CN107044977A - A kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent and preparation method thereof - Google Patents
A kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent and preparation method thereof Download PDFInfo
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- CN107044977A CN107044977A CN201611018794.XA CN201611018794A CN107044977A CN 107044977 A CN107044977 A CN 107044977A CN 201611018794 A CN201611018794 A CN 201611018794A CN 107044977 A CN107044977 A CN 107044977A
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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
- G01N21/763—Bioluminescence
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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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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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
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- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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Abstract
The invention discloses a kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent, the kit includes：The coated magnetic particle working solution of tyrosine phosphatase, the tyrosine phosphatase working solution of the purifying of acridinium ester label, tyrosine phosphatase enzyme antibody calibration product, preexciting liquid, the exciting liquid of purifying.The invention also discloses a kind of preparation method of tyrosine phosphatase antibody chemical luminescence immunity detection reagent in addition.Kit of the present invention is compared with available reagent box, with easy to operate, sensitivity height, the features such as detection range is wide.
Description
Technical field
It is specifically, anti-the invention provides a kind of tyrosine phosphatase the present invention relates to in-vitro diagnosis field of immunodetection
Body chemiluminescence immune detection reagent kit and preparation method thereof.
Background technology
Tyrosine phosphatase is a kind of islet-cell tumour GAP-associated protein GAP（One of tyrosine phosphatase sample molecule families into
Member）, it is islet cell antibodies（ICA）One of main composition, 4 parts are divided into structure：Signal peptide, extracellular structure, list
One membrane spaning domain and intracellular domain.Tyrosine phosphatase enzyme antibody（IA-2A）It is used as a kind of important islet autoantibody, youngster
The frequency occurred in virgin and adolescent patient is that the frequency occurred in 50-70%, adult patient is 30-50%.IA-2 and GAD mono-
Sample type i diabetes major antigen, the process of disease is relevant with the minuent of the antibody.
The common methods of clinical detection tyrosine phosphatase enzyme antibody have IIF, enzyme linked immunosorbent assay, but
These methods are all existed in place of some shortcomings.
First, IIF
The general principle of the method is to form antigen antibody complex with after the antigen binding in specific antibody and slide, after
Combined with fluorescence antibody with antigen antibody complex, form antigen-antibody fluorescent composition.Under fluorescence microscope, according to compound
The luminous situation of thing determines detected antibody.This method is evaluated：Because the fluorescence combined on antigen antibody complex resists
Body increases, and the fluorescent brightness sent is strong, thus its sensitiveness is strong.But its deficiency is also apparent：
(1) operate relative complex, it is necessary to which the fluorescence microscope of price costly, is difficult to promote, also not in many basic hospitals
It is applied to very much the more laboratory of specimen amount；
(2) background in fluoremetry is higher, can only carry out qualitative detection, immunofluorence technic, which is used for quantitative determination, to be had necessarily
It is difficult；
(3) result judgement needs experienced professional, and the objectivity of analysis result is not enough；
2nd, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but there is also following weak points for this method：
(1) 12 × 8 types, 6 × 8 types, 8 × 12 types or the hole Special micro porous plate of complete plate 96 is used to be 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 independent, single part is carried out
Detection；
(2) reagent type used in 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) the corresponding mark to detection information is lacked, can only be by checking that the mark of kit external packing box just will appreciate that or know
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, operating process is extremely cumbersome and multiple more
It is miscellaneous, easily occur bust, the degree of accuracy of testing result and precision are poor.
The content of the invention
Current tyrosine phosphatase antibody test technology 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 shortcoming described above
Scope is wide, reappearance is high, can quantify, tyrosine phosphatase antibody kit simple to operate and preparation method thereof.The present invention
Chemical luminescence immune analysis reagent box is prepared first, is mainly included：The coated magnetic particle of tyrosine phosphatase, tyrosine phosphatase
Coated acridinium ester and tyrosine phosphatase enzyme antibody calibrates product；Then using Full-automatic chemiluminescence immunoassay analysis meter to calibration
Product are detected, draw standard curve, are built in computer software, test actual sample, and it is dense to calculate sample according to sample luminous value
Degree；Performance finally is carried out to tyrosine phosphatase enzyme antibody automatic chemiluminescence immunoassay system（Sensitivity, linear, precision
Degree, interference）Evaluation.
It is of the invention compared with current technology, with advantages below：
1st, present invention selection acridinium ester is as marker material, and 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 0.08 IU/mL,
8 times are at least improved compared to other tyrosine phosphatase antibody detection method sensitivity；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity that the present invention is selected is wide, can reach 10-4000 IU/mL, its
The inspection range of linearity of its tyrosine phosphatase enzyme antibody chemistry hair detection method is 20-2000 IU/mL；
4th, the acridinium ester chemiluminescent immunoassay system repeatability that the present invention is selected is high, criticizes interior and difference between batch 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 tyrosine phosphatase enzyme antibody canonical plotting that embodiment 3 is obtained.
