CN110456044A - A kind of kit for prostatitis detection - Google Patents
A kind of kit for prostatitis detection Download PDFInfo
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- CN110456044A CN110456044A CN201910768127.0A CN201910768127A CN110456044A CN 110456044 A CN110456044 A CN 110456044A CN 201910768127 A CN201910768127 A CN 201910768127A CN 110456044 A CN110456044 A CN 110456044A
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- G—PHYSICS
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- 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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The present invention relates to field of medical examination, more particularly, to preparation, application method and the purposes of a kind of kit for prostatitis detection.Kit of the invention, detection sample is high-efficient, can save plenty of time and manpower；It is linear good, and for the range of linearity up to 0.3~20pg/mL, detection threshold value is low, and sensitivity is higher.And accuracy is good, every sample is continuously detected 20 times with two kinds of kits, is calculated 20 variations and is no more than 5%.And reagent accuracy is higher, and the sample or antigen of kit of the present invention detection high concentration, and HOOK phenomenon does not occur.
Description
Technical field
The present invention relates to field of medical examination, more particularly, to a kind of kit for prostatitis detection preparation,
Application method and purposes.
Background technique
Chronic prostatitis is adult male uropoiesis, one of the most common type in genital system diseases, be mainly in it is between twenty and fifty, in
Year male, puberty morbidity are few.There is investigation display chronic prostatitis to account for the 25% of Urology Surgery male's consulting patients in out-patient department
Or so, there is prostatitis person to suffer from prostatitis more than 10%, 50% male's different times in crowd.
It is prostatitic " goldstandard " that there is presently no diagnosing chronics, can be used for the meaning of the methodology of clinical research very
It is limited, it is only scorching come diagnosis of prostate using very few objective basis less than 5% clinician.Although in the morning of disease
Phase can be by clinical symptoms tentative diagnosis, but must carry out necessary inspection according to modern detection methods, and exclude others
Urological diseases can be determined with after exception.So chronic prostatitis is often a kind of removing property or defective diagnosis,
Classification diagnosis lacks the prostatitic positive feature of other types according to being also method for removing.
The common inspection method in laboratory is mainly urinalysis, examination of prostatic fluid, digital rectal examination, wherein prostatic fluid
Inspection, digital rectal examination have invasion to human body, there is certain pain, and short time consumption is long, and people generally have conflict psychology to this.Cause
It is a kind of noninvasive stabilization of this market demand, simple and efficient, while accurately and reliably biomarker and detection means again, as chronic
Prostatitis detection method avoids the intrusion of patient from checking pain, improves the life of patient for crowd's screening and clinical detection
Quality.
Prostasomes are a kind of subcellular structures by human benign prostatic epithelial cells, contain 440 kinds of protein,
With multiple functions.Since the excretory duct of prostate is opened on the rear wall of prostatic urethra, and many prostatic acinis are straight
It connects and is opened on urethra, therefore Prostasomes are easily accessible in urine and prostatic fluid, tissue is by scorching thin in chronic prostatitis
The infiltration of born of the same parents can release various active materials and chemotactic factor (CF).Research finds forefront in chronic prostatitis syndrome Urine in Patients
Gland corpusculum excretion protein content is apparently higher than content in normal human urine, has significant difference to can be used as a good forefront
Gland specific biomarkers.
The present invention has the characteristics that easy to operate and easy to use relative to product is disclosed currently on the market, saves people
Power material resources；Result is fast (40min) out simultaneously, saves the plenty of time for reviewer；The kit range of linearity is wide, HOOK risk
Application that is small, being more suitable clinically.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of kit for prostatitis detection
Preparation, application method and purposes.Also kit is easy to operate, and detection speed is fast (40min can go out testing result), detection range
Width, HOOK risk is small, clinical use of being more convenient for.
The present invention provides a kind of combinations of reagent comprising labelled antibody mixed liquor, magnetic bead suspension and calibration object；
The labelled antibody mixed liquor include: 0.1%~0.5% (w/v) trace labelling substance markers PSEP antibody,
0.01mol/L~0.1mol/L buffer solution A, 1 ‰~3 ‰ (w/v) preservative A, 1 ‰~3 ‰ (w/v) stabilizer A and 0.5 ‰~
3 ‰ (w/v) surfactant As；
The magnetic bead suspension is by the JSR hydrophobicity magnetic bead, MES buffer, PSEP antibody, surfactant and the envelope that activate
Liquid is protected to be made；Wherein, the envelope protect liquid include: 0.01mol/L~0.1mol/L buffer solution B, 1 ‰~3 ‰ (w/v) preservative B,
1 ‰~3 ‰ (w/v) bovine serum albumin(BSA)s, 5%~15% (w/v) glycerol or sucrose and 0.5 ‰~3 ‰ (w/v) surface-actives
Agent B；
Wherein, the buffer solution A, B independently selected from Tris-NaCl, Bis-tris, PBS, MOPS, MES, Taps extremely
Few one kind；
Described preservative A, B are independently selected from Proclin 300, MIT, Bro, IPBC, NaN3At least one of；
The stabilizer A is selected from least one of casein, isinglass, bovine serum albumin(BSA)；
The surfactant A, B are independently selected from tween-20, Tritonx-100, Silwet L7600
At least one of Surfactant.
