CN106932570A - A kind of kit for detecting PSA PSA and its application - Google Patents
A kind of kit for detecting PSA PSA and its application Download PDFInfo
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- CN106932570A CN106932570A CN201710131799.1A CN201710131799A CN106932570A CN 106932570 A CN106932570 A CN 106932570A CN 201710131799 A CN201710131799 A CN 201710131799A CN 106932570 A CN106932570 A CN 106932570A
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/342—Prostate diseases, e.g. BPH, prostatitis
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Abstract
The present invention relates to a kind of PSA immue quantitative detection reagent box, including following detection reagent：(1) PSA reagents 1；(2) PSA reagents 2；(3) PSA quality-control products, the PSA quality-control products are the phosphate buffer of antigen containing PSA and calf serum；(4) PSA calibration objects, the PSA calibration objects are the phosphate buffer of antigen containing PSA and calf serum.The present invention uses antigen-antibody combination principle, combined with antibody by colloid gold particle and amplified testing result grade ratio, quantitative determination is carried out using automatic clinical chemistry analyzer, in detection range, precipitation strength is directly proportional to PSA concentration in sample, can read the PSA concentration of sample to be tested from matched curve by interpolation method.Higher compared to the kit sensitivity of the invention of other methods, stability is stronger, and cost is extremely low, and the standard of domestic and international equivalent reagent box can be surmounted completely.
Description
Technical field
It is a kind of examination for detecting PSA PSA specifically the present invention relates to technical field of immunoassay
Agent box and its application.
Background technology
PSA PSA (Prostate) is a kind of to contain 237 single chain polypeptide glycoprotein of amino acid, molecule
Amount about 34kDa, belongs to the serine protease race with the effect of chymotrypsin sample with tissue specificity.PSA is main by preceding
The line epithelial cell of row gland is produced, dense in seminal fluid.The major physiological effect of PSA is to decompose the gluey egg in seminal fluid
In vain, there is dilution seminal fluid, increase the effect of Sperm Motility.The PSA for being initially secreted into ducts of prostate gland is a kind of inactive enzyme
Former (proPSA), proenzyme has serine protease after 7 amino acid are fallen in amino cracking.A small amount of PSA can be followed into blood
Ring, the most of PSA into blood circulation is combined rapidly and inactivated with inhibin of protease, mainly with α -1 ACTs (ACT)
Combine (MG) with α -2 macroglobulin, also with the presence of small part by after proteolysis enzyme-deactivating with free state.Free PSA with
And the PSA (PSA-ACT) combined with ACT can be detected by existing immunological method.PSA and PSA- are detected simultaneously
ACT compounds are said t-PSA detection.The parcels of the PSA due to albumen of (MG) is combined with α -2 macroglobulin, is hidden
The effect of covering makes PSA specificity epitopes disappear, therefore cannot be detected with existing immunological method.Serum t-PSA content and prostate
Disease is closely related, and benign prostatic hyperplasis and pernicious prostate cancer can all cause t-PSA to raise, and PSA is then less to be received
The influence of prostate cancer growth.PSA has tissue specificity, but has no tumour-specific, prostatitis, benign prostatic hyperplasis
PSA can be caused to raise with prostate cancer.
Prostate cancer has exceeded cutaneum carcinoma as the most common cancer of North America male, because the lethal case load of cancer ranked second position.
PSA it is most of have the prostate cancer of clinical symptoms in can all raise, therefore monitoring PSA contents are for assessment oncotherapy effect
It is significant.The detection of current PSA all uses immunological method, colloidal gold strip method and chemoluminescence method, immunology
Method sensitivity is not high, and colloidal gold strip sensitivity is not high and needs by eye recognition, and chemoluminescence method is relatively costly.Enter
Mouthful monoclonal antibody because sensitivity is high, precision is high, accuracy is good, flexible and convenient to use etc., commercially position oneself at the forefront.
