CN102901810A - Preparation method of latex particles coated with prostate specific antigen-antibody and PSA enhanced turbidimetric immunophelometry kit - Google Patents
Preparation method of latex particles coated with prostate specific antigen-antibody and PSA enhanced turbidimetric immunophelometry kit Download PDFInfo
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Abstract
The invention relates to a preparation method of latex particles coated with a prostate specific antigen-antibody (PSA) and a PSA-enhanced turbidimetric immunophelometry kit. The latex particles coated with the PSA antibody are prepared by bonding the PSA antibody with latex particles by an optimized physical adsorption method. According to the invention, the kit utilizes the PSA antibody bonded onto the surface of the latex particles and the PSA in the human blood sample to conduct immunoreaction to form the turbidity, and the kit detects the content of the prostate specific antigen-antibody by the increase of the turbidity.
Description
Technical field
The invention provides a kind of preparation method who is coated with the latex particle of prostate specific antigen (PSA) antibody, and a kind of kit that utilizes the immunoturbidimetry method to measure PSA content in the human blood sample.Preparation method of the present invention and detection kit belong to clinical medicine in-vitro diagnosis field.
Background technology
Prostate specific antigen (Prostate specific antigen, PSA) be by the prostate epithelial cell synthesis secretion to seminal fluid, be one of principal ingredient of refining.At eighties of last century end of the sixties, the people such as Hare finds in research immunological contraception process, contains the seminal fluid specific protein of a kind of molecular weight about 34000 in prostatic fluid and seminal fluid.From prostata tissue, extracted in 1979 and purifying this protein.Because this protein only exists in prostata tissue, is named as prostate specific antigen.
PSA mainly contains two kinds of existence forms in serum: a kind of is the PSA(f-PSA of sequestered), account for the 10%-30% of blood-serum P SA total concentration.Another kind is the PSA(PSA-ACT with α 1-ACT (ACT) combination) and with the PSA(PSA-AMG of alpha2-macroglobulin (AMG) combination), account for the 70%-90% of blood-serum P SA total concentration.In the serum of patients with prostate cancer, t-PSA and f-PSA all have rising.PSA has the target organ specificity, and PSA concentration obviously raises in the serum of patients with prostate cancer, so the mensuration of blood-serum P SA is known as the first-selected mark of diagnosing prostate cancer at present.Early stage susceptibility is between 40-75%, and the middle and later periods can reach 75-95%, so it has certain clinical value to diagnosis and the course of disease monitoring of prostate cancer.
For the quantitative detecting analysis of PSA, comprise at present Enzyme Linked Immunoadsorbent Assay (ELISA), radiommunoassay (RIA) and chemiluminescence immune assay (CLIA).The ELISA operating process is complicated, and automaticity is low, and sensing range is narrow, and influence factor is more, easily causes the shortcomings such as false negative and false positive can not satisfy clinical requirement.RIA must use
125The radioelement marks such as I, checkout equipment is complicated, must be with special Instrument measuring, the half life period of its radioactive nuclide is short, can not long preservation, measurement result is unstable, and the while has also brought the radioactivity injury to experiment operator.Although CLIA has improved the susceptibility and the automaticity that detect, usually need expensive Full-automatic chemiluminescence measuring instrument, there is the long and high shortcoming of testing cost detection time.
It is a kind of on-radiation Advances in Homogeneous Immunoassay of setting up in the development of latex agglutination qualitative test basis that the latex intensified transmission immunological turbidimetry detects (PETIA) technology, can carry out accurate quantitative measurement to antigenic substance and the little molecule haptens of various trace, more and more be applied in the clinical labororatory at present.Compare with the CLIA technology, PETIA effectively improves detection speed and reduces testing cost.Yet the quantitative detecting analysis of PSA needs higher sensitivity, and general PETIA technology is difficult to reach with the identical sensitivity of CLIA technology, need to improve to the optimization of PETIA technology to reach the purpose that improves detection PSA sensitivity.
