CN105891193A - Chemiluminescent immune detection kit for respiratory syncytial virus and preparation method thereof - Google Patents
Chemiluminescent immune detection kit for respiratory syncytial virus and preparation method thereof Download PDFInfo
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- CN105891193A CN105891193A CN201610512727.7A CN201610512727A CN105891193A CN 105891193 A CN105891193 A CN 105891193A CN 201610512727 A CN201610512727 A CN 201610512727A CN 105891193 A CN105891193 A CN 105891193A
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- syncytial virus
- respiratory syncytial
- immune detection
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- 241000725643 Respiratory syncytial virus Species 0.000 title claims abstract description 90
- 238000001514 detection method Methods 0.000 title claims abstract description 77
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000006249 magnetic particle Substances 0.000 claims abstract description 33
- 108060003951 Immunoglobulin Proteins 0.000 claims abstract description 21
- 102000018358 immunoglobulin Human genes 0.000 claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims description 43
- 241000700605 Viruses Species 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 27
- 108090000623 proteins and genes Proteins 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 20
- 229940072221 immunoglobulins Drugs 0.000 claims description 19
- 238000003018 immunoassay Methods 0.000 claims description 16
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 15
- 238000002372 labelling Methods 0.000 claims description 14
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 5
- 239000007983 Tris buffer Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 210000002345 respiratory system Anatomy 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- BZSVVCFHMVMYCR-UHFFFAOYSA-N 2-pyridin-2-ylpyridine;ruthenium Chemical compound [Ru].N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1 BZSVVCFHMVMYCR-UHFFFAOYSA-N 0.000 claims description 4
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- 238000002474 experimental method Methods 0.000 abstract description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract 1
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000011033 desalting Methods 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
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- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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- 206010061603 Respiratory syncytial virus infection Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
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- 238000007689 inspection Methods 0.000 description 2
- 238000000504 luminescence detection Methods 0.000 description 2
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- 102000004190 Enzymes Human genes 0.000 description 1
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- 241000287828 Gallus gallus Species 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
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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
- 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
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- Life Sciences & Earth Sciences (AREA)
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- Immunology (AREA)
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- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
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- Analytical Chemistry (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a chemiluminescent immune detection kit for a respiratory syncytial virus and a preparation method thereof. The chemiluminescent immune detection kit for the respiratory syncytial virus comprises a carboxylic magnetic particle and a chemiluminescent maker, wherein the carboxylic magnetic particle is coated by a respiratory syncytial virus recombinant protein, and the chemiluminescent maker is labeled by an anti-human immunoglobulin. The chemiluminescent immune detection kit for the respiratory syncytial virus can employ a full-automatic chemiluminescent immune analyzer as a detection tool to complete the detection of the respiratory syncytial virus; after an experiment, the chemiluminescent immune detection kit for the respiratory syncytial virus has a detection sensitivity as high as 1U/L, which is at least 10 times the sensitivity of a conventional respiratory syncytial virus detection method; and the chemiluminescent immune detection kit for the respiratory syncytial virus has higher detection accuracy.
Description
Technical field
The present invention relates to vitro detection field, particularly relate to a kind of respiratory syncytial virus chemiluminescence immunoassay inspection
Test agent box and preparation method thereof.
Background technology
Respiratory syncytial virus (RSV, is called for short syncytial virus, belongs to Paramyxoviridae) was accompanied first in 1956
It is separated in having the orangutan body of cold symptoms.To cause the modal cause of disease of infantile viral pneumonia, because of its
Cell can form the warm pathological changes of special cell in cultivating, therefore named.The infection of this virus can cause chromic fibrous lung
Inflammation, and bronchiolitis.