In the present invention, surfactant Tritonx-100 is contained in magnetic bead suspension and labelled antibody solution, is improved
Accuracy reduces the effect for jumping hole risk.
In the present invention, the labelled antibody mixed liquor by 0.1% (w/v) trace labelling substance markers PSEP antibody,
0.05mol/L Tris-NaCl, 1 ‰ (w/v) Proclin 300,1 ‰ (w/v) caseins, 1% (w/v) bovine serum albumin(BSA),
It is formed with 5 ‰ (w/v) Tritonx-100.
In the present invention, the trace labelling object is selected from acridinium ester, horseradish peroxidase, luminol or derivatives thereof, different
Luminol or derivatives thereof.
In the present invention, in the magnetic bead suspension, the preparation of the JSR hydrophobicity magnetic bead of activation include: by partial size be 100~
After the JSR hydrophobicity magnetic bead of 200nm is washed 5 times with the MES buffer containing TritonX-100, with the 1- ethyl-of 20mg/ml
(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate activates magnetic particle 1 hour, and the JSR hydrophobicity magnetic bead of activation is made.
The magnetic particle is JSR hydrophobicity magnetic bead, is added with surfactant during coating, increases point of magnetic bead
Property is dissipated, the risk for jumping hole is reduced.
In the present invention, the preparation method of the magnetic bead suspension includes: with the MES buffer washing containing surfactant
The JSR hydrophobicity magnetic bead of activation, then mixes with PSEP antibody, and under surfactant existence condition, room temperature shakes 3h, with end
After only liquid terminates reaction, then fluid-tight is protected with envelope and is closed three times, magnetic bead suspension is made.
In some embodiments, the terminate liquid be 5% ethanol amine.
In the present invention, the envelope protects liquid by 0.05mol/L Tris-NaCl, 1 ‰ (w/v) Proclin 300,1% (w/v)
Bovine serum albumin(BSA), 5% (w/v) sucrose and 0.5 ‰ (w/v) Tritonx-100 composition.
It further include calibration object in reagent combination of the present invention, the calibration object includes: PSEP antigen, 0.01mol/L
~0.1mol/L buffer C, 1 ‰~3 ‰ (w/v) preservative C, 1% (w/v) bovine serum albumin(BSA) and 1 ‰~3 ‰ (w/v) junket
Albumen；
Wherein, the buffer C is selected from least one of Tris-NaCl, Bis-tris, PBS, MOPS, MES, Taps；
The preservative C is selected from Proclin 300, MIT, Bro, IPBC, NaN3At least one of.
In the present invention, the calibration object is by PSEP antigen, 0.05mol/L Tris-NaCl, 1 ‰ (w/v) Proclin 300
With 1 ‰ (w/v) caseins, 1% (w/v) bovine serum albumin(BSA).
In the present invention, the calibration object is the calibration object of gradient concentration, and main includes the PSEP antigen of various concentration.
Specifically, the concentration range of PSEP is 0~20ng/mL in the calibration object.It include multiple and different specific PESP
The standard items of concentration.In the calibration object, it is necessary to the standard items for being 0 comprising PESP concentration.In some embodiments, set in calibration object
The concentration for being equipped with PSEP is respectively 6 parts of calibration objects of 0ng/ml, 0.5ng/ml, 2ng/ml, 6ng/ml, 10ng/ml, 20ng/ml.
The application of reagent combination of the invention in preparation prostatitis detection kit.
In the present invention, the prostatitis detection monitoring chronic prostatitis occurs and/or disease progression.
The present invention also provides a kind of kits, and it includes reagent of the present invention combinations.
The kit has widened detection range, use more conducively clinically using double antibody sandwich method.Institute
The kit stated can allow corresponding Full-automatic chemiluminescence apparatus using Magnetism particulate immuno chemistry luminescence method, easy to use, detection
Used time is short.Reagent of the present invention is screened for the Magnetism particulate immuno chemistry luminescence method detection of PSEP, is mentioned using the present invention
The reagent of confession combines, and can obtain better accuracy, stability and sensitivity, and HOOK risk is lower.
