CN102901810B - 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 immunoturbidimetry method to measure PSA content in 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 prostate epithelial cell synthesis secretion to seminal fluid, be one of principal ingredient of refining.At eighties of last century end of the sixties, in research immunological contraception process, the people such as Hare finds, contains the seminal fluid specific protein of a kind of molecular weight about 34000 in prostatic fluid and seminal fluid.Within 1979, from prostata tissue, extract and purifying this protein.Because this protein only exists in prostata tissue, be 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 target organ specificity, and in serum of patients with prostate cancer, PSA concentration obviously raises, 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 the diagnosis of prostate cancer and course of disease monitoring.
For the quantitative detecting analysis of PSA, comprise at present Enzyme Linked Immunoadsorbent Assay (ELISA), radiommunoassay (RIA) and chemiluminescence immune assay (CLIA).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 meet clinical requirement.RIA must use
125the radioelement marks such as I, checkout equipment is complicated, and it is necessary that with special Instrument measuring, the half life period of its radioactive nuclide is short, can not preserve for a long time, and measurement result is unstable, also to experiment operator, has brought radioactivity injury simultaneously.Although CLIA has improved the susceptibility and the automaticity that detect, conventionally needs expensive Full-automatic chemiluminescence measuring instrument, exist grow and the high shortcoming of testing cost detection time.
It is a kind of on-radiation Advances in Homogeneous Immunoassay that development is set up on latex agglutination qualitative test basis that latex intensified transmission immunological turbidimetry detects (PETIA) technology, can carry out accurate quantitative measurement to the antigenic substance of various trace and little molecule haptens, more and more be applied in clinical labororatory at present.Compare with CLIA technology, PETIA effectively improves detection speed and reduces testing cost.Yet the quantitative detecting analysis of PSA needs higher sensitivity, general PETIA technology is difficult to reach the identical sensitivity of same CLIA technology, need to improve to reach the object that improves detection PSA sensitivity to the optimization of PETIA technology.
Latex enhancing immune than turbid reagent in, it is to prepare most important step in reagent that antigen or antibody are combined with latex, conventionally has physisorphtion and chemical crosslink technique.Chemical crosslink technique is to react with the chemical group on latex surface by antigen or antibody, by 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 protein molecular structure and latex surface, antigen or antibody are adsorbed onto to latex surface, because this is a kind of weak force, protein is easy to get off from particle surface desorb, causes sensitivity decline and reagent stability poor.Antigen in absorption (or antibody) amount is more, difficult drop-off more, and sensitivity is higher, stability better.Because this absorption affinity also can cause some impurity, be also adsorbed onto on latex particle, 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 MES damping fluid;
2) PSA antibody is dissolved in MES damping fluid;
3) by 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 glycine buffer cessation reaction;
5) the centrifugal supernatant that removes;
6) obtain the latex particle that is coated with PSA antibody.
In the preferred embodiment of the present invention, the MES damping fluid in 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 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 MES damping fluid be 0.05M MES pH of buffer 6.
In some embodiments of the present invention, the reaction described in step 3) for carrying out 1-3 hour at 25-40 ℃.In a concrete embodiment of the present invention, the reaction described in step 3) is at 37 ℃, to carry out 2 hours.
In a concrete embodiment of the present invention, the glycine buffer pH7 that the glycine buffer described in step 4) is 0.1M.
In some embodiments of the present invention, the cessation reaction described in step 4) is carried out 0.5-2 hour.The reaction time of cessation reaction can be determined by technician, can stir or not stir, and preferably under stirring condition, carries out, and mixes guaranteeing, stirring rate can be determined according to conventional selection the in this area by technician.Therefore,, in a concrete embodiment of the present invention, the cessation reaction described in step 4) is carried out 1 hour under stirring condition.
In some embodiments of the present invention, described latex particle is polystyrene latex particle.Preferably, described polystyrene latex particle surface is modified with and is selected from a kind of in 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 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; Or also can buy commercial product, such as Hytest, Fitzgerald etc.In a concrete embodiment of the present invention, described PSA monoclonal antibody is prepared 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 and it is characterized in that for measuring the latex enhancing immune turbidimetry kit of human blood sample PSA content, 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 obtaining according to above-mentioned preparation method.