Chinese patent 201210291449.9 discloses a kind of detection kit for prostate specific antigen, including anti-
The coated solid phase carrier of FITC antibody, the anti-PSA antibody of FITC marks, anti-fPSA antibody, the above-mentioned enzyme institute of enzyme mark
The chemical luminous substrate of effect and negative and positive control solution；The method for preparing mentioned reagent box is comprised the following steps：1) with
The coated solid phase carrier of anti-FITC antibody；2) anti-PSA antibody is marked with FITC, the anti-PSA for obtaining FITC marks resists
Body；3) anti-fPSA antibody is marked with enzyme, obtains the anti-fPSA antibody of enzyme mark；4) dispense and assemble.Chinese patent
201110235581.3 disclose free prostate gland specificity antigen quantitative determination reagent kit, comprising FPSA Magneto separate reagents, enzyme
Reactant, increased response agent, dilution, FPSA standard items, FPSA quality-control products clean concentrated liquor and substrate solution.But
In the prior art, on the kit of present invention detection PSA PSA, yet there are no report.
The content of the invention
The purpose of the present invention is directed to deficiency of the prior art, there is provided a kind of PSA quantitative determination examination
Agent box.
It is a further object of the invention to provide the purposes of kit as described above.
To achieve the above object, the present invention is adopted the technical scheme that：
A kind of PSA immue quantitative detection reagent box, including following detection reagent：
(1) No. 1 composition of PSA reagents：
(2) No. 2 compositions of PSA reagents：
Further, the kit also includes PSA quality-control products, and the PSA quality-control products are antigen containing PSA and calf serum
Phosphate buffer, the PSA Antigen extractions are from Seminal plasma.
Further, the kit also includes PSA calibration objects, and the PSA calibration objects are antigen containing PSA and calf serum
Phosphate buffer, the PSA Antigen extractions are from Seminal plasma.
Further, the labeling method of the PSA collaurums is as follows：
(1) 0.1mol/LK is used2CO3Or 0.1mol/LHCl adjusts aurosol to pH6.8；
(2) it is 10000 that titre is added in 100ml aurosols, and volume is the PSA antibody-solutions of 2ml, stirs 2~3 points
Clock；
(3) 5ml 1%PEG20000 solution is added；
(4) it is centrifuged 30 minutes in 10000g, carefully sucks supernatant；
(5) will precipitate and be suspended in buffer solutions of the 20ml containing 0.2~0.5mg/ml PEG20000, after centrifugation, then use
Same buffer solution recovers, and concentration, with 0.5mg/ml Sodium azide anti-corrosions, puts 4 DEG C of preservations with A1cm/540nm=1.5；
(6) aurosol after being coated with can also concentrate and carry out gel chromatography after Sephadex G-200 posts and isolate and purify, with
Cushioning liquid wash-out containing 0.1%BSA.
Further, the preparation method of the aurosol is as follows：
(1) firing of collaurum should be completed in 24 hours；
(2) the desired amount of water is weighed with graduated cylinder, is poured into beaker, rule with marking pen at liquid level on beaker, will
Water is boiled to boiling；
(3) by 1% chlorauric acid solution according to 3:100 ratio is quickly adding into 2 liquid；
(4) 1% sodium citrate solution is pressed 4.5:100 are added in the liquid of the 3 of boiling, are well mixed, and keep boiling 5
Minute is to liquid in stable claret；
(5) stop heating, be cooled to room temperature；
(6) complemented at line with water, collaurum is poured into wide-mouth bottle, sealed with sealed membrane, obtain aurosol.
Further, the LDL of the kit is less than 0.1ng/ml, and repeatability is less than 15%, and difference between batch is less than
15%.
Further, it is 5 kinds of concentration by the sample doubling dilution of (80 ± 16) ng/ml, the sample of each concentration is repeated to examine
Survey 2 times, calculate average value, result average value and dilution ratio are carried out into fitting a straight line, linearly dependent coefficient with least square method
More than or equal to 0.999.
Further, the measured deviation of the kits result is in the range of ± 10%.
Further, the kit is respectively less than 1 ‰ with carcinomebryonic antigen (CEA) and alpha-fetoprotein (AFP) cross reaction.
