CN106855574A - A kind of III procollagen type N-terminal peptide chemiluminescence immunity detection reagent and preparation method thereof - Google Patents
A kind of III procollagen type N-terminal peptide chemiluminescence immunity detection reagent and preparation method thereof Download PDFInfo
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- CN106855574A CN106855574A CN201611018771.9A CN201611018771A CN106855574A CN 106855574 A CN106855574 A CN 106855574A CN 201611018771 A CN201611018771 A CN 201611018771A CN 106855574 A CN106855574 A CN 106855574A
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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
- G01N33/577—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
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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
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
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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
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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Abstract
The invention discloses a kind of III procollagen type N-terminal peptide chemiluminescence immunity detection reagent, the kit includes：The III coated magnetic particle of procollagen type N-terminal peptide monoclonal antibody, the III coated acridinium ester of procollagen type N-terminal peptide monoclonal antibody, III procollagen type N-terminal peptide calibration product, preexciting liquid, exciting liquid.In addition the invention also discloses a kind of preparation method of III procollagen type N-terminal peptide chemiluminescence immunity detection reagent.Kit of the present invention is easy to operate compared with available reagent box, and sensitivity is high, the advantages of detection range is wide.
Description
Technical field
The present invention relates to in-vitro diagnosis field of immunodetection, specifically, the invention provides a kind of inspection of chemiluminescence immunoassay
Survey III procollagen type N-terminal peptide reagent box and preparation method thereof.
Background technology
Cirrhosis is the disease that a class seriously threatens human life and health, and liver fibrosis is various chronic liver diseases to liver
Harden the only stage which must be passed by of development.Therefore, find and treat liver fibrosis play an important roll early to disease preventing and treating deterioration.III type
Precollagen N end peptide is the important blood serum designated object for being clinically used for diagnosing liver fibrosis at present, for early diagnosis liver fibrosis
And prognosis evaluation has important clinical meaning.
The common methods of the current procollagen type N-terminal peptide of clinical detection III have enzyme linked immunosorbent assay, radio immunoassay,
Enzyme-catalyzed chemical luminescence method, but these methods all have some shortcomings part.
First, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but the method there is also following weak points：
(1) 12 × 8 types, 6 × 8 types, 8 × 12 types or the hole Special micro porous plate of complete plate 96 are used as antigen coat apparatus and anti-
Container is answered, 12 batches, 6 batches, 8 batches or whole plate first use can only be divided into when in use, it is impossible to carried out independent, single
The detection of person-portion；
(2) reagent type used by quantitative determining is more, and each detection reagent will be contained with reagent bottle, and often be made
It is required for changing imbibition nozzle during with a kind of reagent being filled into the micropore of microwell plate respectively, not only reagent bottle species is more, filling
The operation of reagent is also extremely cumbersome；
(3) lack the corresponding mark to detection information, can only just will appreciate that or know by checking the mark of kit external packing box
The product batch number and term of validity information of detection reagent are known, and the information known is uncontrolled in detection process, with very big
Randomness；
(4) detection reagent easily causes the cross pollution between various reagents and shadow in open space in detection process
Ring the accuracy of testing result；
(5) use hand-manipulated detection process, the dosage of reagent or sample is not bery accurate, and operating process is extremely cumbersome and multiple more
It is miscellaneous, bust is susceptible to, the degree of accuracy of testing result and precision are poor；
(6) in the quantity configuration of detection project reagent set and using item number × 48/96 person-portion is above, if necessary to examine
10 projects are surveyed, then the configuration of reagent and the use of number must be 10 × 48/96 person-portions, if only a sample needs detection 10
Individual different project, it is also desirable to configure the reagent of 10 × 48/96 person-portions, haves the shortcomings that inadequate economical rationality.
2nd, radio immunoassay
Radioimmunoassay method radioactive pollution is big, load procedure is cumbersome, complex operation, operating time are long, measurement result not
Stabilization, the weak point such as the reagent holding time is short, kit operating automation degree is low, necessary instrument is expensive.
3rd, chemoluminescence method
Chemoluminescence method can be divided into direct chemiluminescence and enzyme-catalyzed chemical luminescence by principle of luminosity.
