CN107102133A - A kind of islet cell antibody chemiluminescence immunity detection reagent and preparation method thereof - Google Patents
A kind of islet cell antibody chemiluminescence immunity detection reagent and preparation method thereof Download PDFInfo
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- CN107102133A CN107102133A CN201611018793.5A CN201611018793A CN107102133A CN 107102133 A CN107102133 A CN 107102133A CN 201611018793 A CN201611018793 A CN 201611018793A CN 107102133 A CN107102133 A CN 107102133A
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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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- 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
- G01N33/543—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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Abstract
The invention discloses a kind of islet cell antibody chemiluminescence immunity detection reagent, the kit includes：The coated magnetic particle working solution of islet cells, the anti-human igg working solution of acridinium ester label, islet cell antibodies calibration product, preexciting liquid, the exciting liquid of purifying.The invention also discloses a kind of preparation method of islet cell antibody chemiluminescence 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
The present invention relates to in-vitro diagnosis field of immunodetection, specifically, the invention provides a kind of islet cell antibodies
Learn electrochemiluminescent immunoassay detection kit and preparation method thereof.
Background technology
Islet cell antibodies（Islet cell antibody, ICA）It is that a kind of cytoplasmic components to islet cells work
Specific antibody, belong to IgG immunoglobulins.ICA in serum can react with β cells and produce CDCC.
Insulin-dependent diabetes mellitus（Ins μ Lin-dependent diabetes mellitus, IDDM）Or I type sugar
It is a kind of progressive chronic injury disease to urinate disease, and it can damage the generation and secretion of insulin, and change metabolism of blood glucose mechanism.
Insulin is a kind of hormone for being synthesized and being secreted by islet cells or Langarhans cells.And in IDDM patient, its body
Interior autoantibody can cause the immune injury of islet cells, so as to destroy the normal complex functionality of insulin, it is such just
Normal autoimmune disease can be contacted by heredity and noxious material, and virus infection and various stress are triggered.
ICA is hypersensitivity, the high specific index for diagnosing insulin-dependent diabetes mellitus.The anti-ICA antibody of normal population
Positive rate be only 0.5%, newly sending out the positives rate of patient in IDDM is up to 60~90%, the anti-ICA antibody indication of high titre
The high risk of progression of disease.In addition, Virus monitory ICA is the important means of IDDM early diagnosis, while being also diabetes
Correct treatment, observation of curative effect and Index for diagnosis provide valuable index.The common side of μ L μ L clinical detection islet cell antibodies
Method has radioimmunoassays, enzyme linked immunosorbent assay, but these methods all have some shortcomings part.
First, radioimmunoassays
The general principle of this method is first to use radioactive I125 Specific antibody in mark restructuring pancreatic island cell antigen, serum
Antigen antibody complex first is formed with antigen binding, incubation after secondary antibody is added and forms Ag-Ab-secondary antibody compound, after centrifugation
Detection radioactivity is so as to judge the content of specific antibody in serum.Party's law technology is more ripe, but it is not enough also very
Substantially：
（1）Radioactivity has an impact to operator's body；
（2）Behaviour does relatively complicated, it is necessary to which centrifuge and emissivity detection means, many basic hospitals are difficult to promote；
（3）Background is high, and specificity is bad；
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 determining islet cell antibody technology has the following disadvantages：Testing cost is high, detection sensitivity is low, reappearance
Low, complex operation etc..
The present invention discloses that a kind of testing cost is low, sensitivity is high, reappearance precisely in order to overcome shortcoming described above
Islet cell antibodies kit high, simple to operate and preparation method thereof.The method comprises the steps of firstly, preparing chemiluminescence immune assay examination
Agent box, mainly includes：The coated magnetic particle of pancreatic island cell antigen, the anti-human igg of acridinium ester label and islet cell antibodies are fixed
Mark product；Then calibration product are detected using Full-automatic chemiluminescence immunoassay analysis meter, draws standard curve, be built in computer
Software, tests actual sample, and concentration of specimens is calculated according to sample luminous value；Finally to islet cell antibodies Full-automatic chemiluminescence
Immunoassay system carries out 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 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 0.06 U/mL, phase
15 times are at least improved than other determining islet cell antibody method sensitivity；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity that the present invention is selected is wide, can reach 5-300U/mL, other
The inspection range of linearity of islet cell antibodies chemistry hair detection method is 10-200U/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 islet cell antibodies canonical plotting that embodiment 3 is obtained.
Embodiment
Embodiment 1：Islet cell antibody chemiluminescence immunity detection reagent preparation method
（1）It is prepared by the coated nanometer magnetic bead of pancreatic island cell antigen：
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 pancreatic island cell antigens, 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 magnetic particle of pancreatic island cell antigen, every bottle of 5 mL packing be stored in 4 DEG C it is standby.
（2）The preparation of the mouse anti-human igg of acridinium ester label：
50 μ L 25mg/mL mouse anti-human igg is taken, 150 μ L 0.1-0.2 M pH 9.0-9.5 carbonate buffer solution is added,
Mix, 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, with 2 mL's
Zeba is centrifuged to be handled with pure water and TBS buffer solutions respectively first in desalting column desalting processing, desalination processes, is eventually adding
The acridine ester solution of obtained islet cells note, collects the acridine that the liquid in centrifuge tube is in control islet cells mark to preservation
Ester, every bottle of 5 mL packing be stored in 4 DEG C it is standby.