Embodiment
Embodiment 1：Tyrosine phosphatase antibody chemical luminescence immunity detection reagent preparation method
（1）It is prepared by the coated nanometer magnetic bead of tyrosine phosphatase：
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, and add the EDC aqueous solution for 10 mg/mL that 0.5-2mL is newly configured, and activated magnetic beads surface carboxyl groups add 3-5
Mg glutamic acid decarboxylases, suspension 2-10 h, Magneto separate, remove supernatant at room temperature, are 8.0 with the 0.1 M pH containing 2% BSA
Tris buffer solutions are resuspended to 1mg/mL, obtain the coated magnetic particle of tyrosine phosphatase Antibodies Monoclonal antibodies, every bottle of 5mL packing
Be stored in 4 DEG C it is standby.
（2）It is prepared by the tyrosine phosphatase of acridinium ester label：
50 μ L 25mg/mL tyrosine phosphatase is taken, 150 μ L 0.1-0.2 M pH 9.0-9.5 carbonate buffer is added
Liquid, is mixed, and the acridinium ester for then adding the mg/mL of 1-2 μ L 5 is mixed, and lucifuge is reacted at room temperature, is taken out after 1-2 h, is used 2 mL
Zeba centrifugation desalting column desalting processings, handled respectively with pure water and TBS buffer solutions first in desalination processes, finally plus
Enter the acridine ester solution of obtained tyrosine phosphatase note, liquid to the preservation collected in centrifuge tube is in control tyrosine phosphatase
The acridinium ester of mark, every bottle of 5 mL packing be stored in 4 DEG C it is standby.
（3）Tyrosine phosphatase enzyme antibody calibrates the preparation of product：
Use standard items buffer solution（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）By tyrosine phosphatase enzyme antibody
Being configured to concentration is, every bottle of 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 80 μ L mass fractions are 20% is sequentially added2O2), 1.0 grams of sodium azide,
1.5 grams of polysorbas20s, shake up rear lucifuge storage.
(5) preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.6 gram of sodium hydroxide, 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：Tyrosine phosphatase antibody chemical luminescence immunologic detection method：
The present invention is using Full-automatic chemiluminescence immunoassay analysis meter as detection instrument, and methodology pattern of the invention is double antigens sandwich
Method, i.e. instrument sequentially add 50 μ L sample, 50 μ the L coated magnetic particle of tyrosine phosphatase and 50 μ L tyrosine
After the coated acridinium ester of phosphatase, 10 min of reaction, Magneto separate is carried out, reactant mixture is sent into darkroom, sequentially added by instrument
50 μ L chemiluminescence preexcitings liquid, 50 μ L chemiluminescences exciting liquids carry out luminescence-producing reaction, finally record luminous intensity, bent from standard
Line computation goes out the tyrosine phosphatase antibody content of sample.
Embodiment 3：Tyrosine phosphatase antibody chemical luminescence immunity detection reagent performance evaluation
Detection curve is shown in accompanying drawing 1.
The detection of sensitivity for analysis：
With reference to CLSI EP17-A file recommendation experimental programs, tyrosine phosphatase antibody chemical luminescence immunologic function test reagent is calculated
The sensitivity for analysis of box, the sensitivity tried to achieve is 0.08 IU/mL.
Linear detection：
It is that standard items do linear analysis to concentration, calculates linearly dependent coefficient, r=0.9901, in addition, the kit is to tyrosine
The range of linearity of phosphatase antibody sample detection is 5-4000 IU/mL.
Precision is determined：
It is two tyrosine phosphatase antibody samples of IU/mL and 500 IU/mL to take concentration, and each each concentration of sample respectively does 3
It is parallel, detected, calculated in kit batch and difference between batch with three batches of kits, as a result shown in the kit batch and difference between batch
Respectively less than 5%.
Interference is tested：
Taking pooled serum to add chaff interference respectively includes：Bilirubin, hemoglobin, ascorbic acid, glyceride, adding proportion according to
1:20 are carried out, and are determined pooled serum respectively and be with the addition of the measured value of pooled serum after various chaff interferences, calculate therebetween
Deviation, with ± 10% for tolerance interval.As a result show, interference reaches NCCLS file standard, available for clinical real
Test room tyrosine phosphatase enzyme antibody accurate evaluation.
Embodiment 4:The sensitivity for analysis contrast experiment of tyrosine phosphatase antibody chemical luminescence immunity detection reagent point
Concentration is not examined for 0 IU/mL Sample dilution with chemical luminescence detection method and traditional enzyme linked immunosorbent assay
Survey, replication 20 times draws the RLU values of 20 measurement results（Relative light unit）, calculate its average value（M）And standard deviation
（SD）, M+2SD is drawn, the luminous value is substituted into calibration curve calculating obtains corresponding concentration value.Using chemiluminescence detection side
The concentration value that method is obtained is 0.08 IU/mL, relative to traditional IU/mL of enzyme linked immunosorbent assay sensitivity for analysis 0.65, is carried
It is high about 8 times.