To realize above-mentioned second purpose, the present invention is adopted the technical scheme that：
As above the purposes of any kit, is used for：
(1) application in PSA quantitative determination；
(2) application in PSA quantitative determination product is prepared；
(3) quantitative detecting method of PSA is detected：It is 0,0.5,5,15,50 and by aimed concn
The PSA calibration object of 100ng/ml adds automatic clinical chemistry analyzer, with water as zero point, sets up working curve.
Operating procedure：
Computational methods：
Calibration solution absorbance difference (A2-A1) is calculated, and sets up calibration solution absorbance-concentration working curve.
The absorbance difference of sample is calculated, corresponding concentration value is read on working curve.
Quality Control program：Measurement PSA quality-control products, test result can carry out Samples detection in the range of Quality Control.
The invention has the advantages that：
1st, the present invention is molten to collaurum by antibody labeling using homogeneous phase sol particle immunoassay (colloidal gold solution method)
In liquid, patient's blood sample is detected by automatic clinical chemistry analyzer, higher compared to other method sensitivity, stability is stronger, cost
It is extremely low, the standard of domestic and international equivalent reagent box can be surmounted completely.
2nd, kit of the invention uses antigen-antibody combination principle, is combined with antibody by colloid gold particle and ties detection
Fruit etc. carries out quantitative determination than amplifying using automatic clinical chemistry analyzer.In detection range, PSA in precipitation strength and sample
Concentration is directly proportional, therefore can just read the PSA concentration of sample to be tested from matched curve by interpolation method.
3rd, kit of the invention stable performance within the effect phase, LDL, repeatability, linear, accuracy, batch between
Difference, analysis specificity meet the requirements.
Specific embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention.In addition, it is to be understood that after the content for having read record of the present invention, art technology
Personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Fixed scope.
Embodiment
First, reagent constituents of the present invention
PSA (PSA) immue quantitative detection reagent box (colloidal gold method) is made up of following four component, each group
Mitogenetic production. art is summarized as follows：
(1) No. 1 (PSA-R1) composition of PSA reagents：
(2) No. 2 (PSA-R2) compositions of PSA reagents：
Anti-psa antibody extracts from the mouse ascites of PSA Hybridoma Cell Cultures supernatant or infection.
(3) PSA quality-control products (PSA-QC)：Content is the phosphate-buffered of the antigens of PSA containing finite concentration and calf serum
Liquid (concentration 0.02mol/L, PH=7.4), PSA Antigen extractions are from Seminal plasma.The nominal concentration of Quality Control is 4ng/ml and 30ng/
Ml, the concentration range of every batch is slightly different.
(4) PSA calibration objects (PSA-STD)：Phosphate buffer (the concentration of the antigens of PSA containing finite concentration and calf serum
0.02mol/L, PH=7.4) it is formulated, PSA Antigen extractions are from Seminal plasma.Aimed concn is respectively 0,0.5,5,15,50 and
100ng/ml.This kit calibration object concentration can trace to the source to the Stanford Reference standard/WHO96/670
(90%PSA-ACT+10%free PSA).For setting up standard curve.
2nd, specifically used method is as follows：
【It is applicable instrument】
This kit is in Hitachi 7060, Olympus400, Siemens's Dimension xpand automatic clinical chemistry analyzers
Upper checking is qualified.
【The method of inspection】
Calibration procedure：Single-point may be selected or multiple spot calibration mode sets up working curve.
One point standard：Liquid calibration solution, directly uses.
Multiple spot is calibrated：(for automatic biochemical analyzer, calibration mode selection Nonlinear or log-logit 4P methods), by mesh
Mark concentration is 0,0.5,5,15,50 and the PSA calibration object of 100ng/ml adds automatic clinical chemistry analyzer,
With water as zero point, working curve is set up.
Operating procedure：
Computational methods：
Calibration solution absorbance difference (A2-A1) is calculated, and sets up calibration solution absorbance-concentration working curve.
The absorbance difference of sample is calculated, corresponding concentration value is read on working curve.
Quality Control program：Measurement PSA quality-control products, test result can carry out Samples detection in the range of Quality Control.