Latex enhancing immune than turbid reagent in, it is most important step in the preparation reagent that antigen or antibody are combined with latex, and physisorphtion and chemical crosslink technique are arranged usually.Chemical crosslink technique is the chemical group reaction by antigen or antibody and latex surface, with antigen or antibody linked on latex.Because the reaction conditions of chemical crosslink technique is comparatively strong and the reaction time is longer, the loss of activity of antigen or antibody is larger.So physisorphtion is a kind of reaction milder, method fast.Physisorphtion is by the interaction between the hydrophobic grouping on the hydrophobic grouping on the protein molecular structure and latex surface, antigen or antibody are adsorbed onto the latex surface, because this is a kind of weak force, protein is easy to get off from the particle surface desorb, causes sensitivity decline and reagent stability poor.Antigen in the absorption (or antibody) amount is more, difficult drop-off more, and then sensitivity is higher, stability better.Because this absorption affinity also can cause some impurity also to be adsorbed onto on the latex particle, has further affected the combination of antigen (or antibody) to particle.Therefore, still need to improve the preparation method of the latex particle that is coated with antigen (or antibody).
Summary of the invention
According to an aspect of the present invention, relate to a kind of preparation method who is coated with the latex particle of prostate specific antigen (PSA) antibody, it is characterized in that, comprise step:
1) latex particle is dissolved in the MES damping fluid;
2) PSA antibody is dissolved in the MES damping fluid;
3) with step 2) potpourri that obtains joins immediately in the potpourri that step 1) obtains and reacts;
4) in the reaction system of step 3), add the glycine buffer cessation reaction;
5) the centrifugal supernatant that removes;
6) acquisition is coated with the latex particle of PSA antibody.
In preferred implementation of the present invention, the MES damping fluid in the step 1) also contains glycerine.In some embodiments of the present invention, it is 0.01% to 0.5% glycerine that the MES damping fluid in the step 1) also contains content.In a concrete embodiment, contain 0.15% glycerine.
In a concrete embodiment of the present invention, step 1) and step 2) described in the MES damping fluid be 0.05M MES pH of buffer 6.
In some embodiments of the present invention, the described reaction of step 3) is for to carry out under 25-40 ℃ 1-3 hour.In a concrete embodiment of the present invention, the described reaction of step 3) is to carry out under 37 ℃ 2 hours.
In a concrete embodiment of the present invention, the described glycine buffer of step 4) is the glycine buffer pH7 of 0.1M.
In some embodiments of the present invention, the described cessation reaction of step 4) was carried out 0.5-2 hour.The reaction time of cessation reaction can be determined by the technician, can stir or not stir, and preferably carries out under stirring condition, mixes guaranteeing, stirring rate can be determined according to conventional selection the in this area by the technician.Therefore, in a concrete embodiment of the present invention, the described cessation reaction of step 4) was carried out under stirring condition 1 hour.
In some embodiments of the present invention, described latex particle is the polystyrene latex particle.Preferably, described polystyrene latex particle surface is modified with and is selected from a kind of in the following chemical group: sulfate, sulfonic group, carboxyl, amino, hydroxyl, hydrazide group, chloromethyl.In a concrete embodiment of the present invention, described latex particle is the polystyrene latex particle that sulfonic group is modified.Preferably, polystyrene latex particle diameter scope is 50-250nm.
In some embodiments of the present invention, described PSA antibody is PSA polyclonal antibody or PSA monoclonal antibody, and preferably, described PSA antibody is the PSA monoclonal antibody.Preferably, described PSA monoclonal antibody can by the method for common bibliographical information, include but not limited to hybridoma technology, and obtain; Perhaps also can buy commercial product, such as Hytest, Fitzgerald etc.In a concrete embodiment of the present invention, described PSA monoclonal antibody prepares by hybridoma cell technology.
