CN106645711A - Anti-Sm antibody IgG determining kit (chemiluminiscence method) and preparation method thereof - Google Patents
Anti-Sm antibody IgG determining kit (chemiluminiscence method) and preparation method thereof Download PDFInfo
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- CN106645711A CN106645711A CN201611018796.9A CN201611018796A CN106645711A CN 106645711 A CN106645711 A CN 106645711A CN 201611018796 A CN201611018796 A CN 201611018796A CN 106645711 A CN106645711 A CN 106645711A
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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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- 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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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses an anti-Sm antibody IgG determining kit (chemiluminiscence method). The kit comprises anti-Sm antibody coated magnetic particles, mouse anti-human IgG coated acridinium ester, a sample diluent, a test diluent, an anti-Sm antibody IgG calibration product, preexciting liquid and exciting liquid. In addition, the invention further discloses a method for preparing the anti-Sm antibody IgG determining kit (chemiluminiscence method). The kit, compared with an existing kit, has the advantages of being simple and convenient to operate, high in sensitivity and wide in detection range and the like.
Description
Technical field
The present invention relates to in-vitro diagnosis field of immunodetection, specifically, the invention provides a kind of anti-Sm antibody IgG is determined
Kit(Chemoluminescence method)And preparation method thereof.
Background technology
Anti- Sm antibody, it is first in this patients serum for being diagnosed as lupus erythematosus with patient's name (Smith) name
It is secondary to be found that the antibody, therefore name.Later substantial amounts of report confirms in lupus erythematosus patients serum, there is this kind of antibody,
Belong to a class antinuclear antibodies.Main protein antigen in Sm systems be B, B ', D, E protein, 11~29kD of molecular weight, additionally
There are the protein such as A, A ', B ", C, F, G and 68kD, 150kD.These protein and the small nuclear rna s containing guanosine very abundant
(snRNAs)That is UsnRNAs forms compound.At present at least 13 kinds UsnRNAs, i.e. U1~U13 are found in mammal
snRNAs.Sm antigens constitute micronuclear ribonucleoprotein by U1, U2, U5, U4/U6 with above-mentioned protein(U1、U2、U5、U4/U6
snRNP), it is in heterogeneous nRNA(hn RNA)To ripe mRNA(mRNA)Conversion in play an important role.DNA transcriptions
HnRN is by encoding fragment extron(exons)With the non-coding fragment introne of interruption insertion(introns)Composition, hnRNA to
When mRNA is converted, noncoding sequence is being cut away in the RNA parts of U1, U2, U5, U4/U6 RNP molecules(Introne), reconnect
The RNA sequence of coding(Extron)In play an important role.Through this kind of montage, ripe mRNA is produced, into cytoplasm, in core
Albumen is translated on sugared body.Having proven to anti-Sm antibody can enter lymphocyte living, rear may disturb it being combined with target antigen
Function, makes cell breed suppressed, secrete cytokines(IFN-γ, IL-2 etc.)Reduce, and inducing cell apoptosis.
It is now recognized that anti-Sm antibody is as anti-dsDNA, there are high degree of specificity, and whether active stage to SLE, resist
Sm can be positive, therefore can be used as the significant antibody of SLE.
The main method of clinical detection anti-Sm antibody IgG be enzyme linked immunosorbent assay, but the method exist it is following not
Foot part:
(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)Quantitative determination reagent type used 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, in open space, easily causes the cross pollution between various reagents and shadow in detection process
Ring the accuracy of testing result;
(5)Dosage more than detection process using manual operations, reagent or sample is not bery accurate, and operating process is extremely loaded down with trivial details and multiple
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 anti-Sm antibody IgG detection techniques are suffered from the drawback that:Testing cost is high, detection sensitivity is low, detection is linear
Narrow range, reappearance be 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 anti-Sm antibody IgG that scope is wide, reappearance is high, can be quantitative, simple to operate determines kit(Chemoluminescence method)And its prepare
Method.The method comprises the steps of firstly, preparing chemical luminescence immune analysis reagent box, mainly includes:The magnetic particle of Sm antigen coats, mouse are anti-human
The coated acridinium esters of IgG and anti-Sm antibody IgG calibration objects;Then using Full-automatic chemiluminescence immunoassay analysis meter to calibration object
Detected, drawn calibration curve, be built in computer software, tested actual sample, concentration of specimens is calculated according to sample luminous value;
Finally resisting Sm IgG antibody automatic chemiluminescence immunoassay systems carries out performance(Sensitivity, linear, precision, interference
Property)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 0.33AU/mL, phase
3 times are at least improve than the sensitivity of other anti-Sm antibody IgG detection methods;
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity width that the present invention is selected, can reach 3-165 ng/mL, other
Anti-Sm antibody IgG 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 is the anti-Sm antibody IgG canonical plottings that embodiment 3 is obtained.
