CN108344869A - A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application - Google Patents
A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application Download PDFInfo
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- CN108344869A CN108344869A CN201810110258.5A CN201810110258A CN108344869A CN 108344869 A CN108344869 A CN 108344869A CN 201810110258 A CN201810110258 A CN 201810110258A CN 108344869 A CN108344869 A CN 108344869A
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
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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
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
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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
- G01N33/68—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
Abstract
The present invention relates to a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application, the kit includes：It is indirectly connected with the magnetic particle reagents of AntiHBsAg antibody 1,2 solution of HBsAg antibody of chemiluminescent labels label,Chemoluminescent substrate,Cleaning solution,The AntiHBsAg antibody 1 includes at least the immune response fragment of two kinds of antibody or antibody,And including at least a kind of anti-S protein monoclonal antibody 1,The AntiHBsAg antibody 2 includes at least the immune response fragment of two kinds of antibody or antibody,And including at least a kind of anti-S protein monoclonal antibody 2,The epitope for the HBsAg that the anti-S protein monoclonal antibody 1 is directed to anti-S protein monoclonal antibody 2 is different,The kit of the present invention still belongs to the first in the world,There is highly sensitive and accuracy when detecting HBsAg,Greatly improve the Detection capability of HBsAg mutant strains,Significantly improve clinical blood transfusion,The safety of operation etc..
Description
Technical field
The present invention relates to a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its applications, belong to vitro detection
Technical field.
Background technology
Hepatitis B surface antigen (HBsAg) is the serologic marker object occurred first after hepatitis B (HBV) infects, is clinical
The most HBV infection blood serum designated object of upper application, is the key index of the generally acknowledged judgement HBV infections of WHO.The hbv replication period is fast
And polymerase fallibility is matched and the extensive use of hepatitis B vaccine, antiviral therapy, HBsAg is mutated in chronic HBV infection patient
It is very common.HBsAg mutation can cause patient's HBsAg testing results to be negative, but there are still HBV in its serum or liver
DNA, i.e., invisible HBV infection (OBI).If OBI patient is as blood donor or liver transfer operation donor, it will to peace of transfusing blood and perform the operation
It is complete to constitute major hidden danger.Meanwhile HBsAg mutation can accelerate occult hepatitis B to be in progress to hepatic sclerosis or liver cancer, and cause and exempt from
Epidemic disease inhibit after HBV reactivations, lead to detection reagent missing inspection, vaccine protection failure, antiviral therapy unsuccessfully etc., to progression of disease
Great influence is caused with curative effect judgement.Therefore, how research improves the Detection capability to HBsAg mutant, is of great significance.
Kit currently used for HBsAg screenings mainly has enzyme linked immunological kit, chemiluminescence immunoassay kit etc., in
State's blood station blood screening generally uses enzyme linked immunological kit, but ELISA reagent in the market generally takes monoclonal antibody
The pattern of capture plus monoclonal antibody detection, since binding site is single, if the site upper surface antigen in combination occurs to dash forward
Become, it will cause antigens to decline or even cannot combine to the binding force of corresponding antibodies, be easy to cause missing inspection；Chemoluminescence method has
The large-scale Grade A hospital of very high sensitivity and specificity, American-European blood station and hospital and China mostly uses chemiluminescence immunoassay reagent
Box, and enzyme linked immunological kit is just gradually substituted, become mainstream, advanced Reagent Company such as Abbott Laboratories and Roche Holding Ag are in the world
The detectability for improving mutant strain upgrades chemiluminescence immunoassay reagent, and reagent captures antibody by two kinds of monoclonals substantially
Clone's detection antibody to be added to constitute, the epitope that polyclonal antibody is directed to is more, even if some site of antigen mutates, more grams
Grand antibody still can be combined with other epitopes of mutant antigen, and capture antibody and be made of two kinds of monoclonal antibodies, to mutant strain
Detection capability it is preferable, but use currently on the market two kinds capture antibody are both for S protein, and the HBsAg of broad sense
It is made of three kinds of albumen：Major surface protein (S protein), hepatitis b large surface antigen protein are (by S1, hepatitis B before hepatitis B surface antigen
Tri- kinds of albumen of S2 and hepatitis B surface antigen S, which connect, before surface antigen folds), hepatitis b surface antigen protein is (by hepatitis B table
Two kinds of albumen of S2 and hepatitis B surface antigen S, which connect, before the antigen of face folds), if some mutation of HBV lead to S protein not table
It reaches, S protein, hepatitis b large surface antigen protein and hepatitis b surface antigen protein then cannot be captured antibody capture, cause
The mutant strain of hepatitis B surface antigen cannot effectively be detected.