In some embodiments of the present invention, between step 5) and step 6), can also comprise the step with glycine buffer washing latex particle.Preferably, described glycine buffer is the 100mM glycine buffer pH7 that contains 0.9% sodium chloride, 50mM EDTA, 0.5%BSA, 0.5% polysorbas20,0.09% sodium azide.
According to a further aspect in the invention, the present invention also provides a kind of latex enhancing immune turbidimetry kit for measuring human blood sample PSA content, it is characterized in that, comprises R1 reagent and R2 reagent; Described R1 reagent comprises damping fluid, stabilizing agent, set accelerator and antiseptic; Described R2 reagent comprises the latex particle that is coated with PSA antibody, damping fluid, stabilizing agent and the antiseptic that obtains according to above-mentioned preparation method.
In preferred implementation of the present invention, kit can also comprise calibration object.Preferably, calibration object is comprised of PSA, damping fluid, stabilizing agent and antiseptic.
In some embodiments of the present invention, latex particle is the polystyrene latex particle.Preferably, described polystyrene latex particle surface is modified with and is selected from a kind of in the following chemical group: sulfate, sulfonic group, carboxyl, amino, hydroxyl, hydrazide group, chloromethyl.
In some embodiments of the present invention, the latex particle diameter range is 50-250nm, and concentration is 0.05-0.5%.In a specific embodiment of the present invention, the latex particle diameter is 100nm, and the coated polystyrene latex granule density of PSA monoclonal antibody is 0.25% in the final R2 reagent.
In some embodiments of the present invention, described PSA antibody is PSA polyclonal antibody or PSA monoclonal antibody.
Of the present invention one preferred embodiment in, described PSA antibody is the PSA monoclonal antibody.Preferably, described PSA monoclonal antibody can obtain by the method for common bibliographical information; Perhaps also can buy commercial product, such as Hytest, Fitzgerald etc.In a concrete embodiment of the present invention, described PSA monoclonal antibody prepares by hybridoma cell technology.Selected PSA monoclonal antibody at first will be passed through epitope analysis, and the PSA of affirmation and sequestered and mating type all can react.
Make R1 reagent by in specific damping fluid, adding inorganic salts, set accelerator, protein and antiseptic.Wherein require the surge capability of damping fluid for regulating the pH scope between 7-9, can select various damping fluids, preferred HEPES, glycocoll, Tris etc. compare with other damping fluids, have that surge capability is strong, solubleness is high, good biological fitness and a reactionlessness.
Therefore, in some embodiments of the present invention, described damping fluid is selected from 3-[N, N-two (hydroxyethyl) amino]-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, 4-(2-hydroxyethyl)-in 1-piperazine propane sulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-HCl damping fluid, phosphate buffer, glycine buffer and the barbitol buffer solution one or more, working concentration is selected from 10-200mM.Wherein HEPES surge capability in specific pH scope is strong, therefore more preferably HEPES, and its concentration range is 10-100mM, the pH scope is 7-8.
In some embodiments of the present invention, described stabilizing agent be selected from EDTA, the 0.1-1% concentration range of sodium chloride, the 2-50mM of bovine serum albumin(BSA), the 0.5-1% concentration range of 0.1-5% concentration range Tween-20,1-10% concentration range glycerine one or more.Preferably, stabilizing agent is selected bovine serum albumin(BSA), and final concentration is 0.1-1%.Sodium chloride concentration is 0.7-0.9%, and Sodium azide concentration is 0.05-0.1%.
In some embodiments of the present invention, described antiseptic is selected from one or more in Sodium azide, thimerosal, phenol and the ethyl mercury sodium thiosulfate, and working concentration is selected from 0.02%-0.1%.
In some embodiments of the present invention, described set accelerator is selected from a kind of among PEG-4000, PEG-6000, PEG-8000, PEG-10000, glucosan T-40, glucosan T-70, the glucosan T-500, and the working concentration scope is selected from 1-10%.Preferably, set accelerator is selected PEG-6000, can accelerate immune response speed, shortens detection time, and its final concentration is 2-6%.