In the preferred embodiment 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 polystyrene latex particle.Preferably, described polystyrene latex particle surface is modified with and is selected from a kind of in following chemical group: sulfate, sulfonic group, carboxyl, amino, hydroxyl, hydrazide group, chloromethyl.
In some embodiments of the present invention, latex particle diameter range is 50-250nm, and concentration is 0.05-0.5%.In a specific embodiment of the present invention, latex particle diameter is 100nm, and in final R2 reagent, the coated polystyrene latex granule density of PSA monoclonal antibody is 0.25%.
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 PSA monoclonal antibody.Preferably, described PSA monoclonal antibody can obtain by the method for common bibliographical information; Or also can buy commercial product, such as Hytest, Fitzgerald etc.In a concrete embodiment of the present invention, described PSA monoclonal antibody is prepared by hybridoma cell technology.First selected PSA monoclonal antibody will pass through epitope analysis, and confirmation all can be reacted with the PSA of sequestered and mating type.
By add inorganic salts, set accelerator, protein and antiseptic in specific damping fluid, make R1 reagent.Wherein require the surge capability of damping fluid for regulating pH scope between 7-9, can select various damping fluids, preferably HEPES, glycocoll, Tris etc., compare with other damping fluids, has 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-bis-(hydroxyethyl) amino]-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, 4-(2-hydroxyethyl) one or more in-1-piperazine propane sulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-HCl damping fluid, phosphate buffer, glycine buffer and barbitol buffer solution, working concentration is selected from 10-200mM.Wherein HEPES surge capability within the scope of specific pH is strong, therefore more preferably HEPES, and its concentration range is 10-100mM, pH scope is 7-8.
In some embodiments of the present invention, described stabilizing agent be selected from 0.1-5% concentration range bovine serum albumin(BSA), the sodium chloride of 0.5-1% concentration range, the Tween-20 of the EDTA of 2-50mM, 0.1-1% concentration range, the glycerine of 1-10% concentration range 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 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 in PEG-4000, PEG-6000, PEG-8000, PEG-10000, glucosan T-40, glucosan T-70, glucosan T-500, and 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%.
By add PSA concentration in specific damping fluid, be respectively 0,4,10,23,45,90ng/mL makes multiple spot calibration object, wherein require the surge capability of damping fluid for regulating 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 within the scope of specific pH is strong, therefore more preferably HEPES, and its concentration range is 10-200mM, pH scope is 7-8.Wherein contain specific protein stabiliser if bovine serum albumin(BSA) concentration is 0.1-1%, contain specific antiseptic if Sodium azide concentration is 0.05-0.1%, contain specific antioxidant as ethylenediamine tetraacetic acid concentration be 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;
Polystyrene latex particle 0.25%, the glycine buffer pH7 of 100mM, 0.9% sodium chloride, 0.5%BSA, 50mM EDTA, 0.5% polysorbas20,0.09% sodium azide that R2:PSA antibody is coated, 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 preparation method obtains of the latex particle that is coated with PSA antibody of optimization, in specific reaction system, there is antigen-antibody reaction and form certain turbidity, can be detected based on spectrophotometric Biochemical Analyzer, by the rising degree of system turbidity after assaying reaction, carried out PSA quantitative test.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, in this, also improved the utilization factor of antibody simultaneously, reduce reagent cost.
Technical scheme of the present invention compared with prior art, has following features:
1) can be applied on automated chemical analyser, detection speed is fast;
2) detection specificity is strong, can detect the prostate specific antigen of sequestered and mating type simultaneously, 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 meet clinical practice needs.
Accompanying drawing explanation
Fig. 1. the kit that the present invention is prepared and chemiluminescence method kit detect the correlativity of clinical sample.
Embodiment
To further illustrate the present invention by following non-limiting example below, 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, such modification also falls into scope of the present invention.
The experiment material that following experimental technique is used if no special instructions, all can easily be obtained from commercial company.
If no special instructions, run through in instructions, " % " represents weight/volume percent.
