CN104991071A - ELISA kit of PSA and application thereof - Google Patents
ELISA kit of PSA and application thereof Download PDFInfo
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- CN104991071A CN104991071A CN201510292841.9A CN201510292841A CN104991071A CN 104991071 A CN104991071 A CN 104991071A CN 201510292841 A CN201510292841 A CN 201510292841A CN 104991071 A CN104991071 A CN 104991071A
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- psa
- monoclonal antibody
- solution
- enzyme linked
- linked immunological
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Abstract
Belonging to the field of biological medicine, the invention in particular relates to an enzyme linked immunosorbent assay (ELISA) kit for detection of free PSA (prostate specific antigen). The kit is composed of a PSA standard substance, a control solution, a free PSA monoclonal antibody coated enzyme label plate, a horse radish peroxidase (HRP) labeled PSA monoclonal antibody solution and an auxiliary agent.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to ELISA kit and the application thereof of PSA.
Background technology
Prostate specific antigen (prostate specific antigen, PSA) is a kind of antigen relevant to prostate cancer, and belong to glycoprotein material, molecular weight is about 34KD, and pH is 6.8 ~ 7.5, and isoelectric point is 6.9, and the half life period is 2.2 scholar 0.8 day.PSA is present in the middle of prostate endoplasmic reticulum and prostate epithelial cell and secretion, all containing PSA in normal prostatic and pathology prostata tissue.It is the good tumor markers of generally acknowledged diagnosing prostate cancer, and the specificity of its diagnosing prostate cancer is 82%-97%.Prostate cancer accounts for 10% ~ 20% in male sex's all types cancer, is the modal cancerous swelling of the male sex.This disease progression is slow, is the major cancers of threat more than 50 years old male sex's life, accounts for the second of deaths in men rate in western countries.Epidemiology survey shows, along with the improvement of China's Living consumption, the aggravation of environmental pollution and the change of dietary structure, the incidence of disease of prostate cancer rises increasingly, has caused great attention clinically.PSA is detected the generaI investigation index as more than the 50 years old male sex by U.S. FDA approved.Research display, PSA is determined on early diagnosis prostate gland cancer and is better than digital rectal examination.The PSA value <4ug/L of normal male.
PSA is in blood to dissociate and to exist in conjunction with two kinds of forms.Wherein PSA (free PSA, F-PSA) only fraction is accounted for, mating type (comlexed PSA, C-PSA) major part is accounted for, namely with endogenous protein enzyme inhibitor prostate cancer α 1-ACT (α 1-antichymotrysin, ACT) be combined into PSA-ACT compound, and be combined into PSA-α 2M with another kind of protein inhibitor alpha2-macroglobulin (α 2-macroglobulin, α 2M).In seminal fluid, PSA also forms compound with C protein mortifier.Because PSA-α 2M does not have immunocompetence, can not be detected by existing PSA detection method.So the PSA that can quantitatively detect at present has 3 kinds: T-PSA (t-PSA), F-PSA, PSA-ACT.
Measure t-PSA in serum and may be used for screening and early diagnosis prostate gland cancer, it is the good tumor markers of generally acknowledged diagnosing prostate cancer.Research display, t-PSA is determined on early diagnosis prostate gland cancer and is better than digital rectal examination.T-PSA is detected the generaI investigation index as more than the 50 years old male sex by U.S. FDA approved.Use t-PSA as the diagnostic criteria of diagnosing prostate cancer is: t-PSA: 0 ~ 4ng/ml is normal; 4 ~ 10ng/mL, the trouble cancer probability of 21 ~ 25%; >10ng/ml cancer.At the gray area of 4.0 ~ 10ng/ml, detect separately t-PSA and cannot distinguish cancer and hyperplasia of prostate.PSA can provide better biochemical indicator, for distinguishing prostate cancer and the hyperplasia of prostate of t-PSA 4 ~ 10ng/ml.
Summary of the invention
An object of the present invention is to provide a kind of enzyme linked immunological kit detecting PSA, its PSA monoclonal antibody solution marked by PSA standard items, contrast liquid, the ELISA Plate of PSA monoclonal antibody bag quilt, horseradish peroxidase (HRP) and auxiliary reagent form.
