CN107037207A - A kind of c-hepatitis antibody polymer enzyme marker and its preparation and application - Google Patents

A kind of c-hepatitis antibody polymer enzyme marker and its preparation and application Download PDF

Info

Publication number
CN107037207A
CN107037207A CN201710467575.8A CN201710467575A CN107037207A CN 107037207 A CN107037207 A CN 107037207A CN 201710467575 A CN201710467575 A CN 201710467575A CN 107037207 A CN107037207 A CN 107037207A
Authority
CN
China
Prior art keywords
polymer
hepatitis antibody
room temperature
polymer enzyme
enzyme marker
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201710467575.8A
Other languages
Chinese (zh)
Other versions
CN107037207B (en
Inventor
盖中涛
欧兰香
汪运山
王露楠
朱之炜
江长林
丁兴龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong Lab Biological Science & Technology Co Ltd
Original Assignee
Shandong Lab Biological Science & Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shandong Lab Biological Science & Technology Co Ltd filed Critical Shandong Lab Biological Science & Technology Co Ltd
Priority to CN201710467575.8A priority Critical patent/CN107037207B/en
Publication of CN107037207A publication Critical patent/CN107037207A/en
Application granted granted Critical
Publication of CN107037207B publication Critical patent/CN107037207B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a kind of c-hepatitis antibody polymer enzyme marker, it is to be used as polymeric carrier using Dextran T 500, polymer enzyme marker (Poly HRP) is cross-linked into after being activated through hydrazine hydrochloride with HRP, sulfydryl modification c-hepatitis antibody is used simultaneously, then the c-hepatitis antibody after sulfydryl modification and polymer enzyme marker (Poly HRP) glue are joined into composition.The invention also discloses the polymer enzyme stabilizers and dilution that the supporting c-hepatitis antibody polymer enzyme marker is used.Experiment is confirmed, the c-hepatitis antibody polymer enzyme marker of the present invention is added to stablize after aggressiveness enzyme stabilizers and preserved 2 years, and it is used for enzyme linked immunosorbent detection after polymer enzyme diluted and chemiluminescence immunoassay detection is greatly improved than the enzyme marker sensitivity that traditional Over-voltage protection or glutaraldehyde method are marked.

Description

A kind of c-hepatitis antibody polymer enzyme marker and its preparation and application
Technical field
The invention belongs to Immunoenzyme techniques field, and in particular to a kind of c-hepatitis antibody polymer enzyme marker and its preparation side Method and application.
Technical background
Detection HCV (Hepatitis C virus, HCV) cAg can intuitively reflect HCV's Infection state, and shorten the detection window phase of HCV infection.But after HCV infection human body, the content of its cAg in blood It is very low, it need to be detected using highly sensitive method.
In EIA enzyme immunoassay detection, the sensitivity and specificity of antigen or abzyme label to immunoassay have weight The effect wanted, is the key reagents in EIA enzyme immunoassay detection.Horseradish peroxidase (Horseradish Peroxidase, HRP) specific activity is high, stable, and molecular weight is small, and pure enzyme is easily prepared, and is the preparing raw material of the most frequently used albumen enzyme marker now. Traditional horseradish peroxidase labeling antibody, is as crosslinking using the hydroxyl on enzyme and antibody on amino, sulfydryl or glycoprotein Group, antibody is directly linked together with enzyme by sodium periodate or glutaraldehyde method.Because molecular size is different, general one 1~2 HRP molecule of connection is only capable of on individual antibody molecule.Immune response is catalyzed substrate to show by HRP.Using polymer as Carrier links together antibody with HRP, due to the extension of polymer, an antibody can be made to connect more than 10 HRP molecules, often 1 antibody can be catalyzed substrate system by the HRP for 10 times of quantity being connected with antigen-reactive signal and embody, therefore, detection Sensitivity is greatly improved.Dako companies