Enzyme-catalyzed chemical luminescence mainly has horseradish peroxidase（HRP）With two kinds of alkaline phosphatase, but there is certain office
Sex-limited, horseradish peroxidase major defect is：Luminol, also can be by H2O2 in the case of the presence of no horseradish peroxidase
Oxidation itself lights, and background is of a relatively high, influences signal to noise ratio, and kinetics is complicated, and influence factor is more, is as a result not sufficiently stable,
The substrate that sensitivity is high and plateau is long is obtained to be not easy.Alkaline phosphatase major defect is：Substrate reach plateau when
Between long, substrate high cost, cause testing cost high, patient burden weight.
Acridinium ester has detailed advantage, main performance as the direct chemiluminescence of label compared to enzyme-catalyzed chemical luminescence
：Reaction do not need catalyst, as long as alkaline environment can carry out, be swift in response, background luminescence is low, and signal to noise ratio is high, interference because
Element is few, and reagent stability is good, can be simple with two-point calibration, system, exciting liquid low cost, acridinium ester easily and protein bind, and
Photon yield is not reduced after connection.
The content of the invention
Current III procollagen type N-terminal peptide detection technique has the following disadvantages：Testing cost is high, detection sensitivity is low, detection
The range of linearity is narrow, reappearance is low, can not quantify, complex operation etc..
The present invention discloses that a kind of testing cost is low, sensitivity is high, detection is linear precisely in order to overcome the above shortcoming
Scope is wide, reappearance is high, can quantify, III procollagen type N-terminal peptide reagent box simple to operate and preparation method thereof.It is of the invention first
Chemical luminescence immune analysis reagent box is first prepared, is mainly included：The III coated magnetic particle of procollagen type N-terminal peptide monoclonal antibody,
The III procollagen type coated acridinium ester of N-terminal peptide monoclonal antibody and III procollagen type N-terminal peptide calibration product；Then using full-automatic
Chemical illumination immunity analysis instrument detects that drafting standard curve is built in computer software, tests actual sample to calibration product,
Concentration of specimens is calculated according to sample luminous value；Finally III procollagen type N-terminal peptide automatic chemiluminescence immunoassay system is entered
Row performance（Sensitivity, linear, precision, interference）Evaluation.
It is of the invention compared with current technology, with advantages below：
1st, present invention selection acridinium ester is used as marker material, and is applied to chemiluminescence immunoassay system, and the luminescence system is
Direct chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, the reaction does not need the participation of enzyme, more cost-effective；
2nd, the acridinium ester chemiluminescent immunoassay system detection sensitivity that the present invention is selected is high, can reach 0.2 ng/mL, phase
10 times are at least improve than other III procollagen type N-terminal peptide detection method sensitivity；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity that the present invention is selected is wide, can reach 0.2 ng/mL-2000
Ng/mLng/mL, the inspection range of linearity of other III procollagen type N-terminal chemistry of peptides hair detection methods is 20-2000 ng/mL；
4th, the acridinium ester chemiluminescent immunoassay system repeatability that the present invention is selected is high, in batch and difference between batch is within 5%,
This is that other chemiluminescence immunoassay systems are unapproachable；
5th, chemiluminescence immunoassay system of the invention has realized quantifying for sample, soft to testing by built-in standard curve
Part, only needs test sample to directly obtain the concentration value of sample；
6th, chemiluminescence immunoassay system of the invention has realized full-automation, and the addition of reagent and sample has instrument complete entirely
Into operation is easier, reduces artificial error.
Brief description of the drawings
Fig. 1 is the III procollagen type N-terminal peptide canonical plotting that embodiment 3 is obtained.