Embodiment
Embodiment 1: preparation PSA monoclonal antibody
According to Sensabaugh etc., 1990, J.Urology144, described in 1523, separated people PSA from people's seminal fluid blood plasma.In certain time interval, mouse is used in to RAS(RIBI adjuvant system) in 25 μ g people PSA carry out injecting immune 4 times.Inject the last time latter 4 months, the lymphocyte of the spleen separation of the mouse from immune and myeloma cell line SP2/O-Ag14 are merged.Secretion is identified by ELISA method for the hybridoma of the antibody of PSA.Hybridoma that can the separated multiple antibody for the different epi-positions of PSA.Then, the monoclonal antibody that affinity purification is corresponding, then carry out epitope analysis.During epitope analysis, use the biology sensor BIAcore based on surface plasma resonance technology (SPR).The supernatant that detects cell culture, monoclonal antibody is attached to the surface of biology sensor by polyclone goat-anti-mouse-Fc-antibody, monitor the combination of PSA antigen and detected monoclonal antibody and dissociate.Data can be used inherent BIA assessment software, based on simple A+B=AB balance model, analyze, and the antibody obtaining can compare according to apparent dissociation constant separately.Then, selecting suitable antibody to combine 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%, stir as 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%, stir as PSA multiple spot calibration object.
Embodiment 4: preparation R2 reagent
1. adopt common physical crosslinking mode to obtain R2 reagent
(surface is sulfonic group modification to the polystyrene latex solution of diameter 100nm (concentration 10%), purchased from Merck) 0.5mL, the 0.05M MES damping fluid (pH6.0) that adds 4.5mL, after 0.5mg PSA monoclonal antibody is diluted with 5mL0.05M MES damping fluid (pH6.0), join immediately in above-mentioned latex solution, 37 ℃ of reactions 2 hours.Finally add glycine buffer (pH7.0) the stirring 1h of 1mL0.1M to carry out cessation reaction, the centrifugal supernatant that removes, with the glycine buffer (pH7.0) of 20mL100mM, wash 3 times, in the glycine buffer of 100mM, contain 0.9% sodium chloride, 50mM EDTA, 0.5%BSA, 0.5% polysorbas20,0.09% sodium azide, last with the glycine buffer of same 20mL, be dispersed into milky latex suspension again, for R2 reagent, in final R2 reagent, the coated polystyrene latex granule density of PSA monoclonal antibody is 0.25%.
2. adopt the physical crosslinking mode after optimizing to obtain R2 reagent
(surface is sulfonic group modification to the polystyrene latex solution of diameter 100nm (concentration 10%), purchased 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 antibody is diluted with 5mL0.05M MES damping fluid (pH6.0), join immediately in above-mentioned latex solution, 37 ℃ of reactions 2 hours.Finally add glycine buffer (pH7.0) the stirring 1h of 1mL0.1M to carry out cessation reaction, the centrifugal supernatant that removes, with the glycine buffer (pH7.0) of 20mL100mM, wash 3 times, in the glycine buffer of 100mM, contain 0.9% sodium chloride, 50mM EDTA, 0.5%BSA, 0.5% polysorbas20,0.09% sodium azide, last with the glycine buffer of same 20mL, be dispersed into milky latex suspension again, for R2 reagent, in final R2 reagent, the coated polystyrene latex granule density of PSA monoclonal antibody is 0.25%.
The Performance Detection of embodiment 5:PSA kit
1. the usage of detection kit:
Hitachi's 7180 Biochemical Analyzers of take are example: measure wavelength 570nm, first add R1 reagent 150 μ L, 37 ℃ of reactions added PSA calibration object 3 μ L after 30 seconds, react again and after 60 seconds, add R2 reagent 50 μ L, assaying reaction the 10th second, absorbance (the A1 of 70 seconds, 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, corresponding concentration is horizontal ordinate, make " concentration-absorbance difference " calibration curve, get sample to be tested, be measured in the same method the absorbance difference of sample, substitution calibration curve, can calculate the content of PSA in sample to be tested.