Invention content
The technical problem to be solved by the present invention is to：To solve available reagent box to hepatitis B surface antigen mutant strain Detection capability
Relatively low technical problem provides a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit, including：It is indirectly connected with the magnetic of AntiHBsAg antibody 1
Particle reagents, 2 solution of HBsAg antibody of chemiluminescent labels label, Chemoluminescent substrate, cleaning solution, the anti-HBsAg
Antibody 1 includes at least the immune response fragment of two kinds of antibody or antibody, and includes at least a kind of anti-S protein monoclonal antibody 1, institute
AntiHBsAg antibody 2 is stated including at least two kinds of antibody or the immune response fragment of antibody, and includes at least a kind of anti-S protein Dan Ke
The epitope of grand antibody 2, the HBsAg that the anti-S protein monoclonal antibody 1 is directed to anti-S protein monoclonal antibody 2 is different.
Preferably, the AntiHBsAg antibody 1 is 1 by molar ratio:The anti-S protein monoclonal antibody 1 of 0.2-1 and polyclonal
Monoclonal antibody segment form, or by molar ratio be 1:The anti-S protein monoclonal antibody 1 and anti-pre-s2 protein monoclonal antibody of 0.2-1
1 composition, or by molar ratio be 1:0.2-1:The anti-S protein monoclonal antibody 1 of 0.2-1, anti-pre-s2 protein monoclonal antibody 1 and more
Clonal antibody Fab segments form.
Preferably, the AntiHBsAg antibody 2 is 1 by molar ratio:The anti-S protein monoclonal antibody 2 of 0.5-2 and polyclonal
Monoclonal antibody segment form, or by molar ratio be 1:The anti-S protein monoclonal antibody 2 and anti-pre-s2 protein monoclonal antibody of 0.2-1
2 composition, or by molar ratio be 1:0.2-1:The anti-S protein monoclonal antibody 1 of 0.5-2, anti-pre-s2 protein monoclonal antibody 2 and more
What clonal antibody Fab segments composition, the anti-pre-s2 protein monoclonal antibody 1 and anti-pre-s2 protein monoclonal antibody 2 were directed to
The epitope of HBsAg is different.
Preferably, the polyclonal antibody Fab segments are close by G-protein by polyclonal antibody specificity Fab digestion products
With the double purification of column and HBsAg antigen affinity columns and obtain, for diagnostic reagent specificity it is good.
Specifically, the polyclonal antibody Fab segments are prepared and purified by following methods：Polyclonal antibody and specificity
Fab processive enzymes by volume 1:0.5-1 reacts 12-24 hours in Tris-HCl buffer systems, obtains digestion products；Digestion is produced
Object crosses G-protein affinity column and collects outflow albumen P1, after PBS buffer solution fully balances G-protein affinity column, with HCl- glycine elutions
Albumen P2 is obtained, albumen P2 collects outflow albumen P3 by HBsAg antigen affinity columns, and PBS buffer solution fully balances HBsAg antigen parents
After column, albumen P4 is obtained with HCl- glycine elutions, albumen P4 adjusts pH to 7.5-8.0 with Tris-HCl, and dialysing in PBS is
It can.
Preferably, the magnetic particle for being indirectly connected with AntiHBsAg antibody 1 is by the coated magnetic particle of Streptavidin and label
The AntiHBsAg antibody 1 of biotin forms, or glimmering by the coated magnetic particle of anti-fluorescein isothiocynate antibody and label isothiocyanic acid
The AntiHBsAg antibody 1 of light element forms.
Preferably, the coated magnetic particle of Streptavidin is nano level Fe2O3Or Fe304Magnetic particle and organic high score
The compound of sub- material, the high-molecular organic material are preferably glucan；The particle size of the magnetic particle is 0.1-5 μm；
The magnetic particle can also by surface modification by carry one or more activity functional groups, the activity functional groups be-
OH ,-COOH or-NH2。
Preferably, the chemiluminescent labels are selected from luminol and its derivative, different luminol and its derivative, acridine
Ester or acridine sulfonamide.