More specifically, the kit contains calibration card, ginseng needed for instrument can obtain test by the way that calibration card is automatic
Number.
The present invention also provides a kind of methods for assisting prostatitis diagnosis, with reagent combine detection provided by the invention
PSEP content in urine, whether auxiliary judgment suffers from prostatitis according to testing result.
Reagent combination provided by the invention can be used in the double-antibody sandwich detection of prostatitis marker PSEP, cooperation
AutoLumo A2000 series Full-automatic chemiluminescence analyzer, can be realized the automatic detection of PSEP.The reagent is incorporated into
There is one of following advantages less:
1, detection sample is high-efficient, can save plenty of time and manpower；The detection time of the prior art at least needs 3.5
Hour, and the reagent of the application only needs the detection just realized to PESP in 40 minutes, efficiency has obtained significant raising.In environment
Under conditions of stabilization, the detection time of reagent of the present invention can be shortened to 20 minutes or even 20 minutes or less.
2, reagent provided by the invention is linearly good, and for the range of linearity up to 0.3~20pg/mL, detection threshold value is low, sensitivity
It is higher.
3, reagent accurate provided by the invention is good, and every sample is continuously detected 20 times with two kinds of kits, calculates 20
Secondary variation is no more than 5%.
4, reagent accuracy provided by the invention is higher, and the sample or antigen of kit of the present invention detection high concentration, not
HOOK phenomenon occurs.
Detailed description of the invention
Fig. 1 is the range of linearity examination of reagent of the present invention；
Fig. 2 is that the correlation of reagent of the present invention and contrast agents box compares；
Fig. 3 is the HOOK examination of reagent of the present invention.
Specific embodiment
The present invention provides preparation, application method and the purposes of a kind of kit for prostatitis detection, this fields
Technical staff can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replacements
Apparent to those skilled in the art with changing, they are considered as being included in the present invention.Method of the invention
And application is described by preferred embodiment, related personnel can obviously not depart from the content of present invention, spirit and model
Enclose it is interior methods herein and application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.Wherein,
PSEP antigen, coated antibody, labelled antibody are both from Yi Meinuo company.
Below with reference to embodiment, the present invention is further explained:
The method of preparation and use of the kit of 1 prostatitis of embodiment detection
One, the preparation of prostatitis detection kit
Kit of the present invention is made of 3 labelled antibody mixed liquor, magnetic bead suspension and calibration object components.
(1) conventional equipment and reagent
1, the antibody mixed liquor of tracer-labelling (loaded in 12mL magnetic bead bottle)；
2, magnetic bead suspension (loaded in 4mL magnetic bead bottle)；
3, reagent rack (for fixing two components of antibody mixed liquor, magnetic bead suspension of trace labelling substance markers)；
4, calibration object (in the HDPE bottle loaded on 4mL)；
5, calibration card；
(2) selection of labelled antibody buffer and the preparation of mixed liquor
1. the selection of buffer
The buffer (concentration 0.05mol/L) of 6 kinds of buffer systems of Fresh is used, 0.1% (w/v) tracer is added
PSEP antibody, 1 ‰ (w/v) Proclin 300,1 ‰ (w/v) caseins of label substance markers, 0.5 ‰ Tritonx-100,
Labelled antibody mixed liquor is made in 1% bovine serum albumin(BSA), and testing calibration product, calibration object is by PSEP antigen, 0.05mol/L
Tris-NaCl, 1 ‰ (w/v) Proclin 300 and 1 ‰ (w/v) caseins, 1% (w/v) bovine serum albumin(BSA) prepare, calibration
In product S0~S5, the concentration of PSEP is followed successively by 6 parts of 0ng/ml, 0.5ng/ml, 2ng/ml, 6ng/ml, 10ng/ml, 20ng/ml
Calibration object.
The magnetic bead suspension of cooperation are as follows: 0.05mol/L Tris-NaCl, addition 1 ‰ (w/v) Proclin 300,1% (w/
V) bovine serum albumin(BSA), 5% (w/v) sucrose, 0.5 ‰ surfactant Tritonx-100, manufactured envelope protect liquid, and closing is eventually
Magnetic bead after only reacting, manufactured magnetic bead suspension.