The at present detection of respiratory syncytial virus infection mainly has a following several method:
One, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but the method there is also following weak point:
(1) use 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate as antigen bag
By apparatus and reaction vessel, 12 batches, 6 batches, 8 batches or imposite can only be divided in use and once make
With, it is impossible to carry out independent, the detection of single part；
(2) reagent type used by quantitative determination is more, and each detectable will contain with reagent bottle,
And it is required for changing imbibition nozzle when often using a kind of reagent to be filled into respectively in the micropore of microwell plate, not only
Reagent bottle kind is many, and the operation of filling reagent is the most loaded down with trivial details；
(3) the corresponding mark to detection information is lacked, can only be by checking the mark ability of test kit external packing box
Understand or know product batch number and the effect duration information of detectable, and the information known is in detection process
In uncontrolled, there is the biggest randomness；
(4) detectable is in open space during detection, easily causes the intersection between various reagent
Pollute and affect the accuracy of testing result；
(5) detection process uses manual operations, and the dosage of reagent or sample is not bery accurate, and operating process is extremely
Loaded down with trivial details and complicated, it is susceptible to bust, accuracy and the precision of testing result are poor；
(6) in the quantity configuration and use of detection project reagent set, it is item number × 48/96 person-portion, if
Need to detect 10 projects, then configuration and the use number of reagent must be 10 × 48/96 person-portions, if only one
Part sample needs to detect 10 different projects, it is also desirable to the reagent of configuration 10 × 48/96 person-portions, also exists
The shortcoming of economical rationality not.
Two, Routine Test Lab detection isolation of virus
The goldstandard of laboratory diagnosis respiratory syncytial virus infection is the method for virus purification.Use specimen
Time is advisable with premorbid for 5 days, takes patient's nasopharynx cotton swab or coughs up phlegm and carry out virus purification cultivation.This virus
The cell strain such as Hela can not can only be cultivated propagation, about at people and MC such as Hep-2 in Embryo Gallus domesticus internal breeding
Cultivating the cytopathy just occurring that cell boundary line is unclear, be fused into multinucleated giant cell etc. 23 weeks, virus is passed through
Sprout release.But the method for virus purification has serious defect.Because they are cumbersome and time consuming, logical
Often need the long period just can obtain final result, so at clinicing aspect, the effectively treatment of patient is had certain
Limitation.
Three, gene diagnosis
For many RNA viruses, RT-PCR diagnostic method is than traditional virus purification and diagnostic antigen side
Method is the fastest but also sensitive.And RT-PCR can diagnose multiple virus, other diagnostic methods the most simultaneously
As virus purification and immunofluorescence analysis but can not.But the method also has weak point, such as round pcr,
Colleges and universities' property of DNA cloning result in the pollution of denier and both may occur in which false positive, thus makes result distortion.This
Outer virus is as the invader of outer protogene, it is necessary to when illustrating its all or part of nucleotide sequence, just may be used
With design primer or probe, carry out making nucleic acid molecular hybridization and PCR detection.
From the detection method of existing respiratory syncytial virus, EIA, virus purification, RT-PCR diagnose
Although method has an advantage of certain specificity and sensitivity, but operationally need professional and technical personnel,
Special instrument and equipment and specific condition and the shortcoming such as time-consuming.
Acridinium ester is compared above method as the direct chemiluminescence of label and is had detailed advantage, mainly shows
: reaction need not catalyst, as long as alkaline environment can be carried out, is swift in response, and background luminescence is low, letter
Ratio of making an uproar is high, and interference factor is few, and reagent stability is good, can be with two-point calibration, and system is simple, exciting liquid cost
Low, acridinium ester easily and after protein bind, and connection photon productivity do not reduce, have become as syncytial virus and examine
Disconnected new developing direction.
Summary of the invention
Based on this, it is necessary to provide the respiratory syncytial virus chemiluminescence immunoassay that a kind of detection sensitivity is higher
Detection kit and preparation method thereof.
A kind of respiratory syncytial virus chemiluminescence immune detection reagent kit, including: respiratory syncytial virus weight
The coated carboxylated magnetic particle of histone and the chemiluminescent labels of human immunoglobulins's labelling.
In the coated carboxylated magnetic particle of described respiratory syncytial virus recombiant protein, described respiratory syncystial
Virus recombiant protein is 1:25～35 with the ratio of described carboxylated magnetic particle.
In the chemiluminescent labels of described human immunoglobulins's labelling, described human immunoglobulins and institute
The ratio stating chemiluminescent labels is 50:1～10.
The particle diameter of described carboxylated magnetic particle is 0.05 μm～1 μm.
Described chemiluminescent labels is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
Described respiratory syncytial virus chemiluminescence immune detection reagent kit, also includes Chemoluminescent substrate,
Described Chemoluminescent substrate includes A liquid and B liquid.
Described A liquid is H2O2Solution, described B liquid is NaOH solution.
Described respiratory syncytial virus chemiluminescence immune detection reagent kit, also includes respiratory syncytial virus
Calibration product.