Preferably, the Chemoluminescent substrate includes chemiluminescence exciting liquid 1 and chemiluminescence exciting liquid 2, describedization
It learns the exciting liquid 1 that shines and contains inorganic acid and peroxide, the chemiluminescence exciting liquid 2 contains hydroxide and tritonx-100.
The present invention also provides the applications of above-mentioned hepatitis B surface antigen chemiluminescence immune detection reagent kit, according to the following steps
It uses：
Sample to be tested is reacted with the magnetic particle reagents for being indirectly connected with AntiHBsAg antibody 1, it is multiple to form magnetic Ag-Ab
Close object suspension；
The magnetic antigen-antibody complex suspension is placed in magnetic field, it is compound to wash the magnetic Ag-Ab
Object；
The AntiHBsAg antibody 2 of magnetic antigen-antibody complex and chemiluminescent labels label after the washing is anti-
Ying Hou forms magnetic composite suspension；
The magnetic composite suspension is placed in magnetic field, the magnetic composite is washed；
Chemiluminescence exciting liquid 1 is first added into the magnetic composite after washing, adds chemiluminescence exciting liquid 2, examines
Survey chemiluminescence photon intensity.
The beneficial effects of the invention are as follows：
The hepatitis B surface antigen chemiluminescence immune detection reagent kit of the present invention, AntiHBsAg antibody 1 and AntiHBsAg antibody 2
It is the immune response fragment including at least two kinds of antibody or antibody, and includes at least a kind of anti-S protein monoclonal antibody, if by
When anti-S protein monoclonal antibody 1 and anti-pre-s2 protein monoclonal antibody form, S protein caused by being mutated can be effectively prevent not
Missing inspection caused by when can express；If be made of anti-S protein monoclonal antibody 1 and polyclonal antibody Fab segments, Anti-TNF-α
Body Fab segments can be directed to multiple epitopes of HBsAg, while special area of the Fab segments as polyclonal antibody, especially pass through
The polyclonal antibody Fab segments of the double purification of G-protein affinity column and HBsAg antigen affinity columns greatly can avoid to degree more
The false positive that clonal antibody cross reaction is brought, the anti-S protein monoclonal antibody 1 being used in mixed way have high specific and height affine
Power so that with high sensitivity when kit detects HBsAg；If by anti-S protein monoclonal antibody 1, anti-pre-s2 protein list
The immune response fragment of clonal antibody and polyclonal antibody forms, then can further increase the sensitive of kit detection HBsAg
Degree and accuracy；This kit still belongs to the first in the world, and the detection energy of HBsAg mutant strains is greatly improved using the kit
Power significantly improves the safety of clinical blood transfusion, operation etc..
Specific implementation mode
The present invention is described in further detail now.
Embodiment 1
The present embodiment provides a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kits, including：It is indirectly connected with anti-
The magnetic particle reagents of HBsAg antibody 1,2 solution of HBsAg antibody of chemiluminescent labels label, Chemoluminescent substrate, cleaning
Liquid；
The AntiHBsAg antibody 1 is 1 by molar ratio:0.2 anti-S protein monoclonal antibody 1 and polyclonal antibody Fab pieces
Disconnected composition；
The AntiHBsAg antibody 2 is 1 by molar ratio:2 anti-S protein monoclonal antibody 2 and polyclonal antibody Fab segments
Composition；
The polyclonal antibody Fab segments are prepared and purified by following methods：Polyclonal antibody and specificity Fab processive enzymes
By volume 1:0.5 reacts 24 hours in 0.2M, the Tris-HCl buffer systems that pH is 7.2 at 37 DEG C, obtains digestion products；It will
Digestion products cross G-protein affinity column and collect outflow albumen P1, and with 0.2M, the PBS buffer solution that pH is 7.2 fully balances G-protein parent
After column, with 0.1M, the HCl- glycine elutions that pH is 2.7 obtain albumen P2；Albumen P2 is directly received by HBsAg antigen affinity columns
Collection flows out albumen P3, is with 0.1M, pH after the PBS buffer solution that pH is 7.2 fully balances HBsAg antigen affinity columns with 0.2M
