CN106596919A - Kit (chemiluminiscence method) for determining anti-ribonucleoprotein 70 antibody IgG and manufacturing method thereof - Google Patents
Kit (chemiluminiscence method) for determining anti-ribonucleoprotein 70 antibody IgG and manufacturing method thereof Download PDFInfo
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- CN106596919A CN106596919A CN201611018734.8A CN201611018734A CN106596919A CN 106596919 A CN106596919 A CN 106596919A CN 201611018734 A CN201611018734 A CN 201611018734A CN 106596919 A CN106596919 A CN 106596919A
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- China
- Prior art keywords
- ribonucleoprotein
- igg antibody
- kit
- acridinium ester
- liquid
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Classifications
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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
-
- 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
Abstract
The invention discloses a kit (chemiluminiscence method) for determining an anti-ribonucleoprotein 70 antibody IgG. The kit comprises magnetic particles coated by anti-ribonucleoprotein 70 antigen, acridinium ester marked by mouse-anti-human IgG, a sample diluent, a test diluent, an anti-ribonucleoprotein 70 antibody IgG calibration product, pre-exciting liquid and exciting liquid. The invention further discloses a manufacturing method of the kit (chemiluminiscence method) for determining the anti-ribonucleoprotein 70 antibody IgG. Compared with an existing kit, the kit has the advantages of being easy to operate, high in sensitivity and wide in detection range.
Description
Technical field
The present invention relates to in-vitro diagnosis field of immunodetection, specifically, the invention provides a kind of anti-ribonucleoprotein 70
IgG antibody side determines kit（Chemoluminescence method）And preparation method thereof.
Background technology
Small nut nucleoglucoprotein compound（U-snRNP）Particularly important effect is played in the shearing of precursor mRNA.Cell
Most of RNP particles are U1-snRNP in core, and it is by a small fragment RNP rich in uridylic acid（U1-RNP）With concentration albumen
The compound of matter composition.The antibody of anti-ribonucleoprotein 70（RNP70）It is the antibody for 70kDa albumen in U1-RNP, it is seen that in
35% ~ 45% systemic loupus erythematosus（SLE）Patient and 95% ~ 100% MCTD（Mixed connective
Tissue disease, MCTD）Patient, is the diagnostic markers of MCTD.Age of onset from 4 years old to 80 years old, greatly
Most of patients occurs symptom 30-40 year or so, average age 37 years old, and women is common, accounts for 80%.
The common methods of the anti-IgG antibody of ribonucleoprotein 70 of clinical detection have IIF, Enzyme-linked Immunosorbent Assay
Method, but these methods all have some shortcomings part.
First, IIF
The general principle of the method is to form antigen antibody complex after the antigen binding in antibody and slide with specificity, after
Combined with antigen antibody complex with fluorescence antibody, formed antigen-antibody fluorescent composition.Under fluorescence microscope, according to compound
The luminous situation of thing is determining detected antibody.The method is evaluated：Because the fluorescence combined on antigen antibody complex resists
Body increases, and the fluorescent brightness for sending is strong, thus its sensitiveness is strong.But its deficiency is also apparent from：
(1) cannot be according to the non-specific identification of the size discrimination of molecular weight in analysis result；
(2) operation is relative complex, needs price fluorescence microscope costly, is difficult to promote in many basic hospitals, also less
Suitable for the more laboratory of specimen amount；
(3) background in fluoremetry is higher, and immunofluorence technic has certain difficulty for quantitative determination；
(4) result judgement needs experienced professional, and the objectivity of analysis result is not enough；
(5) qualitative detection can only be carried out, it is impossible to quantitative determined.
2nd, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but the method there is also following weak points：
(1) using 12 × 8 types, 6 × 8 types, 8 × 12 types or the hole Special micro porous plate of complete plate 96 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 carry out independent, single part
Detection；
(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 to be filled into respectively in the micropore of microwell plate 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 kit external packing box 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) 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
It is miscellaneous, bust is susceptible to, the degree of accuracy of testing result and precision are 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 detection 10
Different project, it is also desirable to configure the reagent of 10 × 48/96 person-portions, haves the shortcomings that inadequate economical rationality.