In the present invention, the PSA monoclonal antibody of the PSA monoclonal antibody of coated elisa plate and HRP mark is pairing antibody, and obtains by commercial channel.
In one embodiment of the invention, the preparation process of described coated elisa plate is: will wrap and be added each hole of ELISA Plate by with after PSA monoclonal antibody 0.05M carbonate buffer solution (pH value is 9.5) dilution, every hole 100 μ l, attach overnight, plate is washed with 0.05M phosphate buffer (pH value is 9.5), close with confining liquid again and spend the night, dry after drying, namely obtain monoclonal antibody coated elisa plate.Described confining liquid is that pH value is 9.5 containing the sucrose of BSA, 0.1g/L of 1g/L and the 15mmol/L PBS damping fluid of the casein (Casein) of 0.5g/L.
The PSA monoclonal antibody solution of described HRP mark is the 15mmol/L PBS damping fluid of the PEG200 of PSA monoclonal antibody and the 0.2mg/L marked containing the HRP of 0.5mg/L, and pH value is 8.5.
Described auxiliary reagent comprises contrast damping fluid, substrate solution, nitrite ion, reaction terminating liquid and cleaning buffer solution, and each reagent is specially:
Contrast damping fluid is PBS (pH7.4) damping fluid of 15mmol/L;
Substrate solution is 3% superoxol that phosphoric acid-citrate buffer solution (pH7.4) is prepared, and the Sodium Acid Pyrophosphate of 0.1mg/L in solution;
Nitrite ion is the methanol solution of tetramethyl benzidine (TMB), and concentration is 0.1mg/ml;
Reaction terminating liquid is 3mol/L sulfuric acid;
Cleaning buffer solution is the 0.05% polysorbas20 solution that the PBS (pH7.4) of 15mmol/L prepares.
The lowest detectable limit of the enzyme linked immunological kit of PSA of the present invention can reach 0.1ng/mL PSA, and stability is splendid, can preserve 1 year at ambient temperature, can preserve 3 years at 4 DEG C.
Embodiment
Below in conjunction with for example bright the present invention.It is pointed out that following explanation is only illustrating the technical scheme that application claims is protected, any restriction not to these technical schemes.The content that protection scope of the present invention is recorded with appended claims is as the criterion.
Embodiment 1
Kit forms: PSA monoclonal antibody coated elisa plate (96 hole); The PSA monoclonal antibody solution that horseradish peroxidase (HRP) marks 1 bottle, 6ml/ bottle; Contrast damping fluid 1 bottle; PSA standard items 1 bottle, each 1 bottle of substrate solution, nitrite ion, each 5ml/ bottle; Reaction terminating liquid 1 bottle, 5ml/ bottle; Cleaning buffer solution (20X concentrates) 1 bottle, 30ml/ bottle.
The preparation process of PSA monoclonal antibody coated elisa plate is: will wrap and be added each hole of ELISA Plate by with after PSA monoclonal antibody 0.05M carbonate buffer solution (pH value is 9.5) dilution, every hole 100 μ l, attach overnight, plate is washed with 0.05M phosphate buffer (pH value is 9.5), close with confining liquid again and spend the night, dry after drying, namely obtain monoclonal antibody coated elisa plate.Described confining liquid is that pH value is 9.5 containing the sucrose of BSA, 0.1g/L of 1g/L and the 15mmol/L PBS damping fluid of the casein (Casein) of 0.5g/L.
The PSA monoclonal antibody solution of HRP mark is the 15mmol/L PBS damping fluid of the PEG200 of PSA monoclonal antibody and the 0.2mg/L marked containing the HRP of 0.5mg/L, and pH value is 8.5.Detailed process is: with NaIO
4-glycol method carries out the oxidation of HRP, reaches final concentration 15mg/ml.Monoclonal antibody and HRP dialyse 9 hours in alkaline carbonic acid salt buffer, realize the mark of HRP to monoclonal antibody, and reaction terminates rear NaBH4 solution cessation reaction, then to PBS dialysed overnight.With saturated ammonium sulphate, obtain the anti-PSA monoclonal antibody of HRP enzyme mark of purifying.Use the 15mmol/L PBS buffer solution of the PEG200 containing 0.2mg/L to antibody final concentration 0.5mg/L again.