As a result as shown in the table:
Table 1 marks product fluorescent value
| Sample ID | Tris-NaCl | Bis-tris | PBS | MOPS | MES | Taps |
| S0 | 12808 | 11291 | 11633 | 86998 | 114202 | 72491 |
| S1 | 488704 | 492743 | 537450 | 653355 | 695755 | 543385 |
| S2 | 2346139 | 2234113 | 2295036 | 2572590 | 2392798 | 2384159 |
| S3 | 14155163 | 13523974 | 14588002 | 14328851 | 13303229 | 12990161 |
| S4 | 44931980 | 46075490 | 49956623 | 45992594 | 43711339 | 46871873 |
| S5 | 186818090 | 176703735 | 198624820 | 178514218 | 171994628 | 168500337 |
| S1/S0 | 38 | 44 | 46 | 8 | 6 | 7 |
Note: S1/S0 value indicates that buffer is Tris-NaCl, Bis- to the signal-to-noise ratio detected as can be seen from Table 1 in table
The labelled antibody mixed liquor background of tris and PBS is all relatively good, and the dilution background of buffer MOPS, MES, Taps are poor.
2, method is same as above, and the dilution of Tris-NaCl, Bis-tris and PBS buffer system is respectively adopted, each reagent is 37
It is placed under the conditions of DEG C 10 days (heat 10 days), the reagent placed 10 days with 4 DEG C, which is used as, to be compareed.With S0~S1 and five part from clinic
Sample (P1~P5) examination reagent stability, the result of appraisal are as follows:
2 sample fluorescence value of table
Note: S0 is the fluorescent value for the mark product that concentration is 0 in table；
S1 is the fluorescent value for the mark product that concentration is 0.5ng/mL；
P1~5 are the fluorescent value detected to clinical sample；
S1/S0 value indicates the signal-to-noise ratio of detection；
The average value of AV expression ratio.
The dilution of Bis-tris buffering is unsatisfactory for requiring in shelf-stability as can be seen from the above table, and PBS is buffered dilute
Background increases after releasing liquid thermal acceleration, therefore using the labelled antibody mixed liquor of Tris-NaCl buffer.
Preferably, labelled antibody mixed liquor the preparation method comprises the following steps: 1, prepare: the Tris-NaCl buffer of 0.05M, addition
1 ‰ (w/v) Proclin 300,1 ‰ (w/v) caseins, 0.5 ‰ Tritonx-100,1% bovine serum albumin(BSA) are made
Solution.
By the PSEP antibody of horseradish peroxidase-labeled, the solution as above to prepare is obtained according to 0.1% dilution proportion
It is spare to labelled antibody mixed liquor.
(3) preparation of magnetic particle suspension
1, envelope protects the selection and preparation of liquid
The buffer (concentration 0.05mol/L) of 6 kinds of buffer systems of Fresh is used, 1 ‰ (w/v) are added
Proclin 300,1% (w/v) bovine serum albumin(BSA), 5% (w/v) sucrose, 0.5 ‰ surfactant Tritonx-100, system
Liquid is protected at envelope, for closing the magnetic bead after terminating reaction.
Calibration object is by PSEP antigen, 0.05mol/L Tris-NaCl, 1 ‰ (w/v) Proclin 300 and 1 ‰ (w/v) junket
Albumen, 1% (w/v) bovine serum albumin(BSA) prepare, in calibration object S0~S5, the concentration of PSEP is followed successively by 0ng/ml, 0.5ng/
6 parts of calibration objects of ml, 2ng/ml, 6ng/ml, 10ng/ml, 20ng/ml.
Closing three times the preparation method comprises the following steps: will terminate the magnetic bead after reacting and protect fluid-tight with envelope for magnetic bead suspension, is finally protected with sealing
Liquid mixes magnetic bead, and 50 tests/ml magnetic bead suspension is made.
The antibody label mixed liquor of use by the PSEP antibody of 0.1% (w/v) horseradish peroxidase-labeled, 0.05M's
Tris-NaCl buffer, 1 ‰ (w/v) Proclin 300,1 ‰ (w/v) caseins, 0.5 ‰ Tritonx-100,1% (w/
V) bovine serum albumin(BSA) composition.
Testing result is as shown in the table:
The fluorescent value of 3 calibration object of table
| Tris-NaCl | Bis-tris | PBS | MOPS | MES | Taps | |
| S0 | 12167 | 12902 | 10152 | 99679 | 118515 | 59123 |
| S1 | 490158 | 493967 | 551157 | 609860 | 612282 | 473438 |
| S2 | 2375044 | 2302176 | 2565312 | 2572069 | 2577765 | 2452702 |
| S3 | 13851344 | 13564273 | 13967674 | 14446863 | 14256699 | 13752346 |
| S4 | 43926219 | 43083381 | 47559871 | 45173849 | 41668756 | 42553131 |
| S5 | 183930739 | 187781724 | 198705825 | 186131175 | 172124767 | 155762097 |
| S1/S0 | 40 | 38 | 54 | 6 | 5 | 8 |
Buffer is the magnetic bead suspension that Tris-NaCl, Bis-tris and PBS are buffer as can be seen from Table 3
Background is all relatively good, and the dilution background of buffer MOPS, MES, Taps are poor.