are in the patent of invention (US005543332A 1996) of U. S. application, and its key technology is to use Water soluble polymer dextrose falls glycosides and makees polymeric carrier molecule, and multiple bivinyl sulfone monomers are connected thereto with covalent bond, freely Vinyl can combine the labeled thing such as antigen, haptens, antibody, nucleotides of functional group；Chinese patent (patent Application number 03150859.2) a kind of new immunoglobulin and horseradish peroxidase crosslinking technological are disclosed, its crucial skill Art is also based on water miscible polymeric carrier molecule --- dextran, covalently tied by vinyl with multiple bivinyl sulfone monomers It is combined, then " molecule " of the free vinyl of another end then with functional group is reacted, so that by different marks Sub be combined together with labeled molecule of scoring completes molecular labeling.But the vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan) used in the two patents, with compared with Strong toxicity, stimulates eyes, respiratory system and skin.Chinese patent (number of patent application 200710168647.5) discloses one kind The preparation method of polyase marker antibody, its key technology is in the presence of nonionic surfactant, to make soluble horseradish mistake Oxide enzyme (HRP) micellization, with NaIO4First the glycan molecule on decoration HRP surfaces is oxidized to aldehyde radical, then utilizes the ammonia on HRP With aldehyde radical condensation reaction generation polymer (Poly-HRP), but its undisclosed concrete application method occur for base.Through retrieval, sulfydryl is used Modified antibodies, the antibody after modification joins with the glue of polymer enzyme marker (Poly-HRP) and made after purification again after being exposed through azanol A kind of standby document of c-hepatitis antibody polymer enzyme marker has not been reported with product.
The content of the invention
In view of the shortcomings of the prior art, problem to be solved by this invention is to provide a kind of c-hepatitis antibody polymer enzyme mark Thing and preparation method and application.
C-hepatitis antibody polymer enzyme marker of the present invention, it is characterised in that：It is by the hepatitis after sulfydryl modification Antibody joins with polymer enzyme marker (Poly-HRP) glue to be constituted；Wherein, the c-hepatitis antibody after the sulfydryl modification is with diformazan Base formamide (DMF) is N- succinimides S- acetylthio-acetates salt (SATA) solution that solvent compound concentration is 1mg/ml, Take 200ul and add 1ml it is to be coupled using 0.1M PBS be solvent prepare concentration as 5mg/ml, pH7.2 c-hepatitis antibody, room Temperature reaction is made for 1 ± 0.2 hour；The polymer enzyme marker is using Dextran T 500 as polymeric carrier, through hydrazine hydrochloride It is made after activation with horseradish peroxidase (HRP) crosslinking.
The c-hepatitis antibody polymer enzyme marker prepared for permanently effective preservation, the invention also discloses one kind is supporting The polymer enzyme stabilizers that above-mentioned c-hepatitis antibody polymer enzyme marker is used, it is characterised in that：The polymer enzyme stabilizers It is, using the c-hepatitis antibody polymer enzyme marker prepared as matrix plus bovine serum albumin(BSA) (BSA), to make its final concentration of 10mg/ Ml, plus kathon, make its final concentration of 10ul/ml, plus iodo-acetamide, make its whole mass concentration for a ten thousandth, glycerol adding, Its volume ratio is set to be made for 1/3rd.It is experimentally confirmed that the c-hepatitis antibody polymer enzyme marker prepared, adds this supporting enzyme steady Determine after agent, can steadily in the long term at least 2 years.
The invention also discloses the dilution that a kind of supporting above-mentioned c-hepatitis antibody polymer enzyme marker is used, its feature exists In：The dilution be using pH7.2,0.1M PBS as matrix, with volume basis, add 20% NBCS, ten thousand/ Five Procin300, with weight by volume basis, add 5/10000ths aminopyrine, 5/1000ths glucan T2000,2% Sucrose, 1% glycine, 1% BSA be made.
