CN106501506A - A kind of Antiproteinase 3 antibody IgG chemiluminescence immune detection reagent kits and preparation method thereof - Google Patents
A kind of Antiproteinase 3 antibody IgG chemiluminescence immune detection reagent kits and preparation method thereof Download PDFInfo
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- CN106501506A CN106501506A CN201611018730.XA CN201611018730A CN106501506A CN 106501506 A CN106501506 A CN 106501506A CN 201611018730 A CN201611018730 A CN 201611018730A CN 106501506 A CN106501506 A CN 106501506A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/543—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
- G01N33/54326—Magnetic particles
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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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Abstract
The invention discloses a kind of Antiproteinase 3 antibody IgG chemiluminescence immune detection reagent kits, the test kit includes：The magnetic particle of protease 3 antigen coat, the coated acridinium ester of Mus anti-human igg monoclonal antibody, Antiproteinase 3 antibody IgG calibration product, preexciting liquid, exciting liquid.In addition the invention also discloses a kind of preparation method of Antiproteinase 3 antibody IgG chemiluminescence immune detection reagent kits.Test 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 Antiproteinase 3 antibody IgG test kits and preparation method thereof.
Background technology
Protease 3（Proteinase 3, PR3）It is a kind of serine stretch protein in neutrophilic granulocyte endochylema azurophilic granule
Enzyme, molecular weight are about the glycoprotein of 29KDa.PR3 can degrade various kinds of cell epimatrix such as elastin laminin, hemoglobin, IV type glue
The Various Tissues composition such as original.Protease 3 promotes platelet activation by cathepsin G, and inactivates C1 inhibitor.Anti- neutrality
Granulocyte endochylema antibody（Antineut rophiIcytoplasmic antibody, ANCA）It is wegener granulomatosises
（Wegener ' s granulomatosis, WG）The blood serum designated object of patient, especially Antiproteinase 3 antibody.
WG is a kind of granuloma gangraenescenss vasculitises of specific type.ANCA is that WG is most had
The autoantibody of diagnostic significance.The diagnosis of past WG is mainly according to typical clinical manifestation and medical history, but some patients, especially
It is that localised patient is difficult to diagnose, disease inspection is the important means for making a definite diagnosis WG.But once or single position disease inspection feminine gender can not be arranged
Remove WG.Current research think the ANCA specific targeted antigens of most of WG patient be the diagnosis of PR3, PR3 antibody and WG and
Disease activity is closely related.Endochylema type ANCA and PR3 antibody have the Sensitivity and Specificity of height to WG diagnostic significance.External
It is reported in the patient that biopsy turns out to be WG, PR3 antibody specificity is more than 90%.Sensitivity is that have with the activeness of disease
Close.Generally PR3 antibody titre is consistent with disease activity, and activeness WG patient resists when pathological changes not yet have influence on respiratory system
PR3 antibody sensitivities are 65%, and when respiratory system, kidney damage occurs in patient, its sensitivity is up to more than 90%.Active stage titre
Higher, part catabasises titre is very low, and the most patients of complete incidence graph do not detect.The catabasises rising of PR3 antibody titre can
Recurrence can be imply that, and contributes to distinguishing infection and recurrence.
The main method of Clinical detection Antiproteinase 3 antibody IgG be enzyme linked immunosorbent assay, but the method exist following
Weak point：
（1）12 × 8 types, 6 × 8 types, 8 × 12 types or 96 hole Special micro porous plate of complete plate are used as antigen coat apparatus
And reaction vessel, using when can only be divided into 12 batches, 6 batches, 8 batches or imposite first use, it is impossible to carry out independence
, the detection of single part；
（2）Quantitative determination reagent type used is more, and each detectable will be contained with reagent bottle, and is often made
It is required for changing imbibition nozzle to be filled in the micropore of microwell plate respectively during with a kind of reagent, not only reagent bottle species is more, filling
The operation of reagent is also extremely loaded down with trivial details；
（3）Lack the corresponding mark to detection information, can only just will appreciate that by checking the mark of test kit external packing box or know
Product batch number and the effect duration information of detectable is known, and the information that is known is uncontrolled in detection process, with very big
Randomness；
（4）Detectable in open space, easily causes the cross-contamination between various reagents and shadow in detection process
Ring the accuracy of testing result；
（5）Dosage more than detection process using manual operations, reagent or sample is not bery accurate, and operating process is extremely loaded down with trivial details and multiple
Miscellaneous, bust is susceptible to, the accuracy and precision of testing result is poor；
（6）Item number × 48/96 person-portion is in the quantity configuration of detection project reagent set and using on, 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 to detect 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.
