
Prostate specific antigen isoform chemiluminescence immunoassay the most according to claim 1 detects Test kit, it is characterised in that in the chemiluminescent labels of described inhibin labeling of monoclonal antibody, described Prostate specific antigen isoform monoclonal antibody is 50 with the ratio of described chemiluminescent labels: 1～10.
Prostate specific antigen isoform chemiluminescence immunoassay the most according to claim 1 detects Test kit, it is characterised in that the particle diameter of described carboxylated magnetic particle is 0.05 μm～1 μm.
Prostate specific antigen isoform chemiluminescence immunoassay the most according to claim 1 detects Test kit, it is characterised in that described chemiluminescent labels is luminol, different luminol, tris (bipyridine) ruthenium Or acridinium ester.
Prostate specific antigen isoform chemiluminescence immunoassay the most according to claim 1 detects Test kit, it is characterised in that also include that Chemoluminescent substrate, described Chemoluminescent substrate include A liquid With B liquid.
Prostate specific antigen isoform chemiluminescence immunoassay the most according to claim 6 detects Test kit, it is characterised in that described A liquid is H2O2Solution, described B liquid is NaOH solution.
Prostate specific antigen isoform chemiluminescence immunoassay the most according to claim 1 detects Test kit, it is characterised in that also include that prostate specific antigen isoform calibrates product.
Prostate specific antigen isoform chemiluminescence immunoassay the most according to claim 8 detects Test kit, it is characterised in that described prostate specific antigen isoform calibration product are that concentration is respectively The prostate of 0pg/mL, 50pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 1600pg/mL The solution of specific antigen isoform.
10. one kind according to the prostate specific antigen isoform according to any one of claim 1～9 The preparation method of chemiluminescence immune detection reagent kit, it is characterised in that comprise the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, then adds Enter EDC aqueous solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into prostate specific antigen Isoform monoclonal antibody, suspendible 2h～10h under room temperature, Magneto separate uses Tris buffer after removing supernatant Resuspended, obtain the coated carboxylated magnetic particle of prostate specific antigen isoform monoclonal antibody； And
Take prostate specific antigen isoform monoclonal antibody, mix after adding carbonate buffer solution, Mixing after being subsequently adding chemiluminescent labels, remove impurity after lucifuge reaction 1h～2h under room temperature, be inhibited element The chemiluminescent labels of labeling of monoclonal antibody.
CN201610505968.9A 2016-06-30 2016-06-30 Chemiluminiscence immune detection kit for homologous isomer of prostate-specific antigen and preparation method of chemiluminiscence immune detection kit Pending CN105929151A (en)

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PCT/CN2017/080400 WO2018000896A1 (en) 2016-06-30 2017-04-13 Prostate-specific antigen homologous chemiluminiscence immunoassay kit and preparation method thereof

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