The preparation method of c-hepatitis antibody polymer enzyme marker of the present invention, step is：
(1) activation of polymer：Using Dextran T 500 as polymeric carrier, activated through hydrazine hydrochloride
1) with following proportional quantities, 25mg Dextran T 500s are weighed up, in the PBS for being dissolved in 0.5ml 0.1M, pH7.2；
2) 0.5ml 0.1M NaIO are added4, lucifuge reaction 2 ± 0.2 hours at room temperature；
3) desalination is carried out with 0.1M, pH7.2 PBS with PD-10 desalting columns, collects the eluate containing glucan, plus 0.6g Hydrazine hydrochloride, uses 2M NaHCO3PH to 9.0 is adjusted, reaction is gently agitated at room temperature 2 ± 0.2 hours；
4) add 5mg trimethylamine boranes compound (TMAB) again, react at room temperature 2 ± 0.2 hours；
5) desalination is carried out with 0.1M, pH7.2 PBS with PD-10 desalting columns, collects the liquid of first peak, in 2~8 DEG C of guarantors Deposit standby；
(2) preparation and purifying of polymer enzyme marker (Poly-HRP)
1) with following proportional quantities, claim 25mg horseradish peroxidases (HRP), be dissolved in 1ml distilled waters, then add 0.15ml0.1M NaIO4, react at room temperature 15 ± 2min；
2) desalination is carried out with 0.1M, pH7.2 PBS with PD-10 desalting columns, collects HRP eluate, and and step (1) The liquid of collection is mixed, and is reacted at room temperature 2 ± 0.2 hours；
3) 8mg gauge is added by every milliliter of reaction solution, TMAB is added, reacted at room temperature 2 ± 0.2 hours；
4) Glycine crystals are added, it is 0.2M, mistake after reacting at room temperature 2 ± 0.2 hours to make its ultimate density Superdex200 posts, are eluted with 0.1M, pH7.2 PBS, collect the liquid of first peak, are corrected with pipe concentration is concentrated by ultrafiltration For 3ml, as purified Poly-HRP, saved backup in 2~4 DEG C；
(3) sulfydryl modification c-hepatitis antibody
Using DMF be solvent compound concentration as 1mg/ml N- succinimides S- acetylthio-acetates salt (SATA) solution, By following proportional quantities, take 200ul and add 1ml it is to be coupled using 0.1M PBS be the concentration prepared of solvent as 5mg/ml, pH7.2 C-hepatitis antibody, react at room temperature 1 ± 0.2 hour, that is, obtain sulfydryl modification c-hepatitis antibody；
(4) sulfydryl modification c-hepatitis antibody and the glue connection of polymer enzyme marker and purifying
1) using DMF be solvent compound concentration as 1mg/ml N- [ε-maleimide acetyl group oxygen] thiosuccimide Ester (EMCS) solution, polymer enzyme marker (Poly-HRP) made from equal volume amounts addition step (2), room temperature reaction 2 ± 0.2 hour, that is, obtain the poly-HRP of EMCS activation；
2) by sulfydryl modification c-hepatitis antibody made from step (3), the 0.5M of equal volume amounts hydroxylamine hydrochloride, room temperature are added React after 15 ± 2min, desalination is carried out with 0.1M, pH7.2 PBS with PD-10 desalting columns, collect the liquid of first peak, and press etc. Volume is added to step 1) in the poly-HRP that have activated of EMCS that obtain, room temperature reaction 2 ± 0.2 hours；
3) it is by every milliliter of load responsive fluid addition 300ul percent weight in volume concentration prepared by solvent of distilled water 2% 2-MEA, reacts at room temperature 15 ± 2min；
4) again by step 3) every milliliter of load responsive fluid add 150ul using DMF be the concentration prepared of solvent as 80mg/ml N- Superdex200 posts are crossed after ethyl maleimide (NEM) solution, 15 ± 2min of room temperature reaction, are entered with 0.1M, pH7.2 PBS Row elution, collects the liquid of first peak, is corrected to 8ml with pipe concentration is concentrated by ultrafiltration, that is, purified c-hepatitis antibody poly is made Body enzyme marker, being placed in 2~8 DEG C can preserve more than 4 weeks.