Content of the invention
Antiproteinase 3 antibody IgG detection techniques are suffered from the drawback that at present：Testing cost is high, detection sensitivity is low, detection
The range of linearity is narrow, repeatability is low, can not quantitatively, 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
Antiproteinase 3 antibody IgG test kits that scope is wide, repeatability is high, can be quantitative, simple to operate and preparation method thereof.The present invention
Prepare chemical luminescence immune analysis reagent box first, mainly include：The magnetic granule of protease 3 antigen coat, Mus anti-human igg Dan Ke
The coated acridinium ester of grand antibody and Antiproteinase 3 antibody IgG calibration product；Then using Full-automatic chemiluminescence immunoassay analysis meter
Calibration product are detected, standard curve is drawn, computer software is built in, actual sample is tested, is calculated according to sample luminous value
Concentration of specimens；Performance is carried out to Anti-proteinase 3 IgG antibody automatic chemiluminescence immunoassay system finally（Sensitivity, line
Property, precision, interference）Evaluation.
Of the invention compared with current technology, with advantages below：
1st, the present invention selects acridinium ester as marker material, and is applied to chemiluminescence immunoassay system, and the luminescence system is
Directly 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 1AU/mL, compare
The sensitivity of other Antiproteinase 3 antibody IgG detection methods at least improves 10 times；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity width that the present invention is selected, can reach 3-400AU/mL, other
Antiproteinase 3 antibody IgG chemistry send out detection method the inspection range of linearity be 20-135AU/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 the quantitative of sample, soft to testing by built-in standard curve
Part, only needs test sample directly obtain the concentration value of sample；
6th, chemiluminescence immunoassay system of the invention has realized that the interpolation of full-automation, reagent and sample has instrument complete entirely
Into operation is easier, reduces artificial error.
Description of the drawings
Fig. 1 is the Antiproteinase 3 antibody IgG canonical plottings that embodiment 3 is obtained.
Specific embodiment
Embodiment 1：Antiproteinase 3 antibody IgG chemiluminescence immune detection reagent kit preparation methoies
（1）Prepared by the nanometer magnetic bead of protease 3 antigen coat：
Take the carboxylated magnetic particles of 50mg（Particle diameter is 0.05-1um）Suspension, Magneto separate remove supernatant, and it is 5.5 to use 0.02 M, pH
MES buffer is resuspended, the EDC aqueous solutions of 10 mg/mL for adding 0.5-2mL newly to configure, and activated magnetic beads surface carboxyl groups add 3-5
Mg protease 3 antigens, suspension 2-10 h under room temperature, Magneto separate removes supernatant, is 8.0 with the 0.1 M pH containing 2% BSA
Tris buffer is resuspended to 1mg/mL, obtains the magnetic granule of protease 3 antigen coat, every bottle of 5mL subpackage be stored in 4 DEG C standby.
（2）The preparation of the acridinium ester of Mus anti-human igg labeling of monoclonal antibody：
The Mus anti-human igg monoclonal 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, mixes, and the acridinium ester for being subsequently adding 5 mg/mL of 1-2 uL is mixed, and under room temperature, lucifuge reaction, takes after 1-2 h
Go out, with the zeba of 2 mL centrifugation desalting column desalting processing, first respectively with pure water and TBS buffer in desalination processes
Reason, is eventually adding the acridine ester solution of the Mus anti-human igg labeling of monoclonal antibody for obtaining, and collects the liquid in centrifuge tube to preservation
Be in control the acridinium ester of Mus anti-human igg labeling of monoclonal antibody, per bottle of 5 mL subpackage be stored in 4 DEG C standby.
（3）Antiproteinase 3 antibody IgG calibrates the preparation of product：
Use standard substance buffer（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）By Antiproteinase 3 antibody IgG
Concentration is configured to for 0 AU/mL, 5 AU/mL, 20 AU/mL, 50AU/mL, 90AU/mL, 165 AU/mL, per bottle of 0.5 mL subpackage
Lyophilizing, 4 DEG C save backup.