【The explanation of assay】
Sample is please pressed 1 by result as exceeded the range of linearity with physiological saline:1 dilution, measurement result multiplies 2.
Reagent is stored：
Each component all should avoid direct sunlight.Magneto separate reagent should be kept upright, and prevent magnetic particle from drying for a long time and losing
It is living.Magneto separate reagent, reagent 2 such as occur freezing to be continuing with.Reagent should in time return to 2-8 DEG C after, it is to avoid
Long-time room temperature is placed.The reagent term of validity：Kit and each component do not break a seal in 2-8 DEG C of preservation, the term of validity is 12 months.
【Sample requirement】
Following sample empirical tests meet the requirement of this kit：Serum：Take in 5.0ml venous blood to teat glass, be not added with
Anti-coagulants, stands at room temperature, then separates sera components by the way that (3000rpm × 5min) is centrifuged.Sample is stored and processed
Serum specimen is placed under the conditions of room temperature (22 DEG C) and is not to be exceeded 8h.Serum specimen is not to be exceeded 48h in 2 DEG C of -8 DEG C of preservations,
Otherwise should -20 DEG C of preservations.- 20 DEG C preserve serum specimen Storage period and are less than 3 months, and number of freezing and thawing does not influence detection less than 3 times
Result should avoid sample multigelation.
Known chaff interference
If contain following concentration interfering materials in sample, testing result is not influenceed：Hemoglobin<5mg/ml；Total courage
Red pigment<0.5umol/ml；Fat Emulsion<10mg/ml；Liquaemin<100IU/ml.Alkaline phosphatase activities sample (Paget high
Disease, cholestasia etc.) testing result may be inaccurate, should avoid using.Sample can not if there is obvious microorganism pollution
For detecting.Close contact rodent received the patient of mouse resource monoclonal antibody drug therapy, can in its sample
HAMA can be contained, acquired results occur abnormal possibility；Contain other various heterophile antibodies in sample, also may be used
Results abnormity can be caused.
Calibration method：PSA calibration objects A-F is detected as sample, two repeating pipes is at least set, while at least making list
The PSA Quality Controls of pipe.If fitting correlation coefficient>0.9900, while PSA Quality Controls measured value is within a predetermined range, then it is assumed that calibration is just
Really, the standard curve can be stored and used.
Quality control：Every workday is required for carrying out quality control, and each Quality Control point must at least do 1 pipe.If hair
Existing Quality Control measured value can not then carry out the detection of sample not in preset range (referring to Quality Control list).
The calculating of measurement result：Recommendation obtains the recurrence side of calibration object dose-response curve using four parameter logistic fits
Journey, further according to sample to be tested relative luminous intensity can from being returned on regression curve and calculating sample determinand concentration.Measurement
Scope：Measurement range is (0.1-100) ng/ml.When concentration of specimens is less than 0.1ng/ml, it is reported as<0.1ng/ml.Concentration of specimens
It is reported as during higher than 100ng/ml>100ng/ml, if it is desired to understand the definite concentration of sample, normal saline dilution sample can be used
Determined again after (20 times of suggestion dilution).
【Term of reference】PSA contents are not more than 4ng/ml in 95% normal male serum.Due to geographical, ethnic group and age
Etc. difference, it is proposed that the reference value (scope) of oneself is set up in each laboratory.In reference value research, because testing result is not belonging to just
State is distributed, so representing reference range with Percentiles.The percentile of PSA reference values is 95%.【The solution of assay
Release】The testing result of this product is not unique confirmation index of clinical indication, and its clinical meaning need to refer to reference to other detections
Mark and clinical manifestation concrete analysis.Some prostate benign lesions, PSA levels can also be raised in blood.
【The limitation of the method for inspection】
The testing result of this kit is supplemented as the one kind to other clinical information in clinical diagnosis.Detection
When to strictly observe operation sequence, any change of step is likely to influence result.PSA concentration is more than 1000ng/ml
When be possible to HOOK (hook-shaped) effect occur.If contain following concentration interfering materials in sample, testing result is not influenceed：Blood
Lactoferrin<5mg/ml；Total bilirubin<0.5umol/ml；Fat Emulsion<10mg/ml；Liquaemin<100IU/ml.Alkaline phosphatase
Activity sample (Paget diseases, cholestasia etc.) testing result high may be inaccurate, should avoid using.EDTA or citric acid
Sodium anti-freezing blood plasma detection result of specimen may be inaccurate, should avoid using.