The content of the invention
At present the IgG antibody detection technique of anti-ribonucleoprotein 70 is suffered from the drawback that：Testing cost is high, detection sensitivity
It is low, detect linear narrow range, reappearance it 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
The IgG antibody of anti-ribonucleoprotein 70 that scope is wide, reappearance is high, can be quantitative, simple to operate determines kit（Chemiluminescence
Method）And preparation method thereof.The method comprises the steps of firstly, preparing chemical luminescence immune analysis reagent box, mainly includes：Anti- ribonucleoprotein 70
Acridinium ester and the IgG antibody calibration object of anti-ribonucleoprotein 70 that the magnetic particle of IgG antibody antigen coat, mouse anti-human igg are marked；
Then calibration object is detected using Full-automatic chemiluminescence immunoassay analysis meter, draws calibration curve, be built in computer software,
Test actual sample, according to sample luminous value concentration of specimens is calculated；Finally resist the IgG antibody full-automatic chemical of ribonucleoprotein 70
Luminescence immunoassay system carries out performance（Sensitivity, linear, precision, interference）Evaluation.
It is 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 0.34AU/mL, phase
About 3 times are improve than the IgG antibody detection method sensitivity of other anti-ribonucleoproteins 70；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity width that the present invention is selected, can reach 3-165 ng/mL, other
The IgG antibody of anti-ribonucleoprotein 70 chemistry send out detection method the inspection range of linearity be 20-135 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 the quantitative of sample, soft to testing by built-in calibration curve
Part, only needs test sample to directly obtain the concentration value of sample；
6th, chemiluminescence immunoassay system of the invention has realized that the addition of full-automation, reagent and sample has instrument complete entirely
Into operation is easier, reduces artificial error.
Description of the drawings
Fig. 1：For the IgG antibody canonical plotting of anti-ribonucleoprotein 70 that embodiment 3 is obtained.
Specific embodiment
Embodiment 1：The IgG antibody of anti-ribonucleoprotein 70 determines kit（Chemoluminescence method）Preparation method
（1）It is prepared by the nanometer magnetic bead of the IgG antibody antigen coat of anti-ribonucleoprotein 70：
Take the magnetic particle of 50mg carboxylated（Particle diameter is 0.05-1um）Suspension, Magneto separate removes supernatant, uses 0.02 M, pH to be 5.5
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 anti-IgG antibody antigens of ribonucleoprotein 70 of mg, suspension 2-10 h under room temperature, Magneto separate removes supernatant, uses containing 2% BSA
0.1 M pH are that 8.0 Tris buffer solutions are resuspended to 1mg/mL, obtain the magnetic of the IgG antibody antigen coat of anti-ribonucleoprotein 70
Grain, every bottle of 5mL packing be stored in 4 DEG C it is standby.
（2）The preparation of the acridinium ester of mouse anti-human igg mark：
The mouse anti-human igg of 40 uL 5mg/mL is taken, the carbonate buffer solution of 150 uL 0.1-0.2 M pH 9.0-9.5 is added,
Mix, the acridinium ester for being subsequently adding the mg/mL of 1-2 uL 5 is mixed, lucifuge reaction at 4 DEG C is taken out, with the zeba of 2 mL after 12h
Centrifugation desalting column desalting processing, is processed with pure water and TBS buffer solutions respectively first in desalination processes, is eventually adding and is obtained
The anti-human mark of mouse acridine ester solution, collect centrifuge tube in liquid to preserve be in control mouse anti-human igg mark acridinium ester,
Per bottle of 5 mL packing be stored in 4 DEG C it is standby.