C-hepatitis antibody polymer enzyme marker of the present invention is preparing the cAg enzyme linked immunological of HCV Application in detection kit or chemiluminescence detection kit.
The present invention is cross-linked into polymer enzyme mark using Dextran T 500 as polymeric carrier with HRP after being activated through hydrazine hydrochloride Note thing (Poly-HRP) is simultaneously purified；Sulfydryl modification c-hepatitis antibody, the c-hepatitis antibody after modification exposed through azanol after again with polymer The glue connection of enzyme marker and purifying obtain c-hepatitis antibody polymer enzyme marker.If adding polymer enzyme stabilizers, make preparation C-hepatitis antibody polymer enzyme marker can for a long time (more than 2 years) effectively preserve.When it is applied, polymer enzyme dilution is used Carry out 1:2000 to 1:It is used for enzyme linked immunological etc., chemiluminescence immunoassay inspection after 3000 dilutions, it is higher by 3 than common enzyme marker sensitivity To 7 times.
Specifically, the application method of the c-hepatitis antibody polymer enzyme marker is：With the c-hepatitis antibody poly prepared Body enzyme marker is that matrix adds the supporting polymer enzyme stabilizers that it is used, then is preferably carried out with the supporting dilution that it is used 1:The cAg enzyme linked immunosorbent detection or chemiluminescence detection of HCV are can be used to after 2000 ± 200 dilutions.
The beneficial effect that the present invention is embodied is：
Present invention contrast prior art has innovative point：
1. using Dextran T 500 as polymeric carrier, polymer enzyme marker is cross-linked into after being activated through hydrazine hydrochloride with HRP (Poly-HRP)。
2. use sulfydryl modification c-hepatitis antibody, the c-hepatitis antibody after modification exposed through azanol after again with polymer enzyme marker Glue joins.
3. have developed the enzyme stabilizers of polymer, enable the c-hepatitis antibody polymer enzyme marker of preparation is permanently effective to protect Deposit.
4. have developed polymer enzyme dilution, make the c-hepatitis antibody polymer enzyme marker of preparation after the diluted Sensitivity is higher, and background value is lower.
Present invention contrast prior art has remarkable advantage：
Significantly carried with c-hepatitis antibody polymer enzyme labelling technique and polymer enzyme stabilizers, the combination of polymer enzyme dilution The sensitivity of high c-hepatitis antibody polymer enzyme marker and stability, the background value for greatly reducing detection, make specificity more It is good.
Embodiment
In order to be better understood from the present invention, technical scheme is described further with reference to embodiment, but Institute's protection domain of the present invention is not limited only to this.
Embodiment 1：
The polymer enzyme markers step of c-hepatitis antibody is as follows：
The first step：The activation of polymer：Using Dextran T 500 as polymeric carrier, activated through hydrazine hydrochloride
1) 25mg Dextran T 500s are weighed up, are dissolved in 1 × PBS of 0.5ml (0.1M PBS, pH7.2)；
2) 0.5ml 0.1M NaIO are added4, lucifuge reaction 2 hours at room temperature；
3) desalination is carried out with 0.1M PBS (pH7.2) with PD-10 desalting columns, collects the eluate containing glucan, plus 0.6g Hydrazine hydrochloride, uses 2M NaHCO3PH to 9.0 is adjusted, reaction 2 hours is gently agitated at room temperature；
4) add 5mg trimethylamine boranes compound (TMAB), react at room temperature 2 hours；
5) desalination is carried out with 0.1M PBS (pH7.2) with PD-10 desalting columns, collects the liquid of first peak, in 2~8 DEG C of guarantors Deposit standby；
Second step：The crosslinking of polymer and horseradish peroxidase (HRP), i.e. polymer enzyme marker (Poly-HRP) Prepare and purify：
1) 25mg HRP are claimed, 1ml is in the distilled water newly prepared for dissolving, plus 0.15ml0.1M NaIO4, room temperature reaction 15min；
2) desalination is carried out with 0.1M PBS (pH7.2) with PD-10 desalting columns, collects HRP eluate, collected with the first step Liquid mixed, react at room temperature 2 hours；
3) 8mg gauge is added by every milliliter of reaction solution, TMAB is added, reacted at room temperature 2 hours；
4) Glycine crystals are added, ultimate density is 0.2M, room temperature reaction crosses Superdex200 posts after 2 hours, with 0.01M PBS are eluted, and collect the liquid of first peak, and 3ml is corrected to pipe concentration is concentrated by ultrafiltration, as purified Poly-HRP, is saved backup in 2~4 DEG C.