Specific embodiment
Embodiment 1：III procollagen type N-terminal peptide chemiluminescence immunity detection reagent preparation method
（1）It is prepared by the coated nanometer magnetic bead of III procollagen type N-terminal peptide monoclonal antibody：
Take the magnetic particle of 50mg carboxylated（Particle diameter is 0.05-1um）Suspension, Magneto separate removes supernatant, and it is 5.5 to use 0.02 M, pH
MES buffer solutions are resuspended, add the EDC aqueous solution of 10 mg/mL of the new configurations of 0.5-2mL, activated magnetic beads surface carboxyl groups to add 3-5
The procollagen type N-terminal peptide monoclonal antibodies of mg III, suspension 2-10 h, Magneto separate, remove supernatant, with containing 2% BSA's at room temperature
0.1 M pH are that 8.0 Tris buffer solutions are resuspended to 1mg/mL, obtain the III procollagen type N-terminal coated magnetic of peptide monoclonal antibody
Grain, every bottle of 5mL packing be stored in 4 DEG C it is standby.
（2）The preparation of the acridinium ester of III procollagen type N-terminal peptide antibody mark：
The III procollagen type N-terminal peptide antibody of 50 uL 25mg/mL is taken, the carbon of 150 uL 0.1-0.2 M pH 9.0-9.5 is added
Phthalate buffer, is mixed, and the acridinium ester for being subsequently adding the mg/mL of 1-2 uL 5 is mixed, and lucifuge reaction, takes after 1-2 h at room temperature
Go out, desalting column desalting processing is centrifuged with the zeba of 2 mL, first respectively with pure water and TBS buffer solutions in desalination processes
Reason, is eventually adding the acridine ester solution of the III procollagen type N-terminal peptide antibody mark for obtaining, and the liquid collected in centrifuge tube is extremely preserved
Be in control the acridinium ester of III procollagen type N-terminal peptide antibody mark, every bottle of 5 mL packing be stored in 4 DEG C it is standby.
（3）III procollagen type N-terminal peptide calibrates the preparation of product：
Use standard items buffer solution（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）III procollagen type N-terminal peptide is matched somebody with somebody
Concentration is set to for 0 ng/mL, 100 ng/mL, 500 ng/mL, 1000 ng/mL, 1500 ng/mL, 2000 ng/mL, every bottle
0.5 mL packing is lyophilized, and 4 DEG C save backup.
(4) preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, the hydrogen peroxide (H that 80uL mass fractions are 20% is sequentially added2O2), 1.0 grams of sodium azide, 1.5
Gram polysorbas20, shakes up rear lucifuge storage.
(5) preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.6 gram of NaOH, 0.5 gram of PC300,0.5g sodium azide, 1.5 grams of Triton are sequentially added
405, shake up rear lucifuge storage.
Embodiment 2：III procollagen type N-terminal peptide chemiluminescence immunologic detection method：
The present invention is detection instrument with Full-automatic chemiluminescence immunoassay analysis meter, and method of the present invention pattern is sandwich method, i.e.,
Instrument sequentially adds the III coated magnetic particle of procollagen type N-terminal peptide monoclonal antibody and 50 uL of the sample of 20 uL, 50 uL
The III procollagen type N-terminal coated acridinium ester of peptide monoclonal antibody, after 10 min of reaction, carry out Magneto separate, instrument will react mixed
Compound sends into darkroom, and sequentially adding 50uL chemiluminescence preexcitings liquid, 50uL chemiluminescences exciting liquid carries out luminescence-producing reaction, finally
Record luminous intensity, III procollagen type N-terminal peptide content of sample is calculated from standard curve.
Embodiment 3：III procollagen type N-terminal peptide chemiluminescence immunity detection reagent performance evaluation
Detection curve is shown in accompanying drawing 1.
The detection of sensitivity：
With reference to CLSI EP17-A file recommendation experimental programs, III procollagen type N-terminal peptide chemiluminescence immunochromatography reagent is calculated
The sensitivity of box, the sensitivity tried to achieve is 0.2 ng/ mL.
Linear detection：
It is 0 ng/mL, 100 ng/mL, 500 ng/mL, 1000 ng/mL, 1500 ng/mL, 2000 ng/mL standards to concentration
Product do linear analysis, calculate linearly dependent coefficient, r=0.9996, in addition, the kit is to III procollagen type N-terminal peptide sample detection
The range of linearity be 0.2-2000 ng/mL.
Precision is determined：
Concentration is taken for two III procollagen type N-terminal peptide samples of 50 ng/mL and 100 ng/mL, each sample each concentration respectively does 3
It is individual parallel, detected with three batches of kits, calculate in kit batch and difference between batch, as a result show in the kit batch and batch between
Difference is respectively less than 5%.