Specific embodiment
Embodiment 1:Anti-Sm antibody IgG determines kit(Chemoluminescence method)Preparation method
(1)It is prepared by the nanometer magnetic bead of Sm antigen coats:
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
Mg Sm antigens, suspension 2-10 h under room temperature, Magneto separate removes supernatant, is delayed with the Tris that the 0.1 M pH containing 2% BSA are 8.0
Rush liquid and be resuspended to 1mg/mL, obtain the magnetic particle of Sm antigen coats, 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 50 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 under room temperature is taken out, with 2 mL's after 1-2 h
Zeba is centrifuged desalting column desalting processing, is processed with pure water and TBS buffer solutions respectively first in desalination processes, is eventually adding
The acridine ester solution of the mouse anti-human igg mark for obtaining, liquid to the preservation collected in centrifuge tube is 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 anti-Sm antibody IgG calibration objects:
Use standard items buffer solution(50 mM Tris, 5% BSA, 0.15% NaCl, pH 7.4)Anti-Sm antibody IgG is configured to dense
Spend for 0 AU/mL, 20 AU/mL, 200 AU/mL, per bottle of 1 mL packing, 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:Anti-Sm antibody IgG determines kit(Chemoluminescence method)Detection method:
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 add take after 10 times of the sample Sample dilution dilution of 10 uL 10ul, be subsequently adding 50 uL Sm antigen coats magnetic
After particle reaction 10min, Magneto separate is carried out;What the mouse anti-human igg of the test dilution and 50ul that sequentially add 50 uL was marked
Acridinium ester, after 10 min of reaction, carries out Magneto separate, and reactant mixture is sent into darkroom by instrument, sequentially adds 50uL chemiluminescences
Preexciting liquid, 50uL chemiluminescences exciting liquid carry out luminescence-producing reaction, finally record luminous intensity, calculate from calibration curve tested
The anti-Sm antibody IgG content of sample.
Embodiment 3:Anti-Sm antibody IgG 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 anti-Sm antibody IgG and determine kit(Chemoluminescence method)'s
Sensitivity, the sensitivity tried to achieve is 0.33 AU/ 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=0.9996 are calculated, in addition, the range of linearity of the kit antagonism Sm IgG antibody sample detections is 10-200
AU/mL。
Precision is determined:
Concentration is taken for two anti-Sm antibody IgG samples of 30 AU/mL and 150 AU/mL, each sample each concentration is respectively done 3 and put down
OK, detected with three batches of kits, calculated in kit batch and difference between batch, as a result shown in the kit batch and difference between batch is equal
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 clinical labororatory anti-Sm antibody IgG.
Embodiment 4:Anti-Sm antibody IgG determines kit(Chemoluminescence method)Sensitivity comparison experiment
It is respectively the calibration object or sample of 0 AU/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 calibration curve and is calculated corresponding concentration value.Adopt
The concentration value obtained with chemical luminescence detection method is 0.33 AU/ml, relative to traditional enzyme linked immunosorbent assay lowest detection
1 RU/ml is limited, 3 times are improve.
Claims (10)
1. a kind of anti-Sm antibody IgG determines kit(Chemoluminescence method), the kit includes:The nano magnetic of Sm antigen coats
Microballoon, chemiluminescent labels, Sample dilution, test dilution, Chemoluminescent substrate, anti-Sm antibody IgG calibration objects.
2. kit according to claim 1, it is characterised in that the solid phase carrier of the Sm antigen coats is magnetic particle.
3. kit according to claim 1, it is characterised in that the solid phase carrier of the Sm 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 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, the chemiluminescence preexciting liquid is the hydrogen peroxide (H of mass fraction 0.005% ~ 0.5%2O2)
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 anti-Sm antibody IgG calibration objects are slow with standard items
It is 0AU/mL, 20AU/mL, 200AU/mL to rush liquid and anti-Sm antibody IgG is configured into concentration, and 4 DEG C save backup.
10. the system of anti-Sm antibody IgG chemiluminescence immune detection reagent kit of the one kind according to any one of claim 1 ~ 9
Preparation Method, it is characterised in that comprise the steps:
1)The preparation of the magnetic particle of Sm antigen coats:
Tosyl nanometer magnetic bead suspension is taken, Magneto separate removes supernatant, and borate buffer solution buffer solution is resuspended, add Sm to resist
Original, suspension 16-24 h at 37 DEG C, Magneto separate removes supernatant, and PBS is resuspended, suspension 16-24 h at 37 DEG C, Magneto separate,
Supernatant is removed, PBS is resuspended, obtains the magnetic particle of Sm antigen coats;
Optionally, tosyl nanometer magnetic bead is a diameter of 0.1 μm ~ 2.0 μm;
Borate buffer solution concentration is 10mM/L ~ 100mM/L,(pH8.0~ 9.5), liquid containing ammonium sulfate;
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 anti-Sm antibody IgG calibration objects:
It is 0AU/mL, 20AU/mL, 200AU/mL that anti-Sm antibody IgG is configured into concentration with standard items buffer solution, is dispensed, 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 is commercialization sodium azide, PC300, and 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, Tween 80, Triton
X100、Triton 405。
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Cited By (4)
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CN109187940A (en) * | 2018-07-27 | 2019-01-11 | 南京谱利健生物技术有限公司 | The preparation and application of a kind of isotopic tag reagent for Analysis of polysaccharides |
CN111548416A (en) * | 2020-05-12 | 2020-08-18 | 迈克生物股份有限公司 | Kit and composition for detecting anti-double-stranded DNA antibody and application |
CN112710857A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | f-beta-hCG protein detection kit and detection method |
CN116004684A (en) * | 2022-10-26 | 2023-04-25 | 广东优尼德生物科技有限公司 | SM fusion antigen and anti-Sm antibody chemiluminescence detection kit |
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CN111548416A (en) * | 2020-05-12 | 2020-08-18 | 迈克生物股份有限公司 | Kit and composition for detecting anti-double-stranded DNA antibody and application |
CN112710857A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | f-beta-hCG protein detection kit and detection method |
CN116004684A (en) * | 2022-10-26 | 2023-04-25 | 广东优尼德生物科技有限公司 | SM fusion antigen and anti-Sm antibody chemiluminescence detection kit |
CN116004684B (en) * | 2022-10-26 | 2024-03-08 | 广东优尼德生物科技有限公司 | Sm fusion antigen and anti-Sm antibody chemiluminescence detection kit |
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