（3）Islet cell antibodies calibrate the preparation of product：
Use standard items buffer solution（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）Islet cell antibodies are configured
It is 5 U/mL, 20 U/mL, 80 U/mL, 200 U/mL, 800 U/mL, 2000 U/mL, every bottle of 0.5 mL, packing into concentration
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 80 μ L 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 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：Islet cell antibody chemiluminescence immunologic detection method：
The present invention is using Full-automatic chemiluminescence immunoassay analysis meter as detection instrument, and methodology pattern of the invention is indirect method.First
By serum/plasma sample 1：50 times of dilutions, take the μ L of sample 50 after dilution, the 50 μ L coated magnetic particle of islet cells it is micro-
Grain is incubated after 10 min, is carried out Magneto separate cleaning, is added 100 μ L mouse anti-human igg（20ng/mL）, 10min is incubated, then
Carry out Magneto separate cleaning.Reactant mixture after Magneto separate is sent into darkroom by instrument, sequentially adds 50 μ L chemiluminescence preexcitings
Liquid, 50 μ L chemiluminescences exciting liquids carry out luminescence-producing reaction, finally record luminous intensity, sample is calculated from standard curve
Islet cell antibodies content.
Embodiment 3：Islet cell antibody chemiluminescence 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, islet cell antibody chemiluminescence immunity detection reagent is calculated
Sensitivity for analysis, the sensitivity for analysis tried to achieve is 0.06.
Linear detection：
It is that 5 U/mL, 20 U/mL, 40 U/mL, 80 U/mL, 150 U/mL, 300 U/mL standard items do linear point to concentration
Analysis, calculates linearly dependent coefficient, r=0.9786, in addition, the kit is to the range of linearity of islet cell antibodies sample detection
5-300 U/mL。
Precision is determined：
It is two islet cell antibodies samples to take the U/mL of concentration 20 and 150 U/mL, and each each concentration of sample is respectively done 3 and put down
OK, 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 is equal
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 islet cell antibodies accurate evaluation.
Embodiment 4:The sensitivity for analysis contrast experiment of islet cell antibody chemiluminescence immunity detection reagent
Concentration is entered for 0 IU/mL Sample dilution with chemical luminescence detection method and traditional enzyme linked immunosorbent assay respectively
Row detection, replication 20 times draws the RLU values of 20 measurement results（Relative light unit）, calculate its average value（M）And standard
Difference（SD）, M+2SD is drawn, the luminous value is substituted into calibration curve calculating obtains corresponding concentration value.Using chemiluminescence detection
The concentration value that method is obtained is 0.06 U/mL, relative to traditional IU/mL of enzyme linked immunosorbent assay sensitivity for analysis 0.91, is carried
It is high about 15 times.
Claims (10)
1. a kind of islet cell antibody chemiluminescence immunity detection reagent, the kit includes：The islet cells bag of purifying
The magnetic particle working solution of quilt, islet cells working solution, the islet cells of the purifying of the anti-human igg acridinium ester label of acridinium ester label
Antibody calibration product, preexciting liquid, exciting liquid.
2. kit according to claim 1, it is characterised in that the coated solid phase carrier of pancreatic island cell antigen is magnetic
Particulate.
3. kit according to claim 1, it is characterised in that the coated solid phase carrier of pancreatic island cell antigen 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 islet cell antibodies calibration product are slow with standard items
Islet cell antibodies are configured to concentration for 5 U/mL, 20 U/mL, 40 U/mL, 80 U/mL, 150 U/mL, 300 by fliud flushing
U/mL, packing is 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 of the coated magnetic particle of islet cells, the preparation of acridinium ester of islet cells mark, the preparation of Chemoluminescent substrate,
Islet cell antibodies calibrate the preparation of product.
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 pancreatic island cell antigen：
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 pancreatic island cell antigen, at room temperature suspension 2-10 h, Magneto separate, removes supernatant, and Tris buffer solutions are resuspended,
Obtain the coated magnetic particle of pancreatic island cell antigen；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 mouse anti-human igg of acridinium ester label：
Mouse anti-human igg is taken, carbonate buffer solution is added, mixed, acridinium ester is then added and mixes, lucifuge is reacted at room temperature, 1-2 h
After take out, centrifuge desalting column desalting processing, handled respectively with pure water and TBS buffer solutions first in desalination processes, finally
The mouse anti-human igg solution of obtained acridinium ester label is added, liquid to the preservation collected in centrifuge tube is in control islet cells mark
The acridinium ester of note；
3）Islet cell antibodies calibrate the preparation of product：
With standard items buffer solution by islet cell antibodies be configured to concentration for 5 U/mL, 20 U/mL, 40U/mL, 80 U/mL,
150 U/mL, 300 U/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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| CN201611018793.5A CN107102133A (en) | 2016-11-21 | 2016-11-21 | A kind of islet cell antibody chemiluminescence immunity detection reagent and preparation method thereof |
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| CN201611018793.5A CN107102133A (en) | 2016-11-21 | 2016-11-21 | A kind of islet cell antibody chemiluminescence immunity detection reagent and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109100519A (en) * | 2018-09-25 | 2018-12-28 | 迪瑞医疗科技股份有限公司 | Determining islet cell antibody kit and preparation method thereof |
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- 2016-11-21 CN CN201611018793.5A patent/CN107102133A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109100519A (en) * | 2018-09-25 | 2018-12-28 | 迪瑞医疗科技股份有限公司 | Determining islet cell antibody kit and preparation method thereof |
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