2.7 HCl- glycine elutions obtain albumen P4, and albumen P4 1M, the Tris-HCl that pH is 8.0 adjust pH to 7.5-8.0, in
It dialyses in the PBS buffer solution that 0.2M, pH are 7.2；
The chemiluminescent labels are luminol；
The magnetic particle for being indirectly connected with AntiHBsAg antibody 1 is by the coated magnetic particle of Streptavidin and label biotin
AntiHBsAg antibody 1 form；The coated magnetic particle of Streptavidin is nano level Fe2O3Magnetic particle and glucan
The particle size of compound, the magnetic particle is 0.1-5 μm；
The Chemoluminescent substrate includes chemiluminescence exciting liquid 1 and chemiluminescence exciting liquid 2, and the chemiluminescence swashs
1 nitric acid containing 0.1M of lotion and 1g/100mL hydrogen peroxide, 2 sodium hydroxide containing 0.25M of chemiluminescence exciting liquid and 0.1g/
100tritonx-100。
Embodiment 2
The present embodiment provides a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit, the kit with embodiment 1
It is essentially identical, the difference is that：
The AntiHBsAg antibody 1 is 1 by molar ratio:1 anti-S protein monoclonal antibody 1 and polyclonal antibody Fab segments
Composition；
The AntiHBsAg antibody 2 is 1 by molar ratio:0.5 anti-S protein monoclonal antibody 2 and polyclonal antibody Fab pieces
Disconnected composition；
The polyclonal antibody Fab segments are prepared and purified by following methods：Polyclonal antibody and specificity Fab processive enzymes
By volume 1:1 reacts 12 hours in 0.2M, the Tris-HCl buffer systems that pH is 7.2 at 37 DEG C, obtains digestion products；By enzyme
It cuts product and crosses G-protein affinity column and collect and flow out albumen P1, with 0.2M, it is affine that the PBS buffer solution that pH is 7.2 fully balances G-protein
After column, with 0.1M, the HCl- glycine elutions that pH is 2.7 obtain albumen P2；Albumen P2 is directly collected by HBsAg antigen affinity columns
Albumen P3 is flowed out, with 0.2M, after PBS buffer solution that pH is 7.2 fully balances HBsAg antigen affinity columns, with 0.1M, pH 2.7
HCl- glycine elutions obtain albumen P4, albumen P4 1M, the Tris-HCl that pH is 8.0 adjust pH to 7.5-8.0, in 0.2M,
It dialyses in the PBS buffer solution that pH is 7.2；
The chemiluminescent labels are different luminol；
The magnetic particle for being indirectly connected with AntiHBsAg antibody 1 by the coated magnetic particle of anti-fluorescein isothiocynate antibody and
The AntiHBsAg antibody 1 of fluorescein isothiocynate is marked to form；The coated magnetic particle of anti-fluorescein isothiocynate antibody is to receive
The Fe of meter level3O4The particle size of the compound of magnetic particle and glucan, the magnetic particle is 0.1-5 μm.
Embodiment 3
The present embodiment provides a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit, the kit with embodiment 1
It is essentially identical, the difference is that：
The AntiHBsAg antibody 1 is 1 by molar ratio:0.2:1 anti-S protein monoclonal antibody 1, anti-pre-s2 protein Dan Ke
Grand antibody and polyclonal antibody Fab segments composition；
The AntiHBsAg antibody 2 is 1 by molar ratio:0.2:2 anti-S protein monoclonal antibody 1, anti-pre-s2 protein Dan Ke
Grand antibody and polyclonal antibody Fab segments composition；
The polyclonal antibody Fab segments are prepared and purified by following methods：Polyclonal antibody and specificity Fab processive enzymes
By volume 1:1 reacts 12 hours in 0.2M, the Tris-HCl buffer systems that pH is 7.2 at 37 DEG C, obtains digestion products；By enzyme
It cuts product and crosses G-protein affinity column and collect and flow out albumen P1, with 0.2M, it is affine that the PBS buffer solution that pH is 7.2 fully balances G-protein
After column, with 0.1M, the HCl- glycine elutions that pH is 2.7 obtain albumen P2；Albumen P2 is directly collected by HBsAg antigen affinity columns
Albumen P3 is flowed out, with 0.2M, after PBS buffer solution that pH is 7.2 fully balances HBsAg antigen affinity columns, with 0.1M, pH 2.7
HCl- glycine elutions obtain albumen P4, albumen P4 1M, the Tris-HCl that pH is 8.0 adjust pH to 7.5-8.0, in 0.2M,
It dialyses in the PBS buffer solution that pH is 7.2；
The chemiluminescent labels are acridinium ester.