（3）The preparation of the IgG antibody calibration object of anti-ribonucleoprotein 70：
Use standard items buffer solution（50 mM Tris, 5% BSA, 0.15% NaCl, pH 7.4）By the antibody of anti-ribonucleoprotein 70
It is 0AU/mL, 20AU/mL, 200AU/mL that IgG is configured to concentration, and per bottle of 1 mL is dispensed, 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：The IgG antibody chemical luminous immune detection method of anti-ribonucleoprotein 70：
With Full-automatic chemiluminescence immunoassay analysis meter to detect instrument, method of the present invention pattern is indirect method to the present invention, i.e.,
Instrument adds 10 times of the sample Sample dilution of 10 uL to take 10ul after diluting, be subsequently adding the anti-ribonucleoprotein 70 of 50 uL
After the magnetic particle reaction 10min of IgG antibody antigen coat, Magneto separate is carried out；Sequentially add the test dilution of 50 uL with
The acridinium ester of the mouse anti-human igg of 50ul, after 10 min of reaction, carries out Magneto separate, and reactant mixture is sent into darkroom by instrument, successively
Add 50uL chemiluminescence preexciting liquid, 50uL chemiluminescences exciting liquid to carry out luminescence-producing reaction, luminous intensity is finally recorded, from mark
Directrix curve calculates the IgG antibody content of anti-ribonucleoprotein 70 of sample.
Embodiment 3：The IgG antibody of anti-ribonucleoprotein 70 determines kit（Chemoluminescence method）Performance evaluation
Detection curve is shown in accompanying drawing 1.
The detection of sensitivity：
With reference to CLSI EP17-A file recommendation experimental programs, calculate the anti-IgG antibody of ribonucleoprotein 70 and determine kit（Chemistry
Luminescence method）Sensitivity, the sensitivity tried to achieve be 0.34AU/ mL.
Linear detection：
It is that 12.5 AU/mL, 25 AU/mL, 50AU/mL, 100 AU/mL, 200 AUmL standard items do linear analysis to concentration, counts
Linearly dependent coefficient, r=0.9997 are calculated, in addition, the kit resists the linear model of the IgG antibody sample detection of ribonucleoprotein 70
Enclose for 3-165 ng/mL.
Precision is determined：
Concentration is taken for the IgG antibody sample of two anti-ribonucleoproteins of 30 AU/mL and 150 AU/mL 70, each sample each concentration
Respectively do 3 it is parallel, detected with three batches of kits, calculate in kit batch and difference between batch, as a result show 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% as tolerance interval.As a result show, interference reaches the file of NCCLS
Standard, can be used for the accurate evaluation of the anti-ribonucleoprotein IgG of clinical labororatory.
Embodiment 4:The IgG antibody of anti-ribonucleoprotein 70 determines kit（Chemoluminescence method）Sensitivity comparison experiment
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 the RLU values of 20 measurement results for sample（Relative light unit）, calculate it
Mean value（M）And standard deviation（SD）, M+2SD is drawn, the luminous value is substituted into into calibration curve and is calculated corresponding concentration value.Adopt
The concentration value obtained with chemical luminescence detection method is 0.34AU/mL, relative to traditional enzyme linked immunosorbent assay lowest detection
1 U/mL is limited, about 3 times are improve.
Claims (10)
1. a kind of IgG antibody of anti-ribonucleoprotein 70 determines kit（Chemoluminescence method）, the kit includes：Anti- ribose core
The nanometer magnetic microsphere of protein 70 IgG antibody antigen coat, chemiluminescent labels, Sample dilution, test dilution, chemistry are sent out
Light substrate solution, the IgG antibody calibration object of anti-ribonucleoprotein 70.
2. kit according to claim 1, it is characterised in that the IgG antibody antigen coat of the anti-ribonucleoprotein 70
Solid phase carrier be magnetic particle.
3. kit according to claim 1, it is characterised in that the IgG antibody antigen coat of the anti-ribonucleoprotein 70
Solid phase carrier be magnetic particle.
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 the chemiluminescent labels.
6. kit according to claim 1, it is characterised in that the Sample dilution is buffered for the Tris of 50mM/L
Liquid, PH6.5.