(4) preparation of chemiluminescence preexciting liquid：
1.0 liters of purified water are measured, the hydrogen peroxide (H that 80uL mass fractions are 20% is sequentially added2O2), 1.0 grams of Hydrazoic acid,sodium salt, 1.5
Gram polysorbas20, shakes up rear lucifuge storage.
(5) preparation of chemiluminescence exciting liquid：
1.0 liters of purified water are measured, 0.6 gram of sodium hydroxide, 0.5 gram of PC300,0.5g Hydrazoic acid,sodium salt, 1.5 grams of Triton are sequentially added
405, shake up rear lucifuge storage.
Embodiment 2：Antiproteinase 3 antibody IgG chemical luminous immune detection methods：
With Full-automatic chemiluminescence immunoassay analysis meter as detecting instrument, method of the present invention pattern is indirect method to the present invention, i.e.,
Instrument sequentially adds 10 min of magnetic particle reaction of the sample of 5uL, 95uL Sample dilutions, the protease 3 antigen coat of 50 uL
Afterwards, Magneto separate is carried out.The coated acridinium ester of Mus anti-human igg monoclonal antibody of 100uL is added, after 10 min of reaction, magnetic is carried out
Separate, reactant mixture is sent into darkroom, sequentially adds 100uL chemiluminescence preexciting liquid, 200uL chemiluminescences and excite by instrument
Liquid carries out luminescence-producing reaction, finally records luminous intensity, and the Antiproteinase 3 antibody IgG for calculating sample from standard curve contains
Amount.
Embodiment 3：Antiproteinase 3 antibody IgG chemiluminescence immune detection reagent kit performance evaluations
Detection curve is shown in accompanying drawing 1.
The detection of sensitivity：
With reference to CLSI EP17-A file recommendation experimental programs, Antiproteinase 3 antibody IgG chemiluminescence immunoassays chromatography reagent is calculated
The sensitivity of box, the sensitivity that tries to achieve are 1AU/ mL.
Linear detection：
Linear point is done for 5 AU/mL, 20 AU/mL, 50 AU/mL, 90 AU/mL, 165 AU/mL standard substance to concentration
Analysis, calculates linearly dependent coefficient, and r=0.9996, in addition, linear model of the test kit to Anti-proteinase 3 IgG antibody sample detection
Enclose for 3-300AU/mL.
Precision is determined：
Concentration is taken for two Antiproteinase 3 antibody IgG samples of 2 AU/mL and 100 AU mL, each sample each concentration respectively does 3
Individual parallel, detected with three batches of test kits, calculated in test kit batch and difference between batch, as a result show in the test kit batch and batch between
Difference is respectively less than 5%.
Interference is tested：
Taking pooled serum and adding chaff interference respectively includes：Conjugated bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid, sweet
Grease, 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% as tolerance interval.As a result show, interference reaches the file of NCCLS
Standard, can be used for the accurate evaluation of clinical laboratory's vitamin D situation.
Embodiment 4:The Sensitivity comparison experiment of Antiproteinase 3 antibody IgG chemiluminescence immune detection reagent kits
It is the calibration object or sample of 0 AU/mL respectively to concentration with chemical luminescence detection method and traditional enzyme linked immunosorbent assay
This diluent is detected that for sample replication 20 times draws the RLU values of 20 measurement results（Relative light unit）, calculate
Its meansigma methods（M）And standard deviation（SD）, M-2SD is drawn, the luminous value is substituted into calibration curve and is calculated corresponding concentration value.
The concentration value for adopting chemical luminescence detection method to obtain is 1.0 AU/ml, relative to the minimum inspection of traditional enzyme linked immunosorbent assay
Limit 8.2 AU/ml is surveyed, about 8 times are improve.
Claims (10)
1. a kind of Antiproteinase 3 antibody IgG chemiluminescence immune detection reagent kits, the test kit include：Protease 3 antigen bag
Quilt-nanometer magnetic microsphere, chemiluminescent labels, Chemoluminescent substrate, Antiproteinase 3 antibody IgG calibration product.
2. test kit according to claim 1, it is characterised in that the solid phase carrier of the protease 3 antigen coat is magnetic
Microgranule.