2. the R2 reagent diversity ratio that different physical crosslinking modes obtains 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 shows that greatly obtained R2 reagent 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 forming with above-mentioned PSA R1, PSA R2 and PSA calibration object, 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 serum.With the coated magnetic particle of a strain PSA monoclonal antibody, make insolubilized antibody, with another strain PSA monoclonal antibody mark acridinium ester, make label.In reaction cup, add the standard items or test serum and the label that contain PSA, after incubation, form the compound of solid-phase coating antibody-PSA antigen-acridinium ester labelled antibody, fully after washing, add oxygenant (H
2o
2) and NaOH make alkalize environment, in 3-10 minute, measure its luminous intensity, according to typical curve, can calculate the content of PSA in sample, the luminous intensity values of sample raises with the increase of PSA concentration.Kit of the present invention is measured 10 times according to method described in 1, calculates the coefficient of variation of different PSA content pattern detection, with A company chemiluminescence detection kit comparative result as 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 Biao3A company
From upper table 2 and 3, can find out, the repeatability when detecting 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 meet the application of clinical diagnosis.
4. the specific detection of kit of the present invention:
With physiological saline, prepare certain density mating type PSA sample (ACT-PSA and AMG-PSA) and sequestered PSA sample, and may detect with PSA the concentration of 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. kit of the present invention and chemiluminescence method detect the correlativity comparison of clinical sample
With the chemiluminescence detection kit of kit of the present invention and A company, 56 routine samples are detected simultaneously, testing result is shown in Fig. 1, the sample PSA result that the chemiluminescence detection kit of take detects is horizontal ordinate, the result that the kit of the present invention of take detects is done regretional analysis as ordinate, dependent equation is Y=0.997X-0.0128, correlation coefficient r=0.9903, through statistical procedures result, show, it is good that the inventive method detects clinical sample measured value correlativity with chemiluminescence method, can meet the application of clinical diagnosis.
Claims (28)
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 MES damping fluid;
2) prostate specific antigen antibody is dissolved in MES damping fluid;
3) by step 2) potpourri that obtains joins step 1 immediately) react in the potpourri that obtains;
4) to step 3) reaction system in add glycine buffer cessation reaction;
5) the centrifugal supernatant that removes;
6) obtain the latex particle that is coated with prostate specific antigen antibody;
Step 1 wherein) the MES damping fluid described in contains 0.15% glycerine.
2. 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 MES damping fluid be that 0.05MpH value is 6 MES damping fluid.
3. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein step 3) described reaction is for carrying out 1-3 hour at 25-40 ℃.
4. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 3, wherein step 3) described reaction is for to carry out 2 hours at 37 ℃.
5. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein step 4) described glycine buffer is the glycine buffer that 0.1M pH value is 7.
6. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein step 4) described cessation reaction carries out 0.5-2 hour.
7. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 6, wherein step 4) described cessation reaction carries out 1 hour under stirring condition.
8. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein said latex particle is polystyrene latex particle.
9. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 8, wherein said polystyrene latex particle surface is modified with and is selected from a kind of in following chemical group: sulfate, sulfonic group, carboxyl, amino, hydroxyl, hydrazide group and chloromethyl.
10. the preparation method who is coated with the latex particle of prostate specific antigen antibody according to claim 8, wherein said polystyrene latex particle diameter scope is 50-250nm.
11. preparation methods that are 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.
12. preparation methods that are coated with the latex particle of prostate specific antigen antibody according to claim 11, wherein said prostate specific antigen antibody is prostate specific antigen monoclonal antibody.
13. preparation methods that are coated with the latex particle of prostate specific antigen antibody according to claim 1, wherein in step 5) and step 6) between also comprise the step with glycine buffer washing latex particle.
14. preparation methods that are coated with the latex particle of prostate specific antigen antibody according to claim 13, wherein in step 5) and step 6) between by the 100mM pH value that contains sodium chloride, EDTA, BSA, tween and sodium azide, be 7 glycine buffer washs latex particle.
15. preparation methods that are coated with the latex particle of prostate specific antigen antibody according to claim 14, wherein in step 5) and step 6) between by the 100mM pH value that contains 0.9% sodium chloride, 50mM EDTA, 0.5%BSA, 0.5% polysorbas20 and 0.09% sodium azide, be 7 glycine buffer washs latex particle.
16. 1 kinds of latex enhancing immune turbidimetry kits of measuring human blood sample prostatic special antigen content, is characterized in that, comprise 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 obtaining according to the preparation method described in claim 1 to 15 any one.