Be respectively 0,4,10,23,45 by in specific damping fluid, adding PSA concentration, 90ng/mL makes the multiple spot calibration object, wherein require the surge capability of damping fluid for regulating the pH scope between 7-9, can select various damping fluids, preferred HEPES, glycocoll, Tris etc., compare with other damping fluids, have that surge capability is strong, solubleness is high, good biological fitness and a reactionlessness.Wherein HEPES surge capability in specific pH scope is strong, therefore more preferably HEPES, and its concentration range is 10-200mM, the pH scope is 7-8.Wherein contain specific protein stabiliser such as bovine serum albumin(BSA) concentration is 0.1-1%, containing specific antiseptic such as Sodium azide concentration is 0.05-0.1%, and containing specific antioxidant such as ethylenediamine tetraacetic acid concentration is 5-50mM.
Therefore, in a concrete embodiment of the present invention, measure the latex enhancing immune turbidimetry kit of human blood sample PSA content, it is comprised of following component:
R1:0.9% sodium chloride, 3%PEG-6000,0.5%BSA, 50mM EDTA, 0.09% sodium azide, 50mM HEPES pH of buffer 7.2;
The polystyrene latex particle 0.25% of R2:PSA antibody sandwich, the glycine buffer pH7 of 100mM, 0.9% sodium chloride, 0.5%BSA, 50mM EDTA, 0.5% polysorbas20,0.09% sodium azide, wherein said polystyrene latex particle diameter is 100nm;
Calibration object: concentration is respectively 0,4,10,23,45, the PSA of 90ng/mL, 0.9% sodium chloride, 1%BSA, 50mM EDTA, 0.1% sodium azide, 50mM HEPES pH of buffer 7.2.
The present invention is based on the latex particle that the preparation method obtains of the latex particle that is coated with PSA antibody of optimization, in specific reaction system antigen-antibody reaction occuring forms certain turbidity, can be detected based on spectrophotometric Biochemical Analyzer, carried out the PSA quantitative test by the rising degree of system turbidity behind the assaying reaction.The present invention has solved sensitivity and the low problem of automaticity that PSA detects simultaneously, can wide popularization and application.
Particularly, to achieve these goals, the technical scheme that the present invention takes is: by the method for optimizing, the organic solvent that adds certain concentration, more specifically, organic solvent is glycerine, and the R2 reagent reacting specificity of making therefrom and sensitivity is corresponding being improved also, and stability increases thereupon.Although be not limited to concrete theory, can think that method of the present invention improved compatibility, the joint efficiency on PSA antibody and latex particle surface, thereby improve atopic and sensitivity, also improved simultaneously the utilization factor of antibody in this, reduce reagent cost.
Technical scheme of the present invention compared with prior art has following features:
1) can be applied on the automated chemical analyser, detection speed is fast;
2) detection specificity is strong, can detect simultaneously the prostate specific antigen of sequestered and mating type, has very high correlativity with chemiluminescence method;
3) detection sensitivity can reach other just at the chemiluminescence detection kit peer-level of clinical practice, can satisfy the clinical practice needs.
Description of drawings
Fig. 1. the kit that the present invention is prepared and chemiluminescence method kit detect the correlativity of clinical sample.
Embodiment
The below will further specify the present invention by following non-limiting example, and will be as well known to those skilled in the art, without departing from the spirit of the invention, can make many modifications to the present invention, and such modification also falls into scope of the present invention.
The employed experiment material of following experimental technique all can easily be obtained from commercial company if no special instructions.
If no special instructions, run through in the instructions, " % " represents weight/volume percent.