Embodiment 4
The present embodiment provides a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit, the kit with embodiment 3
It is essentially identical, the difference is that：
The AntiHBsAg antibody 1 is 1 by molar ratio:1:0.2 anti-S protein monoclonal antibody 1, anti-pre-s2 protein Dan Ke
Grand antibody and polyclonal antibody Fab segments composition；
The AntiHBsAg antibody 2 is 1 by molar ratio:1 anti-S protein monoclonal antibody 2 and anti-pre-s2 protein monoclonal are anti-
Body forms.
Embodiment 5
The present embodiment provides a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit, the kit with embodiment 3
It is essentially identical, the difference is that：
The AntiHBsAg antibody 1 is 1 by molar ratio:1 anti-S protein monoclonal antibody 1 and anti-pre-s2 protein monoclonal are anti-
Body forms；
The AntiHBsAg antibody 2 is 1 by molar ratio:0.2 anti-S protein monoclonal antibody 2 and anti-pre-s2 protein monoclonal
Antibody forms.
Embodiment 6
The present embodiment provides a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit, the kit with embodiment 3
It is essentially identical, the difference is that：
The AntiHBsAg antibody 1 is 1 by molar ratio:0.2 anti-S protein monoclonal antibody 1 and anti-pre-s2 protein monoclonal
Antibody forms；
The AntiHBsAg antibody 2 is 1 by molar ratio:1:0.5 anti-S protein monoclonal antibody 1, anti-pre-s2 protein Dan Ke
Grand antibody and polyclonal antibody Fab segments composition.
Effect example 1
(it is respectively abbott using the kit of embodiment 1-5, Roche and the import of Abbott Laboratories chemiluminescence immunoassay kit
II screening assay of architect HBsAg quantitative assay and ROCHE Elecsys HBsAg) and
Ying Kexin creates (Chinese medicines quasi-word S10910148), section magnificent (Chinese medicines quasi-word S10910113), ten thousand safe (Chinese medicines quasi-word S10980090) three
The natural mutator-strain (the results are shown in Table 1) of the enzyme linked immunological kit detection HBsAg of domestic manufacturer of family mainstream,
The step of detecting natural mutator-strain using the kit of embodiment 1-5 is as follows：
By the magnetic particle reagents for being indirectly connected with AntiHBsAg antibody 1 of 50 μ L samples to be tested and 120 a concentration of 500ng/mL of μ L
Reaction after ten minutes, forms magnetic antigen-antibody complex suspension；
The magnetic antigen-antibody complex suspension is placed in magnetic field, it is compound to wash the magnetic Ag-Ab
Object；
By the anti-of the acridinium ester label of magnetic antigen-antibody complex and 100 μ L concentration 500ng/mL after the washing
HBsAg antibody 2 reacts after ten minutes, forms magnetic composite suspension；
The magnetic composite suspension is placed in magnetic field, the magnetic composite is washed；
100 μ L chemiluminescences exciting liquids are injected into the magnetic composite after 1,1.5 seconds, injection 100 μ L chemistry hair
Light exciting liquid 2 detects its chemiluminescence photon intensity；
By the chemiluminescence photon intensity compared with Cutoff values, if simple chemical shines, photon intensity is more than cutoff
Value, is judged to the positive, if simple chemical shines, photon intensity is less than cutoff values, is judged to feminine gender, the Cutoff values be 500
Average value+the 2SD of negative sample luminous value.
The step of detecting the natural mutator-strain of HBsAg using the import chemiluminescence immunoassay kit of Roche is as follows：
By 50 μ L samples to be tested, each 50 μ L of 2 kinds of biotinylated anti-hbs monoclonal antibodies and 100 μ L ruthenium compounds
The anti-S protein monoclonal antibody and more anti-reflective of label answer 10min, form sandwich complex；
20 μ L Streptavidin MagneSpheres are added and react 10min with the sandwich complex, forms magnetic composite and suspends
Liquid；
The magnetic composite suspension is placed in magnetic field, the magnetic composite is washed；
100 μ L chemiluminescences exciting liquids are injected into the magnetic composite after 1,1.5 seconds, injection 100 μ L chemistry hair
Light exciting liquid 2 detects its chemiluminescence photon intensity；
By the chemiluminescence photon intensity compared with Cutoff values, if simple chemical shines, photon intensity is more than cutoff
Value, is judged to the positive, if simple chemical shines, photon intensity is less than cutoff values, is judged to feminine gender, the Cutoff values be 500
Average value+the 2SD of negative sample luminous value.