2, method is same as above, and the dilution of Tris-NaCl, Bis-tris and PBS buffer system is respectively adopted in 37 DEG C of conditions
Lower to place 10 days (heat 10 days), the reagent placed 10 days with 4 DEG C, which is used as, to be compareed.With S0~S1 and five part from clinical sample
(P1~P5) examines the stability of reagent, and the result of appraisal are as follows:
4 sample fluorescence value of table
Note: S0 is the fluorescent value for the mark product that concentration is 0 in table；
S1 is the fluorescent value for the mark product that concentration is 0.5ng/mL；
P1~5 are the fluorescent value detected to clinical sample；
S1/S0 value indicates the signal-to-noise ratio of detection；
The average value of AV expression ratio.
The dilution stability of Bis-tris buffering is unsatisfactory for requiring as can be seen from the above table, the dilution heat of PBS buffering
Background rises after acceleration, therefore Tris-NaCl buffer preparation envelope is selected to protect the magnetic particle suspension that liquid obtains.
3, the selection of peridium concentration
Liquid method is protected using envelope as above, respectively according to the peridium concentration of 0.3 μ g/T, 0.4 μ g/T, 0.5 μ g/T, 0.7 μ g/T
Magnetic bead is coated with, and bioactivity is carried out to the magnetic bead being coated with, sample 1~10 is from clinical sample in table.Knot
Fruit is as follows:
5 peridium concentration of table
As can be seen from the table, saturation is reached when peridium concentration is 0.5 μ g/T, therefore selects the peridium concentration of 0.5 μ g/T.
4, the selection of surfactant
The antibody label mixed liquor of use by the PSEP antibody of 0.1% (w/v) horseradish peroxidase-labeled, 0.05M's
Tris-NaCl buffer, 1 ‰ (w/v) Proclin 300,1 ‰ (w/v) caseins, 0.5 ‰ surfactant, 1% (w/v)
Bovine serum albumin(BSA) composition.
The envelope of use protects liquid by the Tris-NaCl buffers of 0.05M, adds the Proclin 300 of 1 ‰ (w/v),
1% (w/v) bovine serum albumin(BSA), 5% (w/v) sucrose, 0.5 ‰ surfactant composition.
The surfactant that antibody marks mixed liquor and envelope to protect in liquid is respectively Tritonx100, Silwet L7600
Surfactant or Tween-20, another kit of the setting without surfactant is as control.In one-time detection, envelope protect liquid with
The surfactant selected in antibody label mixed liquor inhibits.Test sample is two clinical samples of height, and every sample is continuous
Detection 20 times calculates 20 variations, and testing result is as shown in table 6:
6 kit accuracy of table
As can be seen from Table 4, the accuracy of the kit containing surfactant is obviously better than without containing surface-active
The accuracy of agent kit, the surfactant containing TritonX-100 are slightly better than other two kinds of surfactants.
Therefore, screen the magnetic particle suspension of acquisition the preparation method comprises the following steps:
1. taking 0.05MTris-NaCl buffers, the preservative Proclin 300,1% (w/v) of 1 ‰ (w/v) are added
Bovine serum albumin(BSA), 5% (w/v) sucrose, 0.5 ‰ surfactant Tritonx-100 are made envelope and protect liquid.
2. taking the JSR hydrophobicity magnetic bead stoste (partial size is 100~200nm) of 60 μ l, is washed 5 times, washed with MES buffer
In the process plus TritonX-100；
3. 1- ethyl-(3- dimethylaminopropyl) the phosphinylidyne diimmonium salt hydrochlorate with 20mg/ml is small to magnetic particle activation 1
When；
4. being washed the magnetic particle after activation 2 times with the MES buffer of pH4~5, TritonX-100 is added in washing process；
5. PSEP antibody is added in the magnetic particle after washing by the amount according to 0.5 μ g/T, 0.5 ‰ are added
TritonX-100, room temperature concussion 3h are coated with；
6. 5% ethanol amine is added, half an hour is terminated；
7. protecting fluid-tight with envelope to close 3 times, dispensed according to the amount of 50T/mL stand-by；
(4) preparation of calibration object
Taking concentration is one of buffer Tris-NaCl, Bis-tris, PBS, MOPS, MES or Taps of 0.05M, is added
Add the Proclin300 of 1 ‰ (w/v), 1 ‰ (w/v) caseins, 1% (w/v) bovine serum albumin(BSA).