Embodiment
Embodiment 1: preparation PSA monoclonal antibody
According to Sensabaugh etc., 1990, J.Urology144,1523 is described, separates people PSA from people's seminal fluid blood plasma.In certain time interval, mouse is used in the RAS(RIBI adjuvant system) in 25 μ g people PSA carry out injecting immune 4 times.Injected the last time the lymphocyte and myeloma cell line SP2/O-Ag14 fusion that will separate from the spleen of the mouse of immunity rear 4 months.Secretion is identified by the ELISA method for the hybridoma of the antibody of PSA.The hybridoma that can separate multiple antibody for the different epi-positions of PSA.Then, the monoclonal antibody that affinity purification is corresponding is carried out epitope analysis again.During epitope analysis, use the biology sensor BIAcore based on surface plasma resonance technology (SPR).Detect the supernatant of cell culture, monoclonal antibody is attached to the surface of biology sensor by polyclone goat-anti-mouse-Fc-antibody, the combination of monitoring PSA antigen and detected monoclonal antibody and dissociating.Data can be used inherent BIA assessment software, analyze based on simple A+B=AB balance model, and the antibody that obtains can compare according to apparent dissociation constant separately.Then, selecting suitable antibody to make up can be for the preparation of R2 reagent of the present invention.
Embodiment 2: preparation R1 reagent
In the HEPES of 50mM pH7.2 damping fluid, add sodium chloride 0.9%, PEG-60003%, BSA0.5%, EDTA50mM, sodium azide 0.09%, stirring is R1 reagent.
Embodiment 3: preparation PSA calibration object
In the HEPES of 50mM pH7.2 damping fluid, add that concentration is respectively 0,4,10,23,45, the PSA of 90ng/mL, add in addition sodium chloride 0.9%, BSA1%, EDTA50mM, sodium azide 0.1%, stirring is PSA multiple spot calibration object.
Embodiment 4: preparation R2 reagent
1. adopt common physical crosslinking mode to obtain R2 reagent
The polystyrene latex solution of diameter 100nm (concentration 10%) (modify for sulfonic group by the surface, available from Merck) 0.5mL, the 0.05M MES damping fluid (pH6.0) that adds 4.5mL, after 0.5mg PSA monoclonal anti body and function 5mL0.05M MES damping fluid (pH6.0) dilution, join immediately in the above-mentioned latex solution, 37 ℃ of reactions 2 hours.The glycine buffer (pH7.0) that adds at last 1mL0.1M stirs 1h and comes cessation reaction, the centrifugal supernatant that removes, glycine buffer (pH7.0) with 20mL100mM washs 3 times, contain 0.9% sodium chloride, 50mM EDTA, 0.5%BSA, 0.5% polysorbas20,0.09% sodium azide in the glycine buffer of 100mM, last be dispersed into the milky latex suspension with the glycine buffer of same 20mL again, be R2 reagent, the coated polystyrene latex granule density of PSA monoclonal antibody is 0.25% in the final R2 reagent.
2. adopt the physical crosslinking mode after optimizing to obtain R2 reagent
The polystyrene latex solution of diameter 100nm (concentration 10%) (modify for sulfonic group by the surface, available from Merck) 0.5mL, the 0.05M MES damping fluid (pH6.0) that adds 4.5mL, contain 0.15% glycerine, after 0.5mg PSA monoclonal anti body and function 5mL0.05M MES damping fluid (pH6.0) dilution, join immediately in the above-mentioned latex solution, 37 ℃ of reactions 2 hours.The glycine buffer (pH7.0) that adds at last 1mL0.1M stirs 1h and comes cessation reaction, the centrifugal supernatant that removes, glycine buffer (pH7.0) with 20mL100mM washs 3 times, contain 0.9% sodium chloride, 50mM EDTA, 0.5%BSA, 0.5% polysorbas20,0.09% sodium azide in the glycine buffer of 100mM, last be dispersed into the milky latex suspension with the glycine buffer of same 20mL again, be R2 reagent, the coated polystyrene latex granule density of PSA monoclonal antibody is 0.25% in the final R2 reagent.