Interference is tested：
Taking pooled serum and adding chaff interference respectively includes：Combined with bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid, glycerine
Ester, adding proportion is according to 1:20 are carried out, and are determined pooled serum respectively and be with the addition of the survey of pooled serum after various chaff interferences
Value, calculates deviation therebetween, with ± 10% for tolerance interval.Result shows that interference reaches the file of NCCLS
Standard, can be used for the accurate evaluation of clinical labororatory's vitamin D situation.
Embodiment 4:The Sensitivity comparison experiment of III procollagen type N-terminal peptide chemiluminescence immunity detection reagent
It is respectively the calibration object or sample of 0 ng/mL to concentration with chemical luminescence detection method and traditional enzyme linked immunosorbent assay
Dilution is detected that replication 20 times draws 20 RLU values of measurement result for sample（Relative light unit）, calculate it
Average value（M）And standard deviation（SD）, M+2SD is drawn, luminous value substitution calibration curve is calculated corresponding concentration value.Adopt
The concentration value obtained with chemical luminescence detection method is 0.03 ng/ml, relative to traditional enzyme linked immunosorbent assay lowest detection
2.0 ng/ml are limited, about 60 times are improve.
Claims (10)
1. a kind of III procollagen type N-terminal peptide chemiluminescence immunity detection reagent, the kit includes：III procollagen type N-terminal
The coated nanometer magnetic microsphere of peptide monoclonal antibody, chemiluminescent labels, Chemoluminescent substrate, III procollagen type N-terminal peptide are determined
Mark product.
2. kit according to claim 1, it is characterised in that the III procollagen type N-terminal peptide monoclonal antibody coating
Solid phase carrier be magnetic particle.
3. kit according to claim 1, it is characterised in that the III procollagen type N-terminal peptide monoclonal antibody coating
Solid phase carrier for carboxylated particle diameter be 0.05-1um magnetic particles.
4. kit according to claim 1, it is characterised in that the chemiluminescent labels are acridinium ester, acridinium ester
Sulfonamide, acridinium ester toluenesulfonamide, acridinium ester are to methylsulfonamides, acridinium ester trimethyl fluoride sulfonyl amine.
5. kit according to claim 1, it is characterised in that the preferred acridinium ester of chemiluminescent labels.
6. kit according to claim 1, it is characterised in that the Chemoluminescent substrate is excited including chemiluminescence
Liquid, chemiluminescence preexciting liquid.
7. kit according to claim 1, it is characterised in that the chemiluminescence preexciting liquid is mass fraction
0.005% ~ 0.5% hydrogen peroxide (H2O2) solution, exciting liquid is the NaOH of 0.005mol/L ~ 0.025mol/L
(NaOH) solution.
8. kit according to claim 1, it is characterised in that the III procollagen type N-terminal peptide calibration product are to use standard
Savor buffer solution by III procollagen type N-terminal peptide be configured to concentration for 0 ng/mL, 100 ng/mL, 500 ng/mL, 1000 ng/mL,
1500 ng/mL, 2000 ng/mL, 4 DEG C save backup.
9. kit according to claim 1, it is characterised in that the preparation method of the kit, it is characterised in that bag
Include preparation, the acridinium ester of III procollagen type N-terminal peptide antibody mark of the III procollagen type N-terminal coated magnetic particle of peptide monoclonal antibody
Preparation, the preparation of Chemoluminescent substrate, III procollagen type N-terminal peptide calibrate product preparation.