Contrast damping fluid is PBS (pH7.4) damping fluid of 15mmol/L;
Substrate solution is 3% superoxol that phosphoric acid-citrate buffer solution (pH7.4) is prepared, and the Sodium Acid Pyrophosphate of 0.1mg/L in solution;
Nitrite ion is the methanol solution of tetramethyl benzidine (TMB), and concentration is 0.1mg/ml;
Reaction terminating liquid is 3mol/L sulfuric acid;
The 0.05% polysorbas20 solution that the PBS (pH7.4) that cleaning buffer solution (1X) consists of 15mmol/L prepares.
The sensitivity of embodiment 2 kit is investigated
Prepare the PBS damping fluid of PSA standard items variable concentrations respectively, concentration is respectively 0.1ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, the kit adopting embodiment 1 to prepare detects, and to contrast damping fluid as blank, concrete detection method is as follows:
Cleaning buffer solution is prepared: the concentrated cleaning buffer solution adding distil water 20 times dilution provided by kit.
A) antigen-antibody reaction: the antibody bag provided at kit by the micropore of plate in respectively diplopore add 50 μ lAFP-L3 standard solutions and contrast damping fluid, use marking pen to be marked as 1,2 series.37 DEG C of water bath heat preservations 50 minutes.Wash plate and operate 5 times.
B) add the solution containing agglutinin in 1 serial well, in 2 serial wells, add the contrast damping fluid not containing lectin.37 DEG C of water bath heat preservations 50 minutes.Taking-up is pulled, and gets rid of liquid in hole.
C) the AFP monoclonal antibody solution that HRP marks is added each hole, every hole 100 μ l, 37 DEG C of water bath heat preservations 50 minutes.Repeat to wash plate and operate 5 times.
D) chromogenic reaction: every hole adds substrate solution A, each 50 μ l of nitrite ion B successively, 37 DEG C of water bath heat preservations 20 minutes, every hole adds 50 μ l reaction terminating liquids again and terminates reaction.
E) colorimetric: measure OD value and record at 450nm by microplate reader.
F) production standard curve: take standard concentration as horizontal ordinate, the OD value that standard items measure is ordinate, makes typical curve; Calculate typical curve regression coefficient R
2, work as R
2during > 0.99, this measures effectively;
Calculate the ratio of AFP-L3 standard items and blank, when ratio is greater than 2, illustrate that kit can measure the AFP-L3 standard items of this concentration, least concentration is the sensitivity of kit, and parallel experiment is averaged for five times, and concrete outcome is as follows:
0.1ng/ml PSA standard items and contrast damping fluid OD
450absorbance
| 0.1ng/mlPSA standard items | Blank damping fluid | OD ratio | |
| OD 450Absorbance | 0.012 | 0.003 | 4.0 |
Obtain absorbance data according to variable concentrations standard items to return, obtaining regression equation is y=0.106x+0.0086; R
2=0.98.Upper table data show that kit of the present invention is under 0.1ng/ml concentration, and sensitivity is good, and linearly splendid.
Embodiment 3 stabilization of kit is investigated
After kit embodiment 1 prepared places 6 months and 12 months respectively at 20 DEG C, measure the sensitivity of test paper according to the method for embodiment 2, and regretional analysis is carried out to data, calculate R
2value.
In the present embodiment, comparative example is set as follows:
Comparative example 1: the preparation method of kit is with embodiment 1, and difference is only not add PEG200 in the PSA monoclonal antibody solution that HRP marks.
Comparative example 2: the preparation method of kit is with embodiment 1, and difference is only that in the PSA monoclonal antibody solution that HRP marks, PEG200 replaces with hyclone (FBS), the same PEG200 of concentration.
Comparative example 3: the preparation method of kit is with embodiment 1, and difference is only that the confining liquid used in coated elisa plate preparation is the 15mmol/L PBS damping fluid of the BSA containing 1g/L, and pH value is 9.5.
Comparative example 4: the preparation method of kit is with embodiment 1, and difference is only that the confining liquid used in coated elisa plate preparation is the 15mmol/L PBS damping fluid of the casein (Casein) of BSA and 0.5g/L containing 1g/L, and pH value is 9.5.