Claims (10)
1. a kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent, the kit includes：The tyrosine of purifying
The coated magnetic particle working solution of phosphatase, the tyrosine phosphatase working solution of the purifying of acridinium ester label, tyrosine phosphatase resist
Body calibration product, preexciting liquid, exciting liquid.
2. kit according to claim 1, it is characterised in that the coated solid phase carrier of tyrosine phosphatase is magnetic
Particulate.
3. kit according to claim 1, it is characterised in that the coated solid phase carrier of tyrosine phosphatase is carboxylic
The particle diameter of base 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 0.005mol/L ~ 0.025mol/L sodium hydroxide
(NaOH) solution.
8. kit according to claim 1, it is characterised in that the tyrosine phosphatase enzyme antibody calibration product are to use standard
Savor buffer solution by tyrosine phosphatase enzyme antibody be configured to concentration for 5 IU/mL, 25 IU/mL, 100 IU/mL, 500 IU/mL,
1000 IU/mL, 4000 IU/mL, packing are lyophilized, and 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 the preparation, the preparation of the acridinium ester of tyrosine phosphatase mark, chemical luminous substrate of the coated magnetic particle of tyrosine phosphatase
The preparation of product is calibrated in the preparation of liquid, tyrosine phosphatase enzyme antibody.
10. the preparation method of kit according to claim 1 and claim 9, it is characterised in that comprise the following steps：
1）The preparation of the coated magnetic particle of tyrosine phosphatase：
The nanometer magnetic bead suspension of carboxylated is taken, Magneto separate removes supernatant, and MES buffer solutions are resuspended, and add the EDC aqueous solution, activate magnetic
Bead surface carboxyl, adds tyrosine phosphatase, at room temperature suspension 2-10 h, Magneto separate, removes supernatant, and Tris buffer solutions are resuspended,
Obtain the coated magnetic particle of tyrosine phosphatase；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）It is prepared by the tyrosine phosphatase of acridinium ester label：
Tyrosine phosphatase is taken, carbonate buffer solution is added, mixed, acridinium ester is then added and mixes, lucifuge is reacted at room temperature, 1-
Taken out after 2 h, centrifuge and handled respectively with pure water and TBS buffer solutions first in desalting column desalting processing, desalination processes, most
The acridine ester solution of obtained tyrosine phosphatase mark is added afterwards, and liquid to the preservation collected in centrifuge tube is in control tyrosine
The acridinium ester of phosphatase enzyme mark；
3）Tyrosine phosphatase enzyme antibody calibrates the preparation of product：
With standard items buffer solution by tyrosine phosphatase enzyme antibody be configured to concentration for 5 IU/mL, 25 IU/mL, 100 IU/mL,
500 IU/mL, 1000 IU/mL, 4000 IU/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 ~ 100 μ L 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 X-100, Triton X -405；
5）The preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters 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 lives
Property agent, shake up the storage of rear lucifuge；Optionally, preservative is commercialization sodium azide, PC300, and surfactant is polysorbas20, told
Temperature 80, Triton X-100, Triton X-405.
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| CN2016206805333 | 2016-06-30 | ||
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261597A (en) * | 2019-06-06 | 2019-09-20 | 三诺生物传感股份有限公司 | A kind of IA-2 autoantibody detection strip |
| 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 |
| US20210247392A1 (en) * | 2018-07-02 | 2021-08-12 | Siemens Healthcare Diagnostics Inc. | Direct immunoassay measurement of autoantibodies |
| CN113917142A (en) * | 2021-09-06 | 2022-01-11 | 博奥赛斯(重庆)生物科技有限公司 | Kit for chemiluminescence immunoassay of tyrosine phosphatase autoantibody magnetic particles, preparation method and detection method |
| CN114252592A (en) * | 2021-11-22 | 2022-03-29 | 广州万孚生物技术股份有限公司 | Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof |
| CN114924071A (en) * | 2022-03-29 | 2022-08-19 | 北京世纪沃德生物科技有限公司 | Anti-tyrosine phosphatase antibody determination kit |
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| CN114252592A (en) * | 2021-11-22 | 2022-03-29 | 广州万孚生物技术股份有限公司 | Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof |
| CN114252592B (en) * | 2021-11-22 | 2024-05-31 | 广州万孚生物技术股份有限公司 | Soluble fms-like tyrosine kinase-1 detection kit and preparation method and application thereof |
| CN114924071A (en) * | 2022-03-29 | 2022-08-19 | 北京世纪沃德生物科技有限公司 | Anti-tyrosine phosphatase antibody determination kit |
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