PSEP antigen is added to according to a certain percentage in calibration object dilution, be made concentration be respectively 0ng/ml,
The calibration object of 0.5ng/ml, 2ng/ml, 6ng/ml, 10ng/ml, 20ng/ml, packing are stand-by.
During aforementioned authentication, calibration object is by PSEP antigen, 0.05mol/L Tris-NaCl, 1 ‰ (w/v) Proclin
300 and 1 ‰ (w/v) caseins, 1% (w/v) bovine serum albumin(BSA) prepare, in calibration object S0~S5, the concentration of PSEP is successively
For 6 parts of calibration objects of 0ng/ml, 0.5ng/ml, 2ng/ml, 6ng/ml, 10ng/ml, 20ng/ml.
Two, the application method of the detection prostatitis kit of step 1 preparation
(1) reagent packaging carries
Reagent packet before upper machine, lightly reverses reagent packet repeatedly to mix the ingredient in novel agent packet, avoids generating for the first time
Bubble.The reagent packet having already turned on is not reversed.
(2) it calibrates
1, instrument obtains the required parameter of test by scanning reagent packet bar code automatically.
2, calibration object is transferred to sample container, is placed in the mating sample rack of instrument, is inputted in instrument software interface
Targeted message selection " RUN " starts to test, and generates calibration curve.
(3) it tests
After setup test sample correct placement, clicks start button and carry out pattern detection program, instrument will execute detection behaviour
Make (each sample application amount is 50ul, and the antibody mixed liquor sample-adding amount of tracer-labelling is 100ul, is incubated 30 minutes).
(4) result calculates
This kit uses four parameter fitting modes, using calibration object concentration value as x-axis, with the log value of calibration object luminous value
Calibration curve is established for y-axis, corresponding concentration value is back-calculated according to the luminous value of sample to be tested.Instrument automatic operation system can pass through
The luminous value that the calibration curve and test sample of storage obtain calculates sample test result automatically.
(5) reference interval
This kit reference interval is≤1.5ng/mL (500 normal population samples of detection, using method of percentiles determination
Reference interval, PSEP measured value≤1.5ng/mL of 95% normal population).It is recommended that each laboratory is according to oneself physical condition and connects
Touching crowd establishes nominal reference section.
2 reagent carrier aircraft stability assessment of embodiment (reagent dismantles rear stability)
A set of reagent prepared is taken, is formulated as follows
1), labelled antibody mixed liquor the preparation method comprises the following steps: 1, prepare: the Tris-NaCl buffer of 0.05M, addition 1 ‰
(w/v) 300 Proclin, 1 ‰ (w/v) caseins, 0.5 ‰ Tritonx-100,1% bovine serum albumin(BSA) are made molten
Liquid.By the PSEP antibody of horseradish peroxidase-labeled, the solution as above to prepare is marked according to 0.1% dilution proportion
Remember that antibody mixed liquor is spare.
2), magnetic particle suspension the preparation method comprises the following steps:
1. taking 0.05MTris-NaCl buffers, the preservative Proclin 300,1% (w/v) of 1 ‰ (w/v) are added
Bovine serum albumin(BSA), 5% (w/v) sucrose, 0.5 ‰ surfactant Tritonx-100 are made envelope and protect liquid.
2. taking the JSR hydrophobicity magnetic bead stoste (partial size is 100~200nm) of 60 μ l, is washed 5 times, washed with MES buffer
In the process plus TritonX-100；
3. 1- ethyl-(3- dimethylaminopropyl) the phosphinylidyne diimmonium salt hydrochlorate with 20mg/ml is small to magnetic particle activation 1
When；
4. being washed the magnetic particle after activation 2 times with the MES buffer of pH4~5, TritonX-100 is added in washing process；
5. PSEP antibody is added in the magnetic particle after washing by the amount according to 0.5 μ g/T, 0.5 ‰ are added
TritonX-100, room temperature concussion 3h are coated with；
6. 5% ethanol amine is added, half an hour is terminated；
7. protecting fluid-tight with envelope to close 3 times, dispensed according to the amount of 50T/mL stand-by；
3), calibration object is by PSEP antigen, 0.05mol/L Tris-NaCl, 1 ‰ (w/v) Proclin 300 and 1 ‰ (w/v)
Casein, 1% (w/v) bovine serum albumin(BSA) prepare, in calibration object S0~S5, the concentration of PSEP be followed successively by 0ng/ml,
6 parts of calibration objects of 0.5ng/ml, 2ng/ml, 6ng/ml, 10ng/ml, 20ng/ml.