Comparative example 5: the preparation method of kit is with embodiment 1, difference is only that the confining liquid used in coated elisa plate preparation is that pH value is 8.5 containing the sucrose of BSA, 0.1g/L of 1g/L and the 15mmol/L PBS damping fluid of the casein (Casein) of 0.5g/L.
Concrete outcome is as follows:
In addition, preserve after 36 months at 4 DEG C, kit sensitivity and linearly good, with the kit just prepared without significant difference.
Embodiment 4
Application the technology of the present invention prepares the quality testing of AFP-L3 enzyme-linked immune quantitative detection reagent box
Precision: randomly draw 50 box different batches kits, carries out replication with a liver cancer positive quality control serum by specification operation steps.Calculate each measurement result, obtain average, SD and coefficient of variation CV.Precision test result display batch between CV be less than 2%.
Content of the present invention merely illustrates some claimed specific embodiments; one of them or more described technical characteristic can be combined with arbitrary one or more technical scheme in technical scheme; these technical schemes obtained through combination also in the application's protection domain, just as these technical schemes obtained through combination in the disclosure of invention concrete record.
Claims (8)
1. detect an enzyme linked immunological kit for PSA, its PSA monoclonal antibody solution marked by PSA standard items, contrast liquid, the ELISA Plate of PSA monoclonal antibody bag quilt, horseradish peroxidase (HRP) and auxiliary reagent form.
2. enzyme linked immunological kit according to claim 1, it is characterized in that, the preparation process of described coated elisa plate is: will wrap and be added each hole of ELISA Plate by with after PSA monoclonal antibody 0.05M carbonate buffer solution (pH value is 9.5) dilution, every hole 100 μ l, attach overnight, washes plate with 0.05M phosphate buffer (pH value is 9.5), then spends the night with confining liquid is closed, dry after drying, namely obtain monoclonal antibody coated elisa plate.
3. enzyme linked immunological kit according to claim 2, is characterized in that, described confining liquid is that pH value is 9.5 containing the sucrose of BSA, 0.1g/L of 1g/L and the 15mmol/L PBS damping fluid of the casein (Casein) of 0.5g/L.
4. enzyme linked immunological kit according to claim 1, it is characterized in that, the PSA monoclonal antibody solution of described HRP mark is the 15mmol/L PBS damping fluid of the PEG200 of PSA monoclonal antibody and the 0.2mg/L marked containing the HRP of 0.5mg/L, and pH value is 8.5.
5. enzyme linked immunological kit according to claim 1, is characterized in that, contrast damping fluid is PBS (pH7.4) damping fluid of 15mmol/L.
6. enzyme linked immunological kit according to claim 1, is characterized in that, substrate solution is 3% superoxol that phosphoric acid-citrate buffer solution (pH7.4) is prepared, and the Sodium Acid Pyrophosphate of 0.1mg/L in solution.
7. enzyme linked immunological kit according to claim 1, is characterized in that, nitrite ion is the methanol solution of tetramethyl benzidine (TMB), and concentration is 0.1mg/ml.
8. enzyme linked immunological kit according to claim 1, is characterized in that, reaction terminating liquid is 3mol/L sulfuric acid; Cleaning buffer solution is the 0.05% polysorbas20 solution that the PBS (pH7.4) of 15mmol/L prepares.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510292841.9A CN104991071A (en) | 2015-05-29 | 2015-05-29 | ELISA kit of PSA and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510292841.9A CN104991071A (en) | 2015-05-29 | 2015-05-29 | ELISA kit of PSA and application thereof |
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| Publication Number | Publication Date |
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| CN104991071A true CN104991071A (en) | 2015-10-21 |
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| CN201510292841.9A Pending CN104991071A (en) | 2015-05-29 | 2015-05-29 | ELISA kit of PSA and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112710835A (en) * | 2020-12-15 | 2021-04-27 | 北京瀚梅生物科技有限公司 | Reproductive system disease detect reagent box |
-
2015
- 2015-05-29 CN CN201510292841.9A patent/CN104991071A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112710835A (en) * | 2020-12-15 | 2021-04-27 | 北京瀚梅生物科技有限公司 | Reproductive system disease detect reagent box |
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