The step of detecting the natural mutator-strain of HBsAg using the import chemiluminescence immunoassay kit of Abbott Laboratories is as follows：
After ten minutes by 50 μ L samples to be tested and the sub- reagent reaction of 120 μ L Paramagnetic particles, it is compound to form magnetic Ag-Ab
Object suspension；
The magnetic antigen-antibody complex suspension is placed in magnetic field, it is compound to wash the magnetic Ag-Ab
Object；
Magnetic antigen-antibody complex after the washing is reacted 10 with the AntiHBsAg antibody of 100 μ L acridinium ester labels
After minute, magnetic composite suspension is formed；
The magnetic composite suspension is placed in magnetic field, the magnetic composite is washed；
100 μ L chemiluminescences exciting liquids are injected into the magnetic composite after 1,1.5 seconds, injection 100 μ L chemistry hair
Light exciting liquid 2 detects its chemiluminescence photon intensity；
By the chemiluminescence photon intensity compared with Cutoff values, if simple chemical shines, photon intensity is more than cutoff
Value, is judged to the positive, if simple chemical shines, photon intensity is less than cutoff values, is judged to feminine gender, the Cutoff values be 500
Average value+the 2SD of negative sample luminous value.
The natural mutation of HBsAg is detected using the enzyme linked immunological kit of Ying Kexin wounds, China of section, Wan Tai domestic manufacturers mainstream
The step of strain, is as follows：
Concentrated cleaning solution distilled water or deionized water are pressed 1:24 are diluted to work cleaning solution；
In each 50 μ L samples to be tested of Kong Zhongjia of reaction plate, 50 μ L of enzyme conjugates are added, after mixing well, cover sealing compound
Paper is set 37 DEG C of water baths and is incubated 30 minutes；
Taking-up has been incubated the reaction plate finished, and board-washing 5 times pats dry；
Color developing agent A liquid, each 50 μ L of B liquid are added per hole, after gently shaking mixing, covers cellophane tape, sets 37 DEG C and is protected from light and incubate
It educates 15 minutes；
50 microlitres of terminate liquid is added per hole；
It is read with microplate reader, takes wavelength 450nm, read each hole OD values；
By the OD values compared with Cutoff values, if OD values are more than cutoff values, it is judged to the positive, if OD values are less than cutoff
Value, is judged to feminine gender, the Cutoff values are for the average value+2SD of 500 negative sample luminous values；
Testing result of the different kits of table 1 to natural mutator-strain
Note：12 plants of HBsAg natural mutator-strains purchase of the effect example 1 from the Germany laboratories PEI, is chosen to examine
Hepatitis B surface antigen diagnostic reagent all over the world is to the Detection capability of mutant strain.
By table 1 as it can be seen that three kinds of domestic mainstream enzyme linked immunological kits to the recall rate of the natural mutator-strain of above-mentioned HBsAg compared with
It is low, and the enzyme linked immunological kit of each producer is different to the Detection capability of different mutant strains；In comparison, two imports
International mainstream chemiluminescence immunoassay kit is higher than domestic enzyme linked immunological kit to the Detection capability of mutant strain, but still has certain
Missing inspection；And the recall rate of the natural mutator-strain of 12 plants of HBsAg of chemiluminescence immunoassay kit pair of this patent is 100%,
Detectability is extremely strong.
Effect example 2
Since 12 plants of natural mutator-strains that effect example 1 uses only represent the important mutation of HBsAg, but can not react completely
Go out kit to the Detection capability of actual clinical case, then this effect example has screened 500 clinically HBsAg mutation rates
The positive blood sample and 500 healthy human bodies of high patients with chronic liver (including chronic hepatitis, hepatic sclerosis, hepatocarcinoma patient)
Negative blood sample, by the detection method of effect example 1 investigate respectively the kit of embodiment 1-6, Ying Kexin wound, section China,
The enzyme linked immunological kit and Roche of ten thousand safe 3 domestic manufacturer's mainstreams, the import chemiluminescence immunoassay kit of Abbott Laboratories are to blood
The recall rate of HBsAg in liquid sample, as a result such as table 2.