Mentioned reagent is packaged, reagent bottle chewing-gum plug pipette tips bundle open on instrument, respectively on day 1,8 days, 15
It, detecting within 22 days, 30 days same group of sample respectively, (sample from clinic, cryo-conservation after packing, sample breakdown is negligible not
Meter), the deviation of subsequent testing result and first day testing result is calculated separately, the result is as follows:
7 carrier aircraft stability of table
As shown in Table 6, reagent detects identical sample within one month and the result error of detection in first day all exists
Within 10%, therefore reagent carrier aircraft (Kaifeng) stability can reach at least one moon.
3 kit sensitivity of embodiment examination
The kit that Example 2 is recorded, according to the property of CLSI standard " EP17-A: the evaluation of clinical labororatory's detection limit "
The method that can confirm that (foundation) carries out the foundation of LoB, LoD, LOQ.This project cannot can trace to the source without standard substance to SI unit,
LOQ is not applicable, check function sensitivity (FS).
Method for building up is as follows:
LoB is examined with finally determining formula (such as embodiment 2), prepares 5 parts of clinical samples close to 0 value, each sample
It is repeated 3 times, does in total 4 days, obtain 60 data；LoD prepares the range of clinical sample that 5 parts of concentration ranges are 1~4 times of LOB,
Each sample is repeated 3 times, and is done in total 4 days, and 60 data are obtained；FS: using LoD test in data, 5 concentration samples are daily
It surveys 3 times, surveys 4 days in total, each sample obtains 12 as a result, the mean value of each sample, SD and CV% are calculated, closest to 20%
Concentration is Functional Sensitivity, the results are shown in Table 7:
8 kit sensitivity of table examination
| First | Second batch | Third batch | |
| LOB | 0.08ng/mL | 0.09ng/mL | 0.08ng/mL |
| LOD | 0.15ng/mL | 0.17ng/mL | 0.16ng/mL |
| FS | 0.21ng/mL | 0.23ng/mL | 0.23ng/mL |
Examining LOB, LOD, FS of 3 batches of reagents this kit as the result is shown is respectively: 0.09ng/mL, 0.17ng/mL,
0.23ng/mL.It therefore is to be set to 0.1ng/mL, 0.2ng/mL, 0.3ng/mL respectively by LOB, LOD, FS of this kit.
The examination of the 4 kit range of linearity of embodiment
The kit that Example 2 is recorded, according to the side of CLSI standard " EP6-A: the linear assessment of quantitative detecting method "
Method, to be formulated the range of linearity that the reagent prepared establishes this kit eventually.
Method for building up is as follows:
Take 1 high level sample H (> 20pg/mL), 1 low value sample L (< 0.3pg/mL), respectively according to following ratio into
Row is mixed into 11 concentration samples (10H, 9H+1L, 8H+2L, 7H+3L, 6H+4L, 5H+5L, 4H+6L, 3H+7L, 2H+8L, 1H+
9L, 10L), each concentration samples detect 4 times, calculate mean value.Theoretical value and actual measurement mean value are carried out once using EXCEL software
Equation y=ax+b regression analysis, statistic equation factor slope a and correlation r, it is desirable that a=1 ± 0.1, r > 0.99.As a result as attached
Shown in figure Fig. 1, it can be seen that the kit range of linearity of the present invention is up to 0.3~20pg/mL.
5 kit clinical evaluation of embodiment
It is leaked albumen using the Prostasomes of kit described in the embodiment of the present invention 2 and the production of Ang Ke biotech firm
(PSEP) detection kit (enzyme-linked immunization) examination 112, gradient sample in parallel, concentration range 0-20pg/mL.Ang Kesheng
Object kit uses method dilution metering in its specification to the sample for being more than 10pg/mL, and testing result carries out clinical correlation
Analysis and coincidence rate analysis.The results are shown in Table 5 for coincidence rate, and correlation is as shown in attached drawing Fig. 2.
Table 9 intersects the coincidence rate that four fold table method calculates examination reagent and contrast agent
As can be seen from Table 5, kit of the present invention and contrast agent box yin-yang coincidence rate and total coincidence rate are preferable.
Kit and Ang Ke biotinylation kit of the present invention has preferable correlation, R2=it can be seen from attached drawing Fig. 2
0.9928, and magnitude deviation is smaller, clinical concordance is preferable.