3rd step：The modification of c-hepatitis antibody, c-hepatitis antibody and the glue connection of polymer enzyme marker and purifying：
1) sulfydryl modification c-hepatitis antibody：Claim 1mg N- succinimide S- acetylthio-acetates salt (SATA), with 1ml DMF Dissolving, takes 200ul SATA dopes to add 1ml c-hepatitis antibody (concentration is 5mg/ml, 0.1M PBS, pH7.2) room temperatures to be coupled Reaction 1 hour；
2) 1mg N- [ε-maleimide acetyl group oxygen] thiosuccimide ester (EMCS) is weighed, with 1ml dimethyl Formamide (DMF) dissolves, and adds polymer enzyme marker (Poly-HRP) made from equal volume amounts second step, and room temperature reaction 2 is small When, that is, obtain the poly-HRP of EMCS activation；
3) by step 1) c-hepatitis antibody modified, plus 1ml 0.5M hydroxylamine hydrochloride, after room temperature reaction 15min, with PD- 10 desalting columns carry out desalination with 0.1M PBS (pH7.2), collect first peak and simultaneously add through step 2) poly- that have activated of EMCS In HRP, react at room temperature 2 hours；
4) 2-MEA (being prepared with distilled water, now with the current) that 300ul percents weight in volume concentration is 2% is added, React at room temperature 15min；
5) 150ul NEMs (NEM, is dissolved with DMF, and concentration is 80mg/ml) are added, room temperature is anti- Answer and Superdex200 posts are crossed after 15min, eluted with 0.01M PBS, collect the liquid of first peak, pipe is dense with being concentrated by ultrafiltration Contracting is corrected to 8ml, and the polymer enzyme marker of as purified c-hepatitis antibody can be preserved more than 4 weeks in 2~8 DEG C.
Embodiment 2：
1st, the preparation for the polymer enzyme stabilizers that supporting c-hepatitis antibody polymer enzyme marker of the present invention is used and make With：
The polymer enzyme marker of the c-hepatitis antibody prepared for permanently effective preservation, can be in the c-hepatitis antibody prepared Polymer enzyme marker in plus BSA (making its final concentration of 10mg/ml), plus kathon (final concentration 10ul/ml), plus iodo second Acid amides (final concentration of a ten thousandth), plus three/mono- volume glycerine.
The polymer enzyme marker of preparation, is added after this enzyme stabilizers, can steadily in the long term at least 2 years.Related monitoring result It can be seen that embodiment 3.
2nd, the diluent preparation method that supporting c-hepatitis antibody polymer enzyme marker of the present invention is used is as follows：
With the NBCS of volume basis addition 20% in 0.1M PBS (pH7.2), 5/10000ths Procin300, with weight by volume basis, 1%BSA, add 5/10000ths aminopyrine, 5/1000ths glucan T2000, 2% sucrose, 1% glycine.
With above-mentioned polymer enzyme thin liquid to being not added with the progress of the c-hepatitis antibody polymer enzyme marker of polymer enzyme stabilizers 1:High 3 times or so of the common enzyme marker sensitivity of 3000 thinner ratios, has added polymer enzyme stabilizers with the enzyme thin liquid pair of polymer C-hepatitis antibody polymer enzyme marker carry out 1:High 5 times or so of the common enzyme marker sensitivity of 2000 thinner ratios.Correlative measurement Test result is shown in embodiment 3.