【Points for attention】This reagent is only used for in-vitro diagnosis.Please proper storage is indicated in strict accordance with specification and use examination
Agent, any change on step may all influence result.When reagent exceedes the effect phase, please stop using.Different lot number kits
Component does not answer cross-reference.The testing result of this kit only supplies clinical reference, and the clinical diagnosis to disease should combine its disease
Situations such as shape/sign, medical history, other laboratory examinations and therapeutic response, considers.Due to methodology or antibody specificity
Etc. reason, different detections may be obtained to carrying out tumor marker analyte detection with portion sample using the reagent of different manufacturers
As a result, therefore, in diagnosing tumor and monitoring process, with different reagents detect acquired results should not directly be compared to each other, in order to avoid
Cause the medical explanation of mistake；Suggestion laboratory indicates agents useful for same feature in the examining report for issue clinician.Disease
If changing types of agents in monitoring, then should carry out extra continuity detection and with original reagent result carry out it is parallel compare with
Redefine baseline value.Kit serum used in preparation process or blood plasma raw material, though have already been through HBsAg,
The detection of the projects such as HIV1/2-Ab, HCV-Ab is feminine gender, but so far, no any one detection may insure definitely
Safety, therefore these components should be treated as Potential infection source.Should basis《Laboratory-bio-safety General Requirement》Advised
Fixed processing method is implemented.It is ensured that otch or its cutaneous lesion are sufficiently protected during operation.
3rd, the operation standard of collaurum is fired
1. confirm before operating：
Confirm that required glass apparatus inner surface does not exist any stain and flaw before 1.1 operations.
Confirm that water resistance used is not less than 18M Ω before 1.2 productions, and the resting period is no more than 24 hours.
2. production operation：
The firing of 2.1 collaurums should be completed in 24 hours.
2.2 weigh the desired amount of water with graduated cylinder, are poured into beaker, are rule with marking pen at liquid level on beaker, will
Water is boiled to boiling.
2.3 by 1% chlorauric acid solution according to 3:100 ratio is quickly adding into 2.2 liquid.
1% sodium citrate solution is pressed 4.5 by 2.4:100 are added in 2.3 liquid of boiling, are well mixed, and keep boiling 5
Minute is to liquid in stable claret.
2.5 stop heating, are cooled to room temperature.
2.6 are complemented at line with water.Collaurum is poured into wide-mouth bottle, is sealed with sealed membrane, obtain aurosol.
4th, the labeling method of PSA collaurums：
(1) 0.1mol/LK is used2CO3Or 0.1mol/LHCl adjusts aurosol to pH6.8.
(2) it is 10000 that titre is added in 100ml aurosols, and volume is the PSA antibody-solutions of 2ml, stirs 2~3 points
Clock.
(3) 5ml 1%PEG20000 solution is added.
(4) it is centrifuged 30 minutes in 10000g, carefully sucks supernatant (never toppling over).
(5) will precipitate and be suspended in buffer solutions of the 20ml containing 0.2~0.5mg/ml PEG20000, after centrifugation, then use
Same buffer solution recovers, and concentration, with 0.5mg/ml Sodium azide anti-corrosions, puts 4 DEG C of preservations with A1cm/540nm=1.5.
(6) aurosol after being coated with can also concentrate and carry out gel chromatography after Sephadex G-200 posts and isolate and purify, with
Cushioning liquid wash-out containing 0.1%BSA.It is 8.2 generally with aurosol eluent pH.
It should be noted that not answering impure particulate in all solution in operating above, can in advance be located with high speed centrifugation or miillpore filter
Reason.