Described respiratory syncytial virus calibration product be concentration be respectively 1U/L, 10U/L, 100U/L, 500U/L,
The solution of the respiratory syncytial virus of 1000U/L and 2000U/L.
The preparation method of a kind of above-mentioned respiratory syncytial virus chemiluminescence immune detection reagent kit, including such as
Lower step:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, then adds
Enter EDC aqueous solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into respiratory syncytial virus weight
Histone, suspendible 2h～10h under room temperature, Magneto separate is resuspended with Tris buffer after removing supernatant, is breathed
The coated carboxylated magnetic particle of road syncytial virus recombiant protein；And
Take human immunoglobulins, mix after adding carbonate buffer solution, be subsequently adding chemiluminescent labels
Rear mixing, under room temperature, remove impurity after lucifuge reaction 1h～2h, obtains the chemiluminescence of human immunoglobulins's labelling
Label.
This respiratory syncytial virus chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunity
Analyser is detection instrument, completes the detection this respiratory syncytial virus chemiluminescence of respiratory syncytial virus
Immunity detection reagent, through experiment, its detection sensitivity reaches 1U/L, closes relative to traditional respiratory tract
The detection method sensitivity of cellular virus at least improves 10 times, this respiratory syncytial virus chemiluminescence immunoassay
The accuracy of detection of detection kit is higher.
Accompanying drawing explanation
Fig. 1 is the preparation method of the respiratory syncytial virus chemiluminescence immune detection reagent kit of an embodiment
Flow chart；
Fig. 2 is the respiratory syncytial virus canonical plotting that embodiment 3 obtains.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, below in conjunction with the accompanying drawings and
The detailed description of the invention of the present invention is described in detail by specific embodiment.Elaborate in the following description very
Many details are so that fully understanding the present invention.But the present invention can be described here to be much different from
Alternate manner is implemented, and those skilled in the art can do similar changing in the case of intension of the present invention
Entering, therefore the present invention is not limited by following public being embodied as.
The respiratory syncytial virus chemiluminescence immune detection reagent kit of one embodiment, including: respiratory tract closes
The coated carboxylated magnetic particle of cellular virus recombiant protein and the chemiluminescent labeling of human immunoglobulins's labelling
Thing.
Preferably, in the coated carboxylated magnetic particle of respiratory syncytial virus recombiant protein, respiratory syncystial
Virus recombiant protein is 1:25～35 with the ratio of carboxylated magnetic particle.
Preferably, in the chemiluminescent labels of human immunoglobulins's labelling, human immunoglobulins and change
The ratio learning luminous marker is 50:1～10.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm～1 μm.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein,
Chemiluminescent labels is preferably acridinium ester.
In other examples, above-mentioned respiratory syncytial virus chemiluminescence immune detection reagent kit also includes
Chemoluminescent substrate.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH
Solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be 0.25mol/L
NaOH solution.
In other examples, above-mentioned respiratory syncytial virus chemiluminescence immune detection reagent kit also includes
Respiratory syncytial virus calibration product.
Respiratory syncytial virus calibration product be concentration be respectively 1U/L, 10U/L, 100U/L, 500U/L,
The solution of the respiratory syncytial virus of 1000U/L and 2000U/L.
Concrete, respiratory syncytial virus calibration product can use standard substance buffer by respiratory syncytial virus
It is configured to exhaling of concentration respectively 1U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L
Inhale the solution of road syncytial virus.
This respiratory syncytial virus chemiluminescence immune detection reagent kit detects for respiratory syncytial virus
Time, utilize Full-automatic chemiluminescence immunoassay analysis meter that respiratory syncytial virus calibration product are detected, draw
Standard curve, is built in computer software；Then test actual sample, calculate sample according to sample luminous value dense
Degree；Finally respiratory syncytial virus automatic chemiluminescence immunoassay system is carried out performance (sensitivity,
Linearly, precision, interference) evaluation.
This respiratory syncytial virus chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunity
Analyser is detection instrument, completes the detection this respiratory syncytial virus chemiluminescence of respiratory syncytial virus
Immunity detection reagent, through experiment, its detection sensitivity reaches 1U/L, closes relative to traditional respiratory tract
The detection method sensitivity of cellular virus at least improves 10 times, this respiratory syncytial virus chemiluminescence immunoassay
The accuracy of detection of detection kit is higher.