6 kit HOOK of embodiment examination
A high concentration antigen is taken, is diluted according to a certain percentage；Its theoretical value and detected value are counted, as a result such as table
Shown in 4:
10 dilution ratio of table and detected value and theoretical value
The sample or antigen of kit of the present invention detection high concentration, HOOK does not occur it can be seen from table 10 and attached drawing Fig. 3
Phenomenon
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of reagent combination, which is characterized in that including labelled antibody mixed liquor and magnetic bead suspension；
The labelled antibody mixed liquor includes: the PSEP antibody, 0.01mol/ of 0.1%~0.5% (w/v) trace labelling substance markers
L~0.1mol/L buffer solution A, 1 ‰~3 ‰ (w/v) preservative A, 1 ‰~3 ‰ (w/v) stabilizer A and 0.5 ‰~3 ‰ (w/v)
Surfactant A；
The magnetic bead suspension protects liquid by JSR hydrophobicity magnetic bead, MES buffer, PSEP antibody, surfactant and the envelope activated
It is made；Wherein, it includes: 0.01mol/L~0.1mol/L buffer solution B, 1 ‰~3 ‰ (w/v) preservative B, 1 ‰ that the envelope, which protects liquid,
~3 ‰ (w/v) bovine serum albumin(BSA)s, 5%~15% (w/v) glycerol or sucrose and 0.5 ‰~3 ‰ (w/v) surfactant Bs；
Wherein, the buffer solution A, B are independently selected from Tris-NaCl, Bis-tris, PBS, MOPS, MES, Taps at least one
Kind；
Described preservative A, B are independently selected from Proclin 300, MIT, Bro, IPBC, NaN3At least one of；
The stabilizer A is selected from least one of casein, isinglass, bovine serum albumin(BSA)；
The surfactant A, B are independently selected from tween-20, Tritonx-100, Silwet L7600 Surfactant
It is at least one.
2. reagent combination according to claim 1, which is characterized in that the labelled antibody mixed liquor is shown by 0.1% (w/v)
PSEP antibody, 0.05mol/L Tris-NaCl, 1 ‰ (w/v) Proclin 300,1 ‰ (w/v) junket eggs of track label substance markers
White, 1% (w/v) bovine serum albumin(BSA) and 5 ‰ (w/v) Tritonx-100 composition.
3. reagent combination according to claim 1 or 2, which is characterized in that the trace labelling object is selected from acridinium ester, horseradish
Peroxidase, luminol or derivatives thereof, different luminol or derivatives thereof.
4. reagent combination according to claim 1, which is characterized in that in the magnetic bead suspension, the JSR hydrophobicity of activation
The preparation of magnetic bead include: by partial size be 100~200nm JSR hydrophobicity magnetic bead with the MES buffer containing TritonX-100
It is small to magnetic particle activation 1 with 1- ethyl-(3- dimethylaminopropyl) the phosphinylidyne diimmonium salt hydrochlorate of 20mg/ml after washing 5 times
When, the JSR hydrophobicity magnetic bead of activation is made；Add surfactant in the washing process.
5. reagent according to claim 1 or 4 combination, which is characterized in that the preparation method of the magnetic bead suspension includes:
With the JSR hydrophobicity magnetic bead of the MES buffer washing activation containing surfactant, then mixed with PSEP antibody, on surface
Under activating agent existence condition, room temperature shakes 3h, after terminating reaction with terminate liquid, then protects fluid-tight with envelope and closes three times, it is mixed that magnetic bead is made
Suspension.
6. according to claim 1 or 4~6 described in any item reagents combine, which is characterized in that the envelope protects liquid by 0.05mol/
LTris-NaCl, 1 ‰ (w/v) Proclin 300,1% (w/v) bovine serum albumin(BSA)s, 5% (w/v) sucrose and 0.5 ‰ (w/v)
Tritonx-100 composition.
7. reagent combination according to claim 1, which is characterized in that wherein further include calibration object, the calibration object includes:
PSEP antigen, 0.01mol/L~0.1mol/L buffer C, 1 ‰~3 ‰ (w/v) preservative C, 1% (w/v) bovine serum albumin(BSA)
With 1 ‰~3 ‰ (w/v) caseins；
Wherein, the buffer C is selected from least one of Tris-NaCl, Bis-tris, PBS, MOPS, MES, Taps；
The preservative C is selected from Proclin 300, MIT, Bro, IPBC, NaN3At least one of.
8. application of the described in any item reagent combinations of claim 1~7 in preparation prostatitis detection kit.
9. application according to claim 8, which is characterized in that the prostatitis is detected as monitoring chronic prostatitis
Generation and/or disease progression.
10. a kind of prostatitis detection kit comprising the described in any item reagent combinations of claim 1~7.
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