5th, the Quality Identification of collaurum PSA antibody conjugates
(1) measurement of colloid gold particle average diameter：With supporting the nickel screen (copper mesh also can) of film to dip gold mark protein reagent,
Directly observed under transmission electron microscope after natural drying.Or observed after being redyed with acetic acid uranium.Calculate the average straight of 100 gold grains
Footpath.
(2) the OD520nm values of colloidal gold solution are determined：There is maximum between wavelength 510nm~550nm in colloid gold particle
Absorption value peak.Collaurum protein reagent is made 1 with 0.02Mol/L pH8.2 PBS liquid (containing 1%BSA, 0.02%NaN3):20 is dilute
Release, OD520=0.25 or so.The OD520 for being normally applied liquid should be 0.2~0.4.
(3) specificity and sensitivity testing of gold mark PSA solution：Using miillpore filter immunogold silver staining (MF-
IGSSA).By soluble PSA Antigen adsorptions in (filter paper, nitrocellulose membrane, miillpore filter) on carrier, with colloid gold label
PSA solution detects corresponding PSA antigens with direct or indirect decoration method and through silver development, or directly by full-automatic biochemical point
PSA antigens in analyzer detection blood, specificity and sensitiveness to gold mark PSA solution are identified.
This kit uses antigen-antibody combination principle, is combined with antibody by colloid gold particle and puts the ratios such as testing result
Greatly, quantitative determination is carried out using automatic clinical chemistry analyzer.In detection range, precipitation strength is with PSA concentration in sample into just
Than, therefore the PSA concentration of sample to be tested can be just read from matched curve by interpolation method.
6th, quality control and technical parameter
1 quality control
1.1 carry out following experiment using PSA reagents to be measured, and standard curve is done with calibration object, detect following each point：
1.2 calculate accuracy, LDL, linear and repeatability successively.
2 technical parameters
2.1 accuracys
Measurement concentration is with bottle mark concentration deviation in ± 10%；
2.2 LDLs
≤0.1ng/ml；
2.3 is linear
Correlation coefficient r >=0.99
2.4 repeatability
Two sample CV are equal<15%；
2.5 quality-control products
Each quality-control product measured value is in the range of.
7th, tested sample
1. sample is applicable
Suitable for serum and Heparin plasma.
2. the treatment of sample
2.1 serum
Take in 5.0ml venous blood to teat glass, not doping stands in room temperature (18-25 DEG C), by centrifugation point
From sera components, storage.
2.2 blood plasma
In taking 5.0ml venous blood to glass or plastic test tube, with heparin as anti-coagulants, by centrifugal separation plasma part,
Storage.
2.3 high concentration samples
If estimating, sample concentration exceeds 100ng/ml, needs to be diluted with sample diluting liquid before measure.
3. the storage of sample
2~8 DEG C of serum and plasma sample are stablized 24 hours.If needing the long period to preserve, sealed packaging can be protected in -20 DEG C
Deposit 30 days, it is to avoid multigelation.
4. known disturbances factor
Avoid using following several serum or Heparin plasma：The sample of significant hemolysis, serious hyperlipidemia sample, alkali
Acid phosphatase activity sample high (Paget diseases, cholestasia etc.), the sample of serious jaundice.Unusable EDTA and lemon
Sour sodium is the plasma sample of anti-coagulants.
8th, result
Detected on the automatic clinical chemistry analyzers of HITACHI 7180
Performance Evaluation data
1 LDL
1.1 requirement of experiment
0.1ng/ml should be not more than.
1.2 experimental techniques
Detected that replication 20 times draws 20 measurement results as sample with zero-dose calibration object, calculate it and put down
Average (M) and standard deviation (SD), draw M+2SD, according to concentration and detected value between zero-dose calibration object and adjacent calibration object
Carry out 2 regression fits and draw linear function, the RLU values of M+2SD are brought into above-mentioned equation, obtain corresponding concentration value, i.e.,
It is LDL.
1.3 experimental datas
1.4 conclusions
From three batches of reagent testing results, LDL experimental result is not higher than 0.1ng/ml.