The latex enhancing immune turbidimetry kit of 17. mensuration human blood sample prostatic special antigen content according to claim 16, it also comprises prostate specific antigen calibration object.
The latex enhancing immune turbidimetry kit of 18. mensuration human blood sample prostatic special antigen content according to claim 17, wherein said prostate specific antigen calibration object is comprised of prostate specific antigen, damping fluid, stabilizing agent and antiseptic.
The latex enhancing immune turbidimetry kit of 19. mensuration human blood sample prostatic special antigen content according to claim 16, wherein said latex particle is polystyrene latex particle.
The latex enhancing immune turbidimetry kit of 20. mensuration human blood sample prostatic special antigen content according to claim 19, wherein said polystyrene latex particle surface is modified with and is selected from a kind of in following chemical group: sulfate, sulfonic group, carboxyl, amino, hydroxyl, hydrazide group and chloromethyl.
The latex enhancing immune turbidimetry kit of 21. mensuration human blood sample prostatic special antigen content according to claim 16, wherein said latex particle diameter range is 50-250nm, concentration is 0.05-0.5%.
The latex enhancing immune turbidimetry kit of 22. mensuration human blood sample prostatic special antigen content according to claim 16, wherein said prostate specific antigen antibody is prostate specific antigen polyclonal antibody or prostate specific antigen monoclonal antibody.
The latex enhancing immune turbidimetry kit of 23. mensuration human blood sample prostatic special antigen content according to claim 22, wherein said prostate specific antigen antibody is prostate specific antigen monoclonal antibody.
24. according to the latex enhancing immune turbidimetry kit of the mensuration human blood sample prostatic special antigen content described in claim 16 or 18, wherein the described damping fluid in R1, R2 and calibration object is selected from respectively 3-[N, N-bis-(hydroxyethyl) amino] a kind of in-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid-sodium hydrate buffer solution, trishydroxymethylaminomethane-HCl damping fluid, phosphate buffer, glycine buffer and barbitol buffer solution, working concentration is selected from 10-200mM.
25. according to the latex enhancing immune turbidimetry kit of the mensuration human blood sample prostatic special antigen content described in claim 16 or 18, wherein the described stabilizing agent in R1, R2 and calibration object be selected from respectively 0.1-5% concentration range bovine serum albumin(BSA), the sodium chloride of 0.5-1% concentration range, the Tween-20 of the EDTA of 2-50mM, 0.1-1% concentration range and the glycerine of 1-10% concentration range one or more.
26. according to the latex enhancing immune turbidimetry kit of the mensuration human blood sample prostatic special antigen content described in claim 16 or 18, wherein the described antiseptic in R1, R2 and calibration object is selected from respectively a kind of in Sodium azide, thimerosal, phenol and ethyl mercury sodium thiosulfate, and working concentration is selected from 0.02%-0.1%.
27. according to the latex enhancing immune turbidimetry kit of the mensuration human blood sample prostatic special antigen content described in claim 16 or 18, wherein said set accelerator is selected from a kind of in PEG-4000, PEG-6000, PEG-8000, PEG-10000, glucosan T-40, glucosan T-70 and glucosan T-500, and working concentration scope is selected from 1-10%.
The latex enhancing immune turbidimetry kit of 28. mensuration human blood sample prostatic special antigen content according to claim 16, it is comprised of following component:
The HEPES damping fluid that R1:0.9% sodium chloride, 3%PEG-6000,0.5%BSA, 50mM EDTA, 0.09% sodium azide and 50mM pH value are 7.2;
The coated polystyrene latex particle of R2:0.25% prostate specific antigen antibody, glycine buffer, 0.9% sodium chloride, 0.5%BSA, 50mMEDTA, 0.5% polysorbas20 and 0.09% sodium azide that 100mM pH value is 7, wherein said polystyrene latex particle diameter is 100nm;
Prostate specific antigen calibration object: the HEPES damping fluid that 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 pH value of 90ng/mL are 7.2.
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| CN110687286A (en) * | 2019-09-09 | 2020-01-14 | 元升生物科技（上海）有限公司 | Latex enhanced immunoturbidimetry kit |
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