The Performance Detection of embodiment 5:PSA kit
1. the usage of detection kit:
Take Hitachi's 7180 Biochemical Analyzers as example: measure wavelength 570nm, at first add R1 reagent 150 μ L, 37 ℃ of reactions added PSA calibration object 3 μ L after 30 seconds, react again and add R2 reagent 50 μ L after 60 seconds, assaying reaction the 10th second, 70 seconds absorbance (A1, A2), calculate absorbance difference △ A=A2-A1, every pipe replication 2 times, the absorbance difference △ A that each calibration QC is recorded for 2 times is ordinate, and corresponding concentration is horizontal ordinate, makes " concentration-absorbance difference " calibration curve, get sample to be tested, measure the absorbance difference of sample with method, the substitution calibration curve can calculate the content of PSA in the sample to be tested.
2. the R2 reagent diversity ratio that obtains of different physical crosslinking modes is:
Detection mode according to 5.1, the difference of the R2 reagent examination criteria product each point absorbance that the physical crosslinking mode after relatively optimizing and common physical crosslinking mode obtain, absorbance variation show that greatly the R2 reagent that obtains can access higher sensitivity and detect performance.As shown in table 1 below:
The R2 reagent absorbance difference that the different physical crosslinking modes of table 1 obtain
3. low value sample repeatability detects:
With normal saline dilution human serum sample, compound concentration is respectively 1,2,4, the sample of 8ng/mL, and the kit that forms with above-mentioned PSA R1, PSA R2 and PSA calibration object is with the chemical luminescence reagent kit of A company Repeatability relatively.The reagent fundamental analysis principle of A company is to adopt double antibody sandwich method to measure PSA level in the serum.Make insolubilized antibody with the coated magnetic particle of a strain PSA monoclonal antibody, make label with another strain PSA monoclonal antibody mark acridinium ester.In reaction cup, add standard items or test serum and the label that contains PSA, namely form the compound of solid-phase coating antibody-PSA antigen-acridinium ester labelled antibody behind the incubation, fully add oxygenant (H after the washing
2O
2) and NaOH make the alkalize environment, in 3-10 minute, measure its luminous intensity, can calculate the content of PSA in the sample according to typical curve, the luminous intensity values of sample raises with the increase of PSA concentration.Kit of the present invention is measured 10 times according to 1 described method, calculates the coefficient of variation of different PSA content pattern detection, with A company chemiluminescence detection kit comparative result shown in following table 2 and 3:
The low value sample repeatability of table 2 kit of the present invention
The low value sample repeatability of the chemical luminescence reagent kit of table 3A company
| 1ng/mL | 2ng/mL | 4ng/mL | 8ng/mL | |
| 1 | 1.18 | 2.12 | 4.03 | 8.08 |
| 2 | 1.09 | 2.09 | 4.01 | 8.07 |
| 3 | 1.02 | 2.13 | 4.07 | 8.12 |
| 4 | 1.15 | 2.08 | 4.12 | 8.04 |
| 5 | 1.07 | 2.01 | 4.08 | 8.07 |
| 6 | 1.01 | 2.07 | 4.01 | 8.05 |
| 7 | 1.08 | 2.14 | 4.03 | 8.02 |
| 8 | 1.14 | 2.21 | 4.08 | 8.03 |
| 9 | 1.15 | 2.17 | 4.09 | 8.05 |
| 10 | 1.08 | 2.03 | 4.11 | 8.11 |
| Mean value | 1.10 | 2.11 | 4.12 | 8.04 |
| SD | 0.05697 | 0.06151 | 0.04029 | 0.03273 |
| CV | 5.2% | 2.9% | 1.0% | 0.4% |
Can find out that from upper table 2 and 3 repeatability when detecting the low value sample of kit of the present invention is suitable with the chemiluminescence detection kit of A company, has reached with the identical sensitivity of CLIA technology, can satisfy the application of clinical diagnosis.