2 repeatability
2.1 requirement of experiment
15% should be not more than
2.2 experimental techniques
With each duplicate detection of the sample of (4 ± 0.8) ng/ml and (30 ± 6) ng/ml 10 times, 10 measurement results are calculated
Average value M and standard deviation SD, coefficient of variation CV is drawn according to formula CV=SD/M × 100%.
2.3 experimental datas
2.4 conclusions
From three batches of reagent testing results, repeated experiment result not higher than 15%.
3 difference between batch
3.1 requirement of experiment
15.0% should be not more than
3.2 experimental techniques
Detect a concentration in the range of (4 ± 0.8) ng/ml and (30 ± 6) ng/ml respectively with three kits of lot number
Sample, be respectively repeated 10 times, calculate 30 the average value M and standard deviation SD of measurement result, according to formula CV=SD/M × 100%
Draw coefficient of variation CV.
3.3 experimental datas
| Sample | The coefficient of variation |
| 4ng/ml | 6.22% |
| 30ng/ml | 7.49% |
3.4 conclusions
From testing result, difference between batch experimental result not higher than 15%.
4 is linear
4.1 requirement of experiment
The range of linearity：0.1~100ng/ml, in this range of linearity, the correlation coefficient r of kit answers >=0.99
4.2 experimental techniques
It is 5 kinds of concentration by the sample doubling dilution of (80 ± 16) ng/ml, by the sample duplicate detection 2 times of each concentration, meter
Average value is calculated, result average value and dilution ratio fitting a straight line is carried out into least square method, and calculate linearly dependent coefficient r.
4.3 experimental datas
4.4 conclusions
From three batches of reagent testing results, linearly dependent coefficient (r) experimental result is not less than 0.99.
5 accuracys
5.1 requirement of experiment
Detected as sample with international standard substance or by enterprise's calibration object (QB) of international standard substance mark, its survey
The relative deviation for measuring result should be in the range of ± 10%.
5.2 experimental techniques
Compound concentration is about the international standard substance or enterprise's calibration object (QB) of 30ng/ml (tolerance is ± 10%), will
Its as sample to specifications the step of detected, measurement 3 times after, take result average value and be designated as M, according to formula：Measurement
Deviation=(M- theoretical values)/theoretical value × 100%.
5.3 experimental datas
5.4 conclusions
From three batches of reagent testing results, calibrated with international standard substance or by the enterprise of international standard substance series markization
Product (QB) are detected that the relative deviation of its measurement result is in the range of ± 10% as sample.
6 specificity
6.1 requirement of experiment
Kit potential cross reaction thing relevant with following table should be without significant cross reaction.
6.2 experimental techniques
CEA, AFP calibration object diluted are detected into the sample to concentration as shown in the following chart as sample kit
Detectable concentration is obtained, to measure the degree that concentration represents cross reaction with the ratio of addition concentration.
6.3 experimental datas
| First batch data | Second batch data | 3rd batch data | |
| CEA degree of disturbances | Less than 1 ‰ | Less than 1 ‰ | Less than 1 ‰ |
| AFP degree of disturbances | Less than 1 ‰ | Less than 1 ‰ | Less than 1 ‰ |
6.4 conclusions
From three batches of reagent testing results, kit is equal with carcinomebryonic antigen (CEA) and alpha-fetoprotein (AFP) cross reaction
Less than 1 ‰
7 conclusions
From three batches of reagent testing results, kit stable performance within the effect phase, LDL, repeatability, it is linear,
Accuracy, difference between batch, analysis specificity meet the requirements.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of PSA immue quantitative detection reagent box, it is characterised in that the detection kit includes following inspection
Test agent：
(1) No. 1 composition of PSA reagents：
(2) No. 2 compositions of PSA reagents：
2. PSA immue quantitative detection reagent box according to claim 1, it is characterised in that the kit is also
Including PSA quality-control products, the PSA quality-control products are the phosphate buffer of antigen containing PSA and calf serum, and the PSA antigens are carried
Take from Seminal plasma.
3. PSA immue quantitative detection reagent box according to claim 1, it is characterised in that the kit is also
Including PSA calibration objects, the PSA calibration objects are the phosphate buffer of antigen containing PSA and calf serum, and the PSA antigens are carried
Take from Seminal plasma.