10. according to claim 1 and claim 9 kit preparation method, it is characterised in that comprise the following steps：
1）The preparation of the III procollagen type N-terminal coated magnetic particle of peptide monoclonal antibody：
The nanometer magnetic bead suspension of carboxylated is taken, Magneto separate removes supernatant, and MES buffer solutions are resuspended, add the EDC aqueous solution, activate magnetic
Bead surface carboxyl, adds III procollagen type N-terminal peptide monoclonal antibody, at room temperature suspension 2-10 h, and Magneto separate removes supernatant,
Tris buffer solutions are resuspended, obtain the III procollagen type N-terminal coated magnetic particle of peptide monoclonal antibody；Optionally, carboxylated nano magnetic
A diameter of 0.1 μm ~ 2.0 μm of pearl；
MES buffer concentrations are 10mM ~ 100mM, pH 5.5 ~ 8.5；
2）The preparation of the acridinium ester of III procollagen type N-terminal peptide antibody mark：
III procollagen type N-terminal peptide antibody is taken, carbonate buffer solution is added, mixed, be subsequently adding acridinium ester mixing, at room temperature lucifuge
Reaction, takes out after 1-2 h, and desalting column desalting processing is centrifuged, and is carried out with pure water and TBS buffer solutions respectively first in desalination processes
Treatment, is eventually adding the acridine ester solution of the III procollagen type N-terminal peptide antibody mark for obtaining, and the liquid collected in centrifuge tube is extremely protected
Deposit the acridinium ester for being in control III procollagen type N-terminal peptide antibody mark；
3）III procollagen type N-terminal peptide calibrates the preparation of product：
With standard items buffer solution by III procollagen type N-terminal peptide be configured to concentration for 0 ng/mL, 100 ng/mL, 500 ng/mL,
1000 ng/mL, 1500 ng/mL, 2000 ng/mL, packing is lyophilized, and 4 DEG C save backup；
4）The preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, the hydrogen peroxide (H that 0.5 ~ 100uL mass fractions are 20% is sequentially added2O2), 0.5 ~ 5 gram prevent
Rotten agent, 0.5 ~ 5 gram of surfactant, shake up rear lucifuge storage；Optionally, preservative be commercialization sodium azide, PC300,
Surfactant is polysorbas20, Tween 80, Triton X100, Triton 405；
5）The preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.2 ~ 1 gram of NaOH, 0.5 ~ 5 gram of preservative, 0.5 ~ 5 gram of surface is sequentially added and is lived
Property agent, shake up the storage of rear lucifuge；Optionally, preservative is commercialization sodium azide, PC300, and surfactant is polysorbas20, tells
Temperature 80, Triton X100, Triton 405.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201620686185 | 2016-06-30 | ||
| CN2016206861850 | 2016-06-30 |
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| CN106855574A true CN106855574A (en) | 2017-06-16 |
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| CN201611018771.9A Pending CN106855574A (en) | 2016-06-30 | 2016-11-21 | A kind of III procollagen type N-terminal peptide chemiluminescence immunity detection reagent and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444430A (en) * | 2018-12-13 | 2019-03-08 | 郑州安图生物工程股份有限公司 | A kind of kit detecting III procollagen type N-terminal peptide |
| CN113358880A (en) * | 2021-06-10 | 2021-09-07 | 吉林基蛋生物科技有限公司 | Detection kit for III type procollagen N-terminal peptide |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101377509A (en) * | 2008-03-14 | 2009-03-04 | 北京科美东雅生物技术有限公司 | III type precollagen N end peptide chemiluminescence immune analysis quantitative determination reagent kit and preparing method thereof |
| CN102998462A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for procollagen III (PC III), and preparation method of kit |
| CN104897901A (en) * | 2015-05-12 | 2015-09-09 | 西安金磁纳米生物技术有限公司 | Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4 |
-
2016
- 2016-11-21 CN CN201611018771.9A patent/CN106855574A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101377509A (en) * | 2008-03-14 | 2009-03-04 | 北京科美东雅生物技术有限公司 | III type precollagen N end peptide chemiluminescence immune analysis quantitative determination reagent kit and preparing method thereof |
| CN102998462A (en) * | 2012-11-20 | 2013-03-27 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for procollagen III (PC III), and preparation method of kit |
| CN104897901A (en) * | 2015-05-12 | 2015-09-09 | 西安金磁纳米生物技术有限公司 | Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444430A (en) * | 2018-12-13 | 2019-03-08 | 郑州安图生物工程股份有限公司 | A kind of kit detecting III procollagen type N-terminal peptide |
| CN113358880A (en) * | 2021-06-10 | 2021-09-07 | 吉林基蛋生物科技有限公司 | Detection kit for III type procollagen N-terminal peptide |
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Application publication date: 20170616 |
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