4. the specific detection of kit of the present invention:
Prepare certain density mating type PSA sample (ACT-PSA and AMG-PSA) and sequestered PSA sample with physiological saline, and the concentration that may detect with PSA noisy target substance, mainly comprise ACT, AMG, testing result shows that kit of the present invention can detect accurately in conjunction with PSA and sequestered PSA, and can not be subject to the interference of ACT and AMG.Be shown in Table 4:
The detection specificity of table 4 kit of the present invention
5. the correlativity of kit of the present invention and chemiluminescence method detection clinical sample relatively
Chemiluminescence detection kit with kit of the present invention and A company detects simultaneously to 56 routine samples, testing result is seen Fig. 1, the sample PSA result who detects take chemiluminescence detection kit is as horizontal ordinate, the result who detects take kit of the present invention does regretional analysis as ordinate, dependent equation is Y=0.997X-0.0128, correlation coefficient r=0.9903, show through the statistical procedures result, it is good that the inventive method detects clinical sample measured value correlativity with chemical luminescent method, can satisfy the application of clinical diagnosis.
Claims (31)
1. a preparation method who is coated with the latex particle of prostate specific antigen antibody is characterized in that, comprises step:
1) latex particle is dissolved in the MES damping fluid;
2) prostate specific antigen antibody is dissolved in the MES damping fluid;
3) with step 2) potpourri that obtains joins immediately in the potpourri that step 1) obtains and reacts;
4) in the reaction system of step 3), add the glycine buffer cessation reaction;
5) the centrifugal supernatant that removes;
6) acquisition is coated with the latex particle of prostate specific antigen antibody.
2. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein the MES damping fluid described in the step 1) also contains glycerine.
3. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 2, wherein glycerol content is 0.01% to 0.5%.
4. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 3, wherein glycerol content is 0.15%.
5. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein step 1) and step 2) described in the MES damping fluid be 0.05MMES pH of buffer 6.
6. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein the described reaction of step 3) is for to carry out under 25-40 ℃ 1-3 hour.
7. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 6, wherein the described reaction of step 3) is for to carry out 2 hours at 37 ℃.
8. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein the described glycine buffer of step 4) is 0.1M glycine buffer pH7.
9. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein the described cessation reaction of step 4) was carried out 0.5-2 hour.
10. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 9, wherein the described cessation reaction of step 4) was carried out under stirring condition 1 hour.
11. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein said latex particle is the polystyrene latex particle.
12. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 11, wherein said polystyrene latex particle surface is modified with and is selected from a kind of in the following chemical group: sulfate, sulfonic group, carboxyl, amino, hydroxyl, hydrazide group and chloromethyl.
13. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 11, wherein said polystyrene latex particle diameter scope is 50-250nm.
14. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein said prostate specific antigen antibody is prostate specific antigen polyclonal antibody or prostate specific antigen monoclonal antibody.
15. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 14, wherein said prostate specific antigen antibody is the prostate specific antigen monoclonal antibody.
16. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1 wherein also comprises the step with glycine buffer washing latex particle between step 5) and step 6).
17. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 16, wherein said glycine buffer is the 100mM glycine buffer pH7 that contains sodium chloride, EDTA, BSA, tween and sodium azide.
18. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 17, wherein said glycine buffer is the 100mM glycine buffer pH7 that contains 0.9% sodium chloride, 50mM EDTA, 0.5%BSA, 0.5% polysorbas20 and 0.09% sodium azide.
19. a latex enhancing immune turbidimetry kit of measuring human blood sample prostatic special antigen content is characterized in that, comprises R1 reagent and R2 reagent;
Described R1 reagent comprises damping fluid, stabilizing agent, set accelerator and antiseptic;
Described R2 reagent comprises the latex particle that is coated with prostate specific antigen antibody, damping fluid, stabilizing agent and the antiseptic that obtains to 18 each described preparation methods according to claim 1.
