CN106771131A - A kind of AMA M2 type chemiluminescence immune detection reagent kits and preparation method thereof - Google Patents
A kind of AMA M2 type chemiluminescence immune detection reagent kits and preparation method thereof Download PDFInfo
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- CN106771131A CN106771131A CN201611018761.5A CN201611018761A CN106771131A CN 106771131 A CN106771131 A CN 106771131A CN 201611018761 A CN201611018761 A CN 201611018761A CN 106771131 A CN106771131 A CN 106771131A
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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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
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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
- G01N33/577—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 AMA M2 type chemiluminescence immune detection reagent kits, the kit includes：The magnetic particle of M2 antigen coats, the coated acridinium ester of mouse anti-human igg monoclonal antibody, AMA M2 types calibration product, preexciting liquid, exciting liquid.In addition the invention also discloses a kind of preparation method of AMA M2 type chemiluminescence immune detection reagent kits.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 AMA M2 type kits and preparation method thereof.
Background technology
Mitochondrial antibody（AMA）1958 are equal to first in PBC by Maokey（primary
Biliary cirrhosis, PBC）Patients serum find, be it is a kind of without organ specificity also without species specificity itself resist
Body.
PBC is a kind of autoimmune disease, common in women, is due to medium and small bile duct progressive destruction and companion in liver
There is Portal inflammation to sexually revise, be further development of fibrosis, cirrhosis, clinical manifestation is progressive dysfunction of liver.Early stage table
It is now pruitus, drowsiness, dull pain in liver, jaundice, liver ascites, cirrhosis occurs in late period.Be can detect in the serum of PBC patient
Resist mitochondria（AMA）Various autoantibodies such as antibody M2 types, antinuclear antibodies, anti-nuclear Pore Complex antibody, wherein with resist mitochondria
Antibody M2 types（AMA-M2）Being characterized property crucial immunological abnormality.
The target position of AMA-M2 is side chain 2- oxygen acidohydrogenase compound family members, including PDHC
E2 subunits, side chain 2- oxygen acidohydrogenases compound, ketoglutaric dehydrogenase compound and dihydrolipoamide dehydrogenase are combined
Albumen.AMA-M2 antibody tests are the effective means for diagnosing PBC.
The common methods of clinical detection AMA M2 types have Western blot, enzyme linked immunosorbent assay, enzymatic
Luminescence method is learned, but these methods all have some shortcomings part.
First, Western blot
Western blotting is, by Protein transfer to film, then to be detected using antibody.To known expressing protein, can be with accordingly
Antibody detected as primary antibody, to the expression product of new gene, can be by merging the antibody test of part.Its weak point exists
In：
(1) quantitative and semi-quantitative analysis can only be carried out, it is impossible to show that analyte is specifically measured；
(2) complex operation step, the experiment used time is more long；
(3) sensitivity of detection need to be improved.
2nd, 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.
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 AMA M2 type detection techniques have 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, AMA M2 type kits simple to operate and preparation method thereof.The present invention
Chemical luminescence immune analysis reagent box is prepared first, is mainly included：The magnetic particle of M2 antigen coats, mouse anti-human igg monoclonal resist
The coated acridinium ester of body and AMA M2 types calibration product；Then using Full-automatic chemiluminescence immunoassay analysis meter to fixed
Mark product are detected that drafting standard curve is built in computer software, tests actual sample, and sample is calculated according to sample luminous value
Concentration；Finally confrontation mitochondrial antibody M2 type automatic chemiluminescence immunoassay systems carry out performance（Sensitivity, linear, essence
Density, 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 1AU/mL, compares
Other AMA M2 type detection methods sensitivity at least improve 10 times；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity that the present invention is selected is wide, can reach 3-400 AU/mL, other
AMA M2 types chemistry hair detection method the inspection range of linearity be 20-135 AU/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 AMA M2 type canonical plottings that embodiment 3 is obtained.
Specific embodiment
Embodiment 1：AMA M2 type chemiluminescence immune detection reagent kit preparation methods
（1）It is prepared by the coated nanometer magnetic bead of AMA M2 type monoclonal antibodies：
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
Mg M2 antigens, suspension 2-10 h, Magneto separate, remove supernatant at room temperature, slow with the Tris that the 0.1 M pH containing 2% BSA are 8.0
Fliud flushing is resuspended to 1mg/mL, obtains the magnetic particle of M2 antigen coats, every bottle of 5mL packing be stored in 4 DEG C it is standby.
（2）AMA M2 types derive the preparation of the acridinium ester of substance markers：
The mouse 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, 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 mouse anti-human igg labeling of monoclonal antibody for obtaining, and the liquid collected in centrifuge tube is extremely preserved
Be in control the acridinium ester of mouse anti-human igg labeling of monoclonal antibody, every bottle of 5 mL packing be stored in 4 DEG C it is standby.