Embodiment 3：
C-hepatitis antibody polymer enzyme marker prepared by embodiment 1 than normal enzyme mark mode (Over-voltage protection) with marking C-hepatitis antibody enzyme marker compare have higher sensitivity, its use and verification method it is as follows：
With the enzyme dilution in embodiment 2 to be not added with enzyme stabilizers by embodiment 1 mark c-hepatitis antibody polymer The carry out 1 of enzyme marker:3000 dilutions, pair polymer enzyme marker for having added the c-hepatitis antibody of enzyme stabilizers in embodiment 2 is entered Row 1:2000 dilutions, with the c-hepatitis antibody enzyme marker that mark than normal enzyme mark mode (Over-voltage protection) in enzyme linked immunological inspection Contrasting detection is carried out in test agent box, its OD value is recorded, P averages/N averages is calculated, the results are shown in Table 1.
Enzyme dilution used, 1 in A, normal enzyme mark, original reagent box:500 dilutions
B, the inventive method mark polymer enzyme marker, are not added with polymer enzyme stabilizers, used in original reagent box Enzyme dilution, 1:1000 dilutions
C, the inventive method mark polymer enzyme marker, are not added with polymer enzyme stabilizers, with the poly under the present invention Body enzyme diluted, 1:3000
D, the inventive method mark polymer enzyme marker, plus the polymer enzyme stabilizers under the present invention, with the present invention Under polymer enzyme diluted, 1:2000
Table 1：The contrast that hepatitis polymer enzyme marker difference application method is marked with hepatitis normal enzyme
Through experimental tests, with the dilution in identical original reagent box, B group extension rates are 2 times of A groups, Detection results It is also 2.28 times of A groups, illustrates the enzyme mark of the common Over-voltage protection mark of hepatitis polymer enzyme marker remolding sensitivity of invention Remember that thing sensitivity is higher.
C group extension rates are 6 times of A groups, and Detection results are 3.33 times of A groups on the contrary, and D groups are that it has added polymer enzyme steady Determine agent, so its extension rate is lower than C by 1/3rd, but still be the dilution times of A groups because effective enzyme content in unit volume is reduced Several 4 times, Detection results are 5.22 times of A groups.Illustrate to employ the polymer enzyme dilution in the present invention, can further improve Sensitive bottom, reduces background value.
Further contrast is not added with polymer enzyme stabilizers with adding the stablizing effect of the polymer enzyme stabilizers in the present invention, right It is not added with the polymer enzyme diluted of the polymer enzyme stabilizers present invention of the present invention, 1:Supervised after 3000 dilutions Survey, pair added the c-hepatitis antibody polymer enzyme marker of the polymer enzyme stabilizers of the present invention, respectively at 0 month, March, June, 12 The moon, 15 months, 18 months, 21 months, 24 months, with the polymer enzyme diluted of the present invention, 1:It is monitored, contrasts after 2000 dilutions Polymer enzyme stabilizers effect steady in a long-term.It the results are shown in Table 2, table 3.
Table 2：The stablizing effect monitoring of aggressiveness enzyme stabilizers is not added
Table 3：Polymer enzyme stabilizers stablizing effect is monitored
P1 P2 P3 P4 P5 N1 N2 N3 N4 N5 P averages/N averages
0 month 0.243 0.489 0.446 0.711 0.899 0.025 0.033 0.031 0.030 0.020 19.89
March 0.223 0.447 0.495 0.699 0.831 0.022 0.030 0.030 0.030 0.020 20.41
June 0.244 0.469 0.486 0.699 0.879 0.029 0.031 0.033 0.030 0.023 19.02
December 0.251 0.467 0.466 0.719 0.892 0.025 0.031 0.030 0.030 0.022 20.25
15 months 0.253 0.488 0.456 0.742 0.890 0.027 0.032 0.031 0.030 0.021 20.06
18 months 0.229 0.502 0.471 0.721 0.901 0.026 0.030 0.033 0.029 0.024 19.89
21 months 0.247 0.488 0.487 0.702 0.893 0.023 0.033 0.034 0.030 0.025 19.42
24 months 0.232 0.491 0.476 0.721 0.889 0.029 0.033 0.032 0.029 0.022 19.37
Test result indicates that, be not added with polymer enzyme stabilizers after 1 month its detection sensitivity be greatly reduced.Plus the present invention Polymer enzyme stabilizers, after 2 years testing result with the 0th month when indistinction, the experiment after 24 months is not monitored, but can be demonstrate,proved Bright plus the present invention polymer enzyme stabilizers can make the c-hepatitis antibody polymer enzyme marker stablizing effect at least 24 of the present invention Month.