Testing result of the different kits of table 2 to clinical blood sample
By table 3 as it can be seen that in terms of the testing result to 500 positive blood samples, the international mainstream chemistry hair of two imports
Light immune reagent kit is higher than domestic enzyme linked immunological kit to the Detection capability of positive clinical blood sample, but is below this patent
The Detection capability of the chemiluminescence immunoassay kit of embodiment 1-6；In terms of the testing result to 500 positive blood samples, this
The probability that the kit false positive results of patent occur is extremely low.As it can be seen that the HBsAg detection kits of this patent have high sensitivity
And accuracy.
It is enlightenment with above-mentioned desirable embodiment according to the present invention, through the above description, relevant staff is complete
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention
Property range is not limited to the contents of the specification, it is necessary to determine its technical scope according to right.
Claims (10)
1. a kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit, including：It is indirectly connected with the magnetic of AntiHBsAg antibody 1
Grain reagent, 2 solution of HBsAg antibody of chemiluminescent labels label, Chemoluminescent substrate, cleaning solution, which is characterized in that institute
AntiHBsAg antibody 1 is stated including at least two kinds of antibody or the immune response fragment of antibody, and includes at least a kind of anti-S protein Dan Ke
Grand antibody 1, the AntiHBsAg antibody 2 include at least the immune response fragment of two kinds of antibody or antibody, and including at least a kind of anti-
S protein monoclonal antibody 2, the epitope for the HBsAg that the anti-S protein monoclonal antibody 1 and anti-S protein monoclonal antibody 2 are directed to
It is different.
2. hepatitis B surface antigen chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that described anti-
HBsAg antibody 1 is 1 by molar ratio:The anti-S protein monoclonal antibody 1 and polyclonal antibody Fab segments of 0.2-1 forms, or by rubbing
You are than being 1:The anti-S protein monoclonal antibody 1 of 0.2-1 and anti-pre-s2 protein monoclonal antibody 1 composition, or by molar ratio be 1:
0.2-1:Anti- S protein monoclonal antibody 1, anti-pre-s2 protein monoclonal antibody 1 and the polyclonal antibody Fab segments composition of 0.2-1.
3. hepatitis B surface antigen chemiluminescence immune detection reagent kit according to claim 1, which is characterized in that described anti-
HBsAg antibody 2 is 1 by molar ratio:The anti-S protein monoclonal antibody 2 and polyclonal antibody Fab segments of 0.5-2 forms, or by rubbing
You are than being 1:The anti-S protein monoclonal antibody 2 of 0.2-1 and anti-pre-s2 protein monoclonal antibody 2 composition, or by molar ratio be 1:
0.2-1:Anti- S protein monoclonal antibody 1, anti-pre-s2 protein monoclonal antibody 2 and the polyclonal antibody Fab segments composition of 0.5-2,
The epitope for the HBsAg that the anti-pre-s2 protein monoclonal antibody 1 is directed to anti-pre-s2 protein monoclonal antibody 2 is different.
4. hepatitis B surface antigen chemiluminescence immune detection reagent kit according to claim 2 or 3, which is characterized in that institute
Polyclonal antibody Fab segments are stated by polyclonal antibody specificity Fab digestion products by G-protein affinity column and HBsAg antigen parents
It is obtained with the double purification of column.
5. hepatitis B surface antigen chemiluminescence immune detection reagent kit according to claim 4, which is characterized in that described more
Clonal antibody Fab segments are prepared and purified by following methods：Polyclonal antibody and specificity Fab processive enzymes by volume 1:0.5-
1 reacts 12-24 hours in Tris-HCl buffer systems, obtains digestion products；Digestion products are crossed into G-protein affinity column and collect outflow
Albumen P1 after PBS buffer solution fully balances G-protein affinity column, obtains albumen P2, albumen P2 passes through with HCl- glycine elutions
HBsAg antigen affinity columns collect outflow albumen P3, after PBS buffer solution fully balances HBsAg antigen affinity columns, with HCl- glycine
Albumen P4 is eluted to obtain, albumen P4 adjusts pH to 7.5-8.0 with Tris-HCl, dialyses in PBS.