Additionally, this respiratory syncytial virus chemiluminescence immune detection reagent kit also has the advantage that
1, selection acridinium ester is as marker material, and is applied to chemiluminescence immunoassay system, this luminous body
System is direct chemiluminescence, and compared with traditional enzyme-catalyzed chemical luminescence, this reaction need not the participation of enzyme, more
Add cost-effective；
2, select acridinium ester chemiluminescence immunoassay system range of linearity width, can reach 1U/L～
1000U/L, and the inspection range of linearity of the detection method of traditional respiratory syncytial virus be 20U/L～
1000U/L；
3, acridinium ester chemiluminescent immunoassay system repeatability is high, in batch and difference between batch is all within 5%,
This is that other chemiluminescence immunoassay system is unapproachable；
4, chemiluminescence immunoassay system has realized the quantitative of sample, soft to test by built-in standard curve
Part, only needs test sample just can directly obtain the concentration value of sample；
5, chemiluminescence immunoassay system can realize the interpolation of full-automation, reagent and sample instrument entirely
Complete, operate easier, decrease artificial error.
The preparation method of above-mentioned respiratory syncytial virus chemiluminescence immune detection reagent kit as shown in Figure 1,
Comprise the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, then adds
Enter EDC aqueous solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into respiratory syncytial virus weight
Histone, suspendible 2h～10h under room temperature, Magneto separate is resuspended with Tris buffer after removing supernatant, is breathed
The coated carboxylated magnetic particle of road syncytial virus recombiant protein.
The concentration of MES (2-(N-morpholine) ethyl sulfonic acid) buffer be 0.02M, pH be 5.5.
The concentration of Tris buffer is 0.1M and is 8.0 containing 2%BSA, pH.
The concentration of EDC (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides) aqueous solution is
10mg/mL～20mg/mL, EDC are 0.05:0.1～1 with the ratio of carboxylated magnetic particle.
Preferably, in the coated carboxylated magnetic particle of respiratory syncytial virus recombiant protein, respiratory syncystial
Virus recombiant protein is 1:25～35 with the ratio of carboxylated magnetic particle.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm～1 μm.
Take human immunoglobulins, mix after adding carbonate buffer solution, be subsequently adding chemiluminescent labels
Rear mixing, under room temperature, remove impurity after lucifuge reaction 1h～2h, obtains the chemiluminescence of human immunoglobulins's labelling
Label.
Carbonate buffer solution concentration be 0.1M, pH be 9.0～9.5,
The operation of remove impurity is centrifugal desalting column desalination, and concrete operations are: the most respectively by pure water and TBS buffering
Liquid (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0) processes centrifugal desalting column, finally adds
Enter the solution of the coated carboxylated magnetic particle of the respiratory syncytial virus recombiant protein obtained, finally collect from
Liquid in heart pipe.
Preferably, in the chemiluminescent labels of human immunoglobulins's labelling, respiratory syncytial virus is recombinated
Albumen is 50:1～10 with the ratio of chemiluminescent labels.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein,
Chemiluminescent labels is preferably acridinium ester.
The coated carboxylated magnetic particle of respiratory syncytial virus recombiant protein obtained and human immunoglobulins
The chemiluminescent labels combination of labelling i.e. can get the detection examination of above-mentioned respiratory syncytial virus chemiluminescence immunoassay
Agent box.
This respiratory syncytial virus chemiluminescence immune detection reagent kit is in use, in addition it is also necessary to chemiluminescence
Substrate solution and respiratory syncytial virus calibration product.
Chemoluminescent substrate and respiratory syncytial virus calibration product can be prepared voluntarily and obtain.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH
Solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be 0.25mol/L
NaOH solution.
Concrete, respiratory syncytial virus calibration product can use standard substance buffer by respiratory syncytial virus
It is configured to exhaling of concentration respectively 1U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L
Inhale the solution of road syncytial virus.
The preparation method of this respiratory syncytial virus chemiluminescence immune detection reagent kit is simple and convenient, prepares
The detection sensitivity of respiratory syncytial virus chemiluminescence immune detection reagent kit higher, have good should
Use prospect.
It it is below specific embodiment.