20. the latex enhancing immune turbidimetry kit of mensuration human blood sample prostatic special antigen content according to claim 19, it also comprises the prostate specific antigen calibration object.
21. the latex enhancing immune turbidimetry kit of mensuration human blood sample prostatic special antigen content according to claim 20, wherein said prostate specific antigen calibration object is comprised of prostate specific antigen, damping fluid, stabilizing agent and antiseptic.
22. the latex enhancing immune turbidimetry kit of mensuration human blood sample prostatic special antigen content according to claim 19, wherein said latex particle is the polystyrene latex particle.
23. being modified with, the latex enhancing immune turbidimetry kit of mensuration human blood sample prostatic special antigen content according to claim 22, wherein said polystyrene latex particle surface be selected from a kind of in the following chemical group: sulfate, sulfonic group, carboxyl, amino, hydroxyl, hydrazide group and chloromethyl.
24. the latex enhancing immune turbidimetry kit of mensuration human blood sample prostatic special antigen content according to claim 19, wherein said latex particle diameter range is 50-250nm, and concentration is 0.05-0.5%.
25. the latex enhancing immune turbidimetry kit of mensuration human blood sample prostatic special antigen content according to claim 19, wherein said prostate specific antigen antibody are prostate specific antigen polyclonal antibody or prostate specific antigen monoclonal antibody.
26. the latex enhancing immune turbidimetry kit of mensuration human blood sample prostatic special antigen content according to claim 25, wherein said prostate specific antigen antibody is the prostate specific antigen monoclonal antibody.
27. according to claim 19 or the latex enhancing immune turbidimetry kit of 21 described mensuration human blood sample prostatic special antigen content, wherein said damping fluid is selected from 3-[N, N-two (hydroxyethyl) amino]-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, 4-(2-hydroxyethyl)-in 1-piperazine propane sulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-HCl damping fluid, phosphate buffer, glycine buffer and the barbitol buffer solution one or more, working concentration is selected from 10-200mM.
28. according to claim 19 or the latex enhancing immune turbidimetry kit of 21 described mensuration human blood sample prostatic special antigen content, wherein said stabilizing agent be selected from the Tween-20 of EDTA, 0.1-1% concentration range of sodium chloride, the 2-50mM of bovine serum albumin(BSA), the 0.5-1% concentration range of 0.1-5% concentration range and 1-10% concentration range glycerine one or more.
29. according to claim 19 or the latex enhancing immune turbidimetry kit of 21 described mensuration human blood sample prostatic special antigen content, wherein said antiseptic is selected from one or more in Sodium azide, thimerosal, phenol and the ethyl mercury sodium thiosulfate, and working concentration is selected from 0.02%-0.1%.
30. according to claim 19 or the latex enhancing immune turbidimetry kit of 21 described mensuration human blood sample prostatic special antigen content, wherein said set accelerator is selected from a kind of among PEG-4000, PEG-6000, PEG-8000, PEG-10000, glucosan T-40, glucosan T-70 and the glucosan T-500, and the working concentration scope is selected from 1-10%.
31. to the latex enhancing immune turbidimetry kit of 30 each described mensuration human blood sample prostatic special antigen content, it is comprised of following component according to claim 19:
R1:0.9% sodium chloride, 3%PEG-6000,0.5%BSA, 50mM EDTA, 0.09% sodium azide and 50mM HEPES pH of buffer 7.2;
R2: the polystyrene latex particle 0.25% of prostate specific antigen antibody sandwich, the glycine buffer pH7 of 100mM, 0.9% sodium chloride, 0.5%BSA, 50mM EDTA, 0.5% polysorbas20 and 0.09% sodium azide, wherein said polystyrene latex particle diameter is 100nm;
The prostate specific antigen calibration object: concentration is respectively 0,4,10,23,45, prostate specific antigen, 0.9% sodium chloride, 1%BSA, 50mM EDTA, 0.1% sodium azide and the 50mM HEPES pH of buffer 7.2 of 90ng/mL.
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