6. according to claim 1-5 any one of them hepatitis B surface antigen chemiluminescence immune detection reagent kits, feature exists
In the magnetic particle for being indirectly connected with AntiHBsAg antibody 1 is by the coated magnetic particle of Streptavidin and marks the anti-of biotin
HBsAg antibody 1 forms, or by the coated magnetic particle of anti-fluorescein isothiocynate antibody and marks the anti-of fluorescein isothiocynate
HBsAg antibody 1 forms.
7. according to claim 1-6 any one of them hepatitis B surface antigen chemiluminescence immune detection reagent kits, feature exists
In the magnetic particle is nano level Fe2O3Or Fe304The compound of magnetic particle and high-molecular organic material, the magnetic particle
Particle size be 0.1-5 μm, and by surface modification by carry one or more activity functional groups, the active function group
Group is-OH ,-COOH or-NH2。
8. according to claim 1-7 any one of them hepatitis B surface antigen chemiluminescence immune detection reagent kits, feature exists
In the chemiluminescent labels are selected from luminol and its derivative, different luminol and its derivative, acridinium ester or acridine sulphonyl
Amine.
9. according to claim 1-8 any one of them hepatitis B surface antigen chemiluminescence immune detection reagent kits, feature exists
In the Chemoluminescent substrate includes chemiluminescence exciting liquid 1 and chemiluminescence exciting liquid 2, the chemiluminescence exciting liquid 1
Containing inorganic acid and peroxide, the chemiluminescence exciting liquid 2 contains hydroxide and tritonx-100.
10. a kind of application of claim 1-9 any one of them hepatitis B surface antigen chemiluminescence immune detection reagent kit,
It is characterized in that, uses according to the following steps：
Sample to be tested is reacted with the magnetic particle reagents for being indirectly connected with AntiHBsAg antibody 1, forms magnetic antigen-antibody complex
Suspension；
The magnetic antigen-antibody complex suspension is placed in magnetic field, the magnetic antigen-antibody complex is washed；
Magnetic antigen-antibody complex after the washing is reacted with the AntiHBsAg antibody 2 that chemiluminescent labels mark
Afterwards, magnetic composite suspension is formed；
The magnetic composite suspension is placed in magnetic field, the magnetic composite is washed；
Chemiluminescence exciting liquid 1 is first added into the magnetic composite after washing, adds chemiluminescence exciting liquid 2, detectionization
Learn the photon intensity that shines.
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| CN201810110258.5A CN108344869B (en) | 2018-02-05 | 2018-02-05 | Hepatitis B surface antigen chemiluminescence immunoassay kit and application thereof |
| PCT/CN2018/104108 WO2019148836A1 (en) | 2018-02-05 | 2018-09-05 | Hepatitis b surface antigen chemiluminescence immunoassay kit and application thereof |
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|---|---|---|---|
| CN201810110258.5A CN108344869B (en) | 2018-02-05 | 2018-02-05 | Hepatitis B surface antigen chemiluminescence immunoassay kit and application thereof |
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| WO2019148836A1 (en) * | 2018-02-05 | 2019-08-08 | 苏州长光华医生物医学工程有限公司 | Hepatitis b surface antigen chemiluminescence immunoassay kit and application thereof |
| CN110146692A (en) * | 2019-05-28 | 2019-08-20 | 迪瑞医疗科技股份有限公司 | One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit |
| CN111707827A (en) * | 2020-06-28 | 2020-09-25 | 博奥赛斯（天津）生物科技有限公司 | Hepatitis B surface antigen detection kit |
| WO2021134299A1 (en) * | 2019-12-30 | 2021-07-08 | 深圳迈瑞生物医疗电子股份有限公司 | Immunoassay analyzer selectively testing different substances to be tested, method, and kit |
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| CN113960313A (en) * | 2021-12-22 | 2022-01-21 | 上海思路迪医学检验所有限公司 | Exosome ALK fusion protein magnetic immunochemiluminescence detection kit |
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| Publication number | Publication date |
|---|---|
| CN108344869B (en) | 2020-07-03 |
| WO2019148836A1 (en) | 2019-08-08 |
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