Embodiment 1: the preparation of respiratory syncytial virus chemiluminescence immune detection reagent kit
(1) preparation of the coated carboxylated magnetic particle of respiratory syncytial virus recombiant protein:
Take containing carboxylated magnetic particle (MagnaBindTM, the article No. that 50mg particle diameter is 0.05 μm～1 μm
21353) suspension, Magneto separate removes supernatant, is that 5.5MES buffer is resuspended with 0.02M, pH, adds
The EDC aqueous solution of the 10mg/mL that 1mL is newly configured, activated magnetic beads surface carboxyl groups, add 4mg respiratory tract
Syncytial virus recombiant protein (biorbyt, article No. orb48780), suspendible 6h under room temperature, Magneto separate, in removal
Clearly, it is resuspended to 1mg/mL with the Tris buffer that the 0.1M containing 2%BSA, pH are 8.0, is breathed
The coated carboxylated magnetic particle of road syncytial virus recombiant protein, every bottle of 5mL subpackage be stored in 4 DEG C standby.
(2) preparation of the acridinium ester of human immunoglobulins's labelling:
Taking 50 μ L concentration is the human immunoglobulins of 25mg/mL, and adding 150 μ L concentration is 0.1M, pH
It is the carbonate buffer solution of 9.0～9.5, mixing, it is subsequently adding the acridinium ester that 1.5 μ L concentration are 5mg/mL molten
Liquid mixes, lucifuge reaction under room temperature, takes out, be centrifuged desalting column desalting processing with the zeba of 2mL after 1.5h,
Desalination processes processes with pure water and TBS buffer the most respectively, is eventually adding obtain anti-human and exempts from
The acridinium ester solution of epidemic disease globulin labelling, the liquid collected in centrifuge tube is in control anti-human immune globulin to preservation
The acridinium ester of white marker, every bottle of 5mL subpackage be stored in 4 DEG C standby.
(3) preparation of respiratory syncytial virus calibration product:
To breathe with standard substance buffer (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0)
It is 0U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L that road syncytial virus is configured to concentration,
Every bottle of 0.5mL subpackage lyophilizing, 4 DEG C save backup.
Embodiment 2: respiratory syncytial virus chemical luminous immune detection method
It is detection instrument with Full-automatic chemiluminescence immunoassay analysis meter (YHLO, article No. iFlash3000), side
Science of law pattern is the respiratory syncystial disease that indirect immunization, i.e. instrument are sequentially added into the sample of 50 μ L, 50 μ L
The poison coated carboxylated magnetic particle of recombiant protein and the respiratory syncytial virus treatment fluid of 50 μ L, reaction
After 20min, then add human immunoglobulins's acridinium ester of 50 μ L, after reaction 20min, carry out Magneto separate,
Reactant mixture is sent into darkroom by instrument, is sequentially added into luminous substrate A liquid (H2O2) and B liquid (NaOH)
Carry out luminescence-producing reaction, finally record luminous value.
Embodiment 3: respiratory syncytial virus chemiluminescence immune detection reagent kit performance evaluation
Use the method in embodiment 2 that respiratory syncytial virus calibration product are detected, obtain drafting standard
Curve is as shown in Figure 2.
Then to then testing actual sample, concentration of specimens is calculated according to sample luminous value.
The detection of sensitivity:
With reference to CLSI EP17-A file recommendation experimental program, calculate respiratory syncytial virus chemiluminescence immunoassay
The sensitivity of detection kit, the sensitivity tried to achieve is 1U/L.
Linear detection:
It is that 1U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L standard substance do to concentration
Linear analysis, calculates linearly dependent coefficient, and r=0.9996, it addition, this test kit is to respiratory syncytial virus sample
The range of linearity that product examine is surveyed is 1U/L～1000U/L.
Precision measures:
Taking concentration is 50U/L and two respiratory syncytial virus samples of 500U/L, and each concentration of each sample is each
Do 3 parallel, detect with three batches of test kits, calculate test kit criticize interior and difference between batch, result shows this
Test kit is criticized interior and difference between batch and is respectively less than 5%.
Interference is tested:
Take pooled serum to add chaff interference respectively and include: conjugated bilirubin, unconjugated bilirubin, hemoglobin,
Ascorbic acid, glyceride, adding proportion is carried out according to 1:20, measures pooled serum respectively and with the addition of each
After kind chaff interference, the measured value of pooled serum, calculates deviation therebetween, with ± 10% as tolerance interval.Knot
Fruit shows, interference all reaches the file standard of NCCLS, can be used for clinical laboratory's respiratory syncystial sick
The accurate evaluation of poison situation.