7. kit according to claim 1, it is characterised in that the test dilution for 50mM/L MES buffer solutions,
PH6.5。
8. kit according to claim 1, it is characterised in that the Chemoluminescent substrate is excited including chemiluminescence
Liquid, chemiluminescence preexciting liquid, chemiluminescence preexciting liquid, the chemiluminescence preexciting liquid be mass fraction 0.005% ~
0.5% hydrogen peroxide (H2O2) solution, exciting liquid is NaOH (NaOH) solution of 0.005mol/L ~ 0.025mol/L.
9. kit according to claim 1, it is characterised in that the IgG antibody calibration object of the anti-ribonucleoprotein 70 is
It is 0AU/mL, 20AU/mL, 200AU/mL that the IgG antibody of anti-ribonucleoprotein 70 is configured to into concentration with standard items buffer solution, 4 DEG C
Save backup.
10. anti-ribonucleoprotein 70 IgG antibody antigen chemiluminescence immunity of the one kind according to any one of claim 1 ~ 9 kind
The preparation method of detection kit, it is characterised in that comprise the steps：
1）The preparation of the magnetic particle of the IgG antibody antigen coat of anti-ribonucleoprotein 70：
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 the anti-IgG antibody antigen of ribonucleoprotein 70, suspension 2-10 h under room temperature, Magneto separate to remove supernatant,
Tris buffer solutions are resuspended, obtain the magnetic particle of the IgG antibody antigen coat of anti-ribonucleoprotein 70；Optionally, carboxylated nano magnetic
Pearl 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 mouse anti-human igg mark：
Mouse anti-human igg is taken, carbonate buffer solution is added, is mixed, be subsequently adding acridinium ester mixing, lucifuge reaction, 1-2 h under room temperature
After take out, desalting column desalting processing is centrifuged, processed with pure water and TBS buffer solutions respectively first in desalination processes, finally
The acridine ester solution of the mouse anti-human igg mark that addition is obtained, the liquid collected in centrifuge tube is in control mouse anti-human igg mark to preservation
The acridinium ester of note；
3）The preparation of the IgG antibody calibration object of anti-ribonucleoprotein 70：
It is 0AU/mL, 20AU/mL, 200AU/mL that the IgG antibody of anti-ribonucleoprotein 70 is configured to into concentration with standard items buffer solution,
Packing, 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620676833 | 2016-06-30 | ||
| CN2016206768334 | 2016-06-30 |
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| CN106596919A true CN106596919A (en) | 2017-04-26 |
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| CN201611018734.8A Pending CN106596919A (en) | 2016-06-30 | 2016-11-21 | Kit (chemiluminiscence method) for determining anti-ribonucleoprotein 70 antibody IgG and manufacturing method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111272999A (en) * | 2020-02-17 | 2020-06-12 | 珠海丽珠试剂股份有限公司 | Antigen-coupled magnetic particle, preparation method thereof, detection method of anti-U1-snRNP antibody and kit |
| CN115078731A (en) * | 2022-06-08 | 2022-09-20 | 宁波瑞源生物科技有限公司 | Kit for quantitatively determining cytokeratin 19 fragments and preparation method thereof |
| CN115656154A (en) * | 2022-10-26 | 2023-01-31 | 广东优尼德生物科技有限公司 | anti-RNP antibody chemiluminescence detection kit based on recombinant RNP multiple antigens |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111272999A (en) * | 2020-02-17 | 2020-06-12 | 珠海丽珠试剂股份有限公司 | Antigen-coupled magnetic particle, preparation method thereof, detection method of anti-U1-snRNP antibody and kit |
| CN111272999B (en) * | 2020-02-17 | 2022-11-22 | 珠海丽珠试剂股份有限公司 | Antigen-coupled magnetic particle, preparation method thereof, detection method of anti-U1-snRNP antibody and kit |
| CN115078731A (en) * | 2022-06-08 | 2022-09-20 | 宁波瑞源生物科技有限公司 | Kit for quantitatively determining cytokeratin 19 fragments and preparation method thereof |
| CN115656154A (en) * | 2022-10-26 | 2023-01-31 | 广东优尼德生物科技有限公司 | anti-RNP antibody chemiluminescence detection kit based on recombinant RNP multiple antigens |
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