3. test kit according to claim 1, it is characterised in that the solid phase carrier of the protease 3 antigen coat is carboxylic
The particle diameter of base is 0.05-1um magnetic particles.
4. test 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. test kit according to claim 1, it is characterised in that the preferred acridinium ester of the chemiluminescent labels.
6. test kit according to claim 1, it is characterised in that the Chemoluminescent substrate includes that chemiluminescence is excited
Liquid, chemiluminescence preexciting liquid.
7. test 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, sodium hydroxide of the exciting liquid for 0.005mol/L ~ 0.025mol/L
(NaOH) solution.
8. test kit according to claim 1, it is characterised in that the Antiproteinase 3 antibody IgG calibrations product are to use standard
Savor buffer by Antiproteinase 3 antibody IgG be configured to concentration for 0 AU/mL, 5 AU/mL, 20 AU/mL, 50 AU/mL, 90
AU/mL, 165 AU/mL, 4 DEG C save backup.
9. test kit according to claim 1, it is characterised in that the preparation method of the test kit, it is characterised in that bag
Include the preparation of the magnetic particle of protease 3 antigen coat, the preparation of the acridinium ester of Mus anti-human igg labeling of monoclonal antibody, chemistry to send out
The preparation of product is calibrated in the preparation of light substrate solution, Antiproteinase 3 antibody IgG.
10. according to claim 1 and described in claim 9 test kit preparation method, it is characterised in that comprise the following steps：
1）The preparation of the magnetic particle of protease 3 antigen coat：
Carboxylated nanometer magnetic bead suspension is taken, Magneto separate removes supernatant, and MES buffer is resuspended, add EDC aqueous solutions, activate magnetic
Bead surface carboxyl, adds protease 3 antigen, suspension 2-10 h under room temperature, Magneto separate to remove supernatant, and Tris buffer is resuspended,
Obtain the magnetic particle of protease 3 antigen coat；Optionally, carboxylated nanometer magnetic bead is a diameter of 0.1 μm ~ 2.0 μm；
MES buffer concentrations are 10mM ~ 100mM, pH 5.5 ~ 8.5；
2）The preparation of the acridinium ester of Mus anti-human igg labeling of monoclonal antibody：
Mus anti-human igg monoclonal antibody is taken, carbonate buffer solution is added, is mixed, be subsequently adding acridinium ester mixing, lucifuge under room temperature
Reaction, takes out after 1-2 h, is centrifuged desalting column desalting processing, is carried out with pure water and TBS buffer respectively in desalination processes first
Process, be eventually adding the acridine ester solution of the Mus anti-human igg labeling of monoclonal antibody for obtaining, the liquid in centrifuge tube is collected to guarantor
Deposit the acridinium ester for being in control Mus anti-human igg labeling of monoclonal antibody；
3）Antiproteinase 3 antibody IgG calibrates the preparation of product：
With standard substance buffer by Antiproteinase 3 antibody IgG be configured to concentration for 0 AU/mL, 5 AU/mL, 20 AU/mL, 50
AU/mL, 90 AU/mL, 165 AU/mL, subpackage lyophilizing, 4 DEG C save backup；
4）The preparation of chemiluminescence preexciting liquid：
1.0 liters of purified water 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 Hydrazoic acid,sodium salt, PC300,
Surfactant is polysorbas20, Tween 80, Triton X100, Triton 405；
5）The preparation of chemiluminescence exciting liquid：
1.0 liters of purified water are measured, 0.2 ~ 1 gram of sodium hydroxide, 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 Hydrazoic acid,sodium salt, PC300, and surfactant is polysorbas20, Tween 80, Triton
X100、Triton 405.
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| CN201620676545 | 2016-06-30 | ||
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108896538A (en) * | 2018-06-21 | 2018-11-27 | 上海彧成生物科技有限公司 | A kind of creatinine chemiluminescence immune detection reagent kit and each component preparation method |
| WO2020034939A1 (en) * | 2018-08-13 | 2020-02-20 | 博阳生物科技（上海）有限公司 | Chemiluminescence analysis method and application thereof |
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| CN102707055A (en) * | 2012-06-11 | 2012-10-03 | 郑州安图绿科生物工程有限公司 | Kit for joint or single detection of autoimmune liver disease related antibody and detection method of kit |
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