Embodiment 4, the contrast experiment of respiratory syncytial virus chemiluminescence immune detection reagent kit
Be 0 by chemical luminescence detection method and traditional enzyme linked immunosorbent assay to concentration respectively, 50U/L exhales
Inhaling road syncytial virus sample to detect, two kinds of method detection sensitivities are compared, and data are as shown in the table:
As can be seen from the above table, the sensitivity of chemical luminescence detection method relatively enzyme linked immunosorbent assay improves 10
More than Bei.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed,
But therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for this area
Those of ordinary skill for, without departing from the inventive concept of the premise, it is also possible to make some deformation and
Improving, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended
Claim is as the criterion.
Claims (10)
1. a respiratory syncytial virus chemiluminescence immune detection reagent kit, it is characterised in that including: exhale
The chemistry inhaling the road coated carboxylated magnetic particle of syncytial virus recombiant protein and human immunoglobulins's labelling is sent out
Signal thing.
Respiratory syncytial virus chemiluminescence immune detection reagent kit the most according to claim 1, it is special
Levy and be, in the coated carboxylated magnetic particle of described respiratory syncytial virus recombiant protein, described respiratory tract
Syncytial virus recombiant protein is 1:25～35 with the ratio of described carboxylated magnetic particle.
Respiratory syncytial virus chemiluminescence immune detection reagent kit the most according to claim 1, it is special
Levy and be, in the chemiluminescent labels of described human immunoglobulins's labelling, described respiratory syncytial virus
Recombiant protein is 50:1～10 with the ratio of described chemiluminescent labels.
Respiratory syncytial virus chemiluminescence immune detection reagent kit the most according to claim 1, it is special
Levying and be, the particle diameter of described carboxylated magnetic particle is 0.05 μm～1 μm.
Respiratory syncytial virus chemiluminescence immune detection reagent kit the most according to claim 1, it is special
Levying and be, described chemiluminescent labels is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
Respiratory syncytial virus chemiluminescence immune detection reagent kit the most according to claim 1, it is special
Levy and be, also include that Chemoluminescent substrate, described Chemoluminescent substrate include A liquid and B liquid.
Respiratory syncytial virus chemiluminescence immune detection reagent kit the most according to claim 6, it is special
Levying and be, described A liquid is H2O2Solution, described B liquid is NaOH solution.
Respiratory syncytial virus chemiluminescence immune detection reagent kit the most according to claim 1, it is special
Levy and be, also include that respiratory syncytial virus calibrates product.
Respiratory syncytial virus chemiluminescence immune detection reagent kit the most according to claim 8, it is special
Levy and be, described respiratory syncytial virus calibration product be concentration be respectively 1U/L, 10U/L, 100U/L,
The solution of the respiratory syncytial virus of 500U/L, 1000U/L and 2000U/L.
10. one kind according to the respiratory syncytial virus chemiluminescence immunoassay according to any one of claim 1～9
The preparation method of detection kit, it is characterised in that comprise the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, then adds
Enter EDC aqueous solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into respiratory syncytial virus weight
Histone, suspendible 2h～10h under room temperature, Magneto separate is resuspended with Tris buffer after removing supernatant, is breathed
The coated carboxylated magnetic particle of road syncytial virus recombiant protein；And
Take human immunoglobulins, mix after adding carbonate buffer solution, be subsequently adding chemiluminescent labels
Rear mixing, under room temperature, remove impurity after lucifuge reaction 1h～2h, obtains the chemiluminescence of human immunoglobulins's labelling
Label.
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| CN201610512727.7A CN105891193A (en) | 2016-06-30 | 2016-06-30 | Chemiluminescent immune detection kit for respiratory syncytial virus and preparation method thereof |
| PCT/CN2017/080405 WO2018000901A1 (en) | 2016-06-30 | 2017-04-13 | Respiratory syncytial virus chemiluminescence immunoassay kit and preparation method thereof |
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| CN108169475A (en) * | 2017-12-18 | 2018-06-15 | 郑州安图生物工程股份有限公司 | A kind of Respiratory Syncytial Virus(RSV) IgM antibody detection kit |
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| WO2018000901A1 (en) | 2018-01-04 |
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Application publication date: 20160824 |