（3）AMA M2 types calibrate the preparation of product：
Use standard items buffer solution（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）By AMA M2 types
Concentration is configured to for 0AU/mL, 5AU/mL, 20 AU/mL, 50 AU/mL, 90 AU/mL, 165 AU/mL, every bottle of 0.5 mL packing
Lyophilized, 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：AMA M2 type chemical luminous immune detection methods：
The present invention is detection instrument with Full-automatic chemiluminescence immunoassay analysis meter, and method of the present invention pattern is indirect method, i.e.,
After instrument sequentially adds the sample of 5 uL, 95 μ L Sample dilutions, the min of magnetic particle reaction 10 of the M2 antigen coats of 50 uL,
Carry out Magneto separate.The mouse coated acridinium ester of anti-human igg monoclonal antibody of 100uL is added, after 10 min of reaction, magnetic point is carried out
From reactant mixture is sent into darkroom by instrument, sequentially adds 100uL chemiluminescence preexcitings liquid, 200uL chemiluminescence exciting liquids
Luminescence-producing reaction is carried out, luminous intensity is finally recorded, the AMA M2 types for calculating sample from standard curve contain
Amount.
Embodiment 3：AMA M2 type 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, AMA M2 types chemiluminescence immunoassay chromatography reagent is calculated
The sensitivity of box, the sensitivity tried to achieve is 1AU/ mL.
Linear detection：
It is that 5 AU/mL, 20 AU/mL, 50 AU/mL, 90 AU/mL, 165 AU/mL standard items do linear analysis to concentration, calculates
Linearly dependent coefficient, r=0.9996, in addition, the range of linearity of kit confrontation mitochondrial antibody M2 type sample detections is 3-
400 AU/mL。
Precision is determined：
Concentration is taken for two samples of 2 AU/mL and 100AU/mL, each sample each concentration respectively do 3 it is parallel, with three batches of reagents
Box is detected, calculated in kit batch and difference between batch, as a result shown in the kit batch and difference between batch is respectively less than 5%.
Interference is tested：
Taking pooled serum and adding chaff interference respectively includes：It is combined with 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% 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 AMA M2 type chemiluminescence immune detection reagent kits
It is respectively the calibration object or sample of 0AU/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 1.0AU/ml, relative to traditional enzyme linked immunosorbent assay LDL
8.2AU/ml, improves about 8 times.
Claims (10)
1. a kind of AMA M2 type chemiluminescence immune detection reagent kits, the kit includes：M2 antigen coats-
Nanometer magnetic microsphere, chemiluminescent labels, Chemoluminescent substrate, AMA M2 types calibration product.
2. kit according to claim 1, it is characterised in that the solid phase carrier of the M2 antigen coats is magnetic particle.
3. kit according to claim 1, it is characterised in that the solid phase carrier of the M2 antigen coats is carboxylated
Particle diameter is 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 AMA M2 types calibration product are to use standard
Savor buffer solution by AMA M2 types 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. kit according to claim 1, it is characterised in that the preparation method of the kit, it is characterised in that bag
Include preparation, chemiluminescence that the preparation of the magnetic particle of M2 antigen coats, AMA M2 types derive the acridinium ester of substance markers
The preparation of product is calibrated in the preparation of substrate solution, AMA M2 types.
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 magnetic particle of M2 antigen coats：
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 M2 antigens, at room temperature suspension 2-10 h, and Magneto separate removes supernatant, and Tris buffer solutions are resuspended, obtain M2
The magnetic particle of antigen coat；Optionally, a diameter of 0.1 μm ~ 2.0 μm of carboxylated nanometer magnetic bead；MES buffer concentrations are 10mM
~ 100mM, pH 5.5 ~ 8.5；
2）AMA M2 types derive the preparation of the acridinium ester of substance markers：
AMA M2 type derivatives are taken, carbonate buffer solution is added, mixed, be subsequently adding acridinium ester mixing, at room temperature
Lucifuge is reacted, and is taken out after 1-2 h, and desalting column desalting processing is centrifuged, and uses pure water and TBS buffer solutions in desalination processes respectively first
Processed, the acridine ester solution that the AMA M2 types for obtaining derive substance markers is eventually adding, in collection centrifuge tube
Liquid is in control the acridinium ester of AMA M2 types derivative substance markers to preserving；
3）AMA M2 types calibrate the preparation of product：
AMA M2 types are configured to concentration for 0 AU/mL, 5 AU/mL, 20 AU/mL, 50 with standard items buffer solution
AU/mL, 90AU/mL, 165AU/mL, packing are 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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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102955028A (en) * | 2012-06-11 | 2013-03-06 | 郑州安图绿科生物工程有限公司 | Kit for detecting AMA (anti-mitochondrial antibody)-M2 relative to autoimmune liver diseases, and detection method with 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 CN201611018761.5A patent/CN106771131A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102955028A (en) * | 2012-06-11 | 2013-03-06 | 郑州安图绿科生物工程有限公司 | Kit for detecting AMA (anti-mitochondrial antibody)-M2 relative to autoimmune liver diseases, and detection method with 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 |
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