4. PSA immue quantitative detection reagent box according to claim 1, it is characterised in that the PSA colloids
The labeling method of gold is as follows：
(1) 0.1mol/LK is used2CO3Or 0.1mol/LHCl adjusts aurosol to pH6.8；
(2) it is 10000 that titre is added in 100ml aurosols, and volume is the PSA antibody-solutions of 2ml, is stirred 2~3 minutes；
(3) 5ml 1%PEG20000 solution is added；
(4) it is centrifuged 30 minutes in 10000g, carefully sucks supernatant；
(5) will precipitate and be suspended in buffer solutions of the 20ml containing 0.2~0.5mg/ml PEG20000, after centrifugation, then with same
Buffer solution recovers, and concentration, with 0.5mg/ml Sodium azide anti-corrosions, puts 4 DEG C of preservations with A1cm/540nm=1.5；
(6) aurosol after being coated with can also concentrate and carry out gel chromatography after Sephadex G-200 posts and isolate and purify, to contain
The cushioning liquid wash-out of 0.1%BSA.
5. PSA immue quantitative detection reagent box according to claim 4, it is characterised in that the aurosol
Preparation method is as follows：
(1) firing of collaurum should be completed in 24 hours；
(2) the desired amount of water is weighed with graduated cylinder, is poured into beaker, rule with marking pen at liquid level on beaker, water is boiled
To boiling；
(3) by 1% chlorauric acid solution according to 3:100 ratio is quickly adding into 2 liquid；
(4) 1% sodium citrate solution is pressed 4.5:100 are added in the liquid of the 3 of boiling, are well mixed, and keep boiling 5 minutes
To liquid in stable claret；
(5) stop heating, be cooled to room temperature；
(6) complemented at line with water, collaurum is poured into wide-mouth bottle, sealed with sealed membrane, obtain aurosol.
6. PSA immue quantitative detection reagent box according to claim 1, it is characterised in that the kit
LDL is less than 0.1ng/ml, and repeatability is less than 15%, and difference between batch is less than 15%.
7. PSA immue quantitative detection reagent box according to claim 1, it is characterised in that by (80 ± 16)
The sample doubling dilution of ng/ml is 5 kinds of concentration, by the sample duplicate detection 2 times of each concentration, calculates average value, and result is put down
Average and dilution ratio carry out fitting a straight line with least square method, and linearly dependent coefficient is more than or equal to 0.999.
8. PSA immue quantitative detection reagent box according to claim 1, it is characterised in that the kit is surveyed
The measured deviation of amount result is in the range of ± 10%.
9. PSA immue quantitative detection reagent box according to claim 1, it is characterised in that the kit with
Carcinomebryonic antigen (CEA) and alpha-fetoprotein (AFP) cross reaction are respectively less than 1 ‰.
10. purposes of any kits of claim 1-3 in PSA quantitative determination, is used for：
(1) application in PSA quantitative determination；
(2) application in PSA quantitative determination product is prepared；
(3) quantitative detecting method of PSA is detected in sample to be tested：It is 0,0.5,5,15,50 by aimed concn
PSA calibration object with 100ng/ml adds automatic clinical chemistry analyzer, with water as zero point, sets up work bent
Line.
Operating procedure：
Computational methods：
Calibration solution absorbance difference (A2-A1) is calculated, and sets up calibration solution absorbance-concentration working curve.
The absorbance difference of sample is calculated, corresponding concentration value is read on working curve.
Quality Control program：Measurement PSA quality-control products, test result can carry out Samples detection in the range of Quality Control.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710131799.1A CN106932570A (en) | 2017-03-07 | 2017-03-07 | A kind of kit for detecting PSA PSA and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710131799.1A CN106932570A (en) | 2017-03-07 | 2017-03-07 | A kind of kit for detecting PSA PSA and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113866412A (en) * | 2021-09-07 | 2021-12-31 | 山东博科生物产业有限公司 | Sensitive total prostate specific antigen detection kit |
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Application publication date: 20170707 |