Embodiment 4：
The c-hepatitis antibody polymer enzyme marker of the present invention is in hepatitis c core antigen detection kit (chemical hair method) On application and measure of merit：
After the steady agent of polymer enzyme of the c-hepatitis antibody polymer enzyme marker present invention of the present invention, then it is dilute with polymer enzyme Release liquid and carry out 1:The enzyme marker for the c-hepatitis antibody that 2000 dilutions are marked with normal enzyme mark mode (Over-voltage protection) is in chemistry Contrasted on luminescent detection system：It the results are shown in Table 4.
Enzyme dilution used, 1 in A, normal enzyme mark, original reagent box:500 dilutions.
The polymer enzyme marker that D, the inventive method mark are obtained, plus polymer enzyme stabilizers of the invention, then with originally The polymer enzyme diluted 1 of invention:2000.
Table 4：Application of the present invention on chemiluminescence detection system is compared
S/CO P1 P2 P3 P4 P5 N1 N2 N3 N4 N5 P averages/N averages
A 1.78 2.39 12.92 13.56 14.23 0.72 0.81 0.82 0.82 0.86 11.13
D 2.59 4.79 34.96 37.65 38.67 0.27 0.32 0.31 0.33 0.21 82.40
Test result indicates that, c-hepatitis antibody polymer enzyme marker of the invention applied on chemiluminescence detection system than Common enzyme marker wants high more than 7 times.

Claims (6)

1. a kind of c-hepatitis antibody polymer enzyme marker, it is characterised in that：It is by the c-hepatitis antibody and poly after sulfydryl modification Body enzyme marker (Poly-HRP) glue connection is constituted；Wherein, the c-hepatitis antibody after the sulfydryl modification is with dimethylformamide (DMF) it is N- succinimides S- acetylthio-acetates salt (SATA) solution that solvent compound concentration is 1mg/ml, takes 200ul simultaneously Add 1ml it is to be coupled using 0.1M PBS be solvent prepare concentration as 5mg/ml, pH7.2 c-hepatitis antibody, room temperature reaction 1 ± It is made within 0.2 hour；The polymer enzyme marker be using Dextran T 500 as polymeric carrier, after being activated through hydrazine hydrochloride with it is peppery Root peroxidase (HRP) crosslinking is made.
2. the polymer enzyme stabilizers that c-hepatitis antibody polymer enzyme marker described in a kind of supporting claim 1 is used, its feature It is：The polymer enzyme stabilizers are using the c-hepatitis antibody polymer enzyme marker prepared as matrix plus bovine serum albumin(BSA) (BSA), make its final concentration of 10mg/ml, plus kathon, make its final concentration of 10ul/ml, plus iodo-acetamide, make its whole matter Amount concentration is a ten thousandth, and glycerol adding makes its volume ratio be made for 1/3rd.
3. the dilution that c-hepatitis antibody polymer enzyme marker described in a kind of supporting claim 1 is used, it is characterised in that：It is described Dilution is, using pH7.2,0.1M PBS as matrix, with volume basis, to add 20% NBCS, 5/10000ths Procin300, with weight by volume basis, the BSA of addition 1%, 5/10000ths aminopyrine, 5/1000ths glucan T2000,2% sucrose, 1% glycine are made.
4. the preparation method of c-hepatitis antibody polymer enzyme marker described in claim 1, step is：
(1) activation of polymer：Using Dextran T 500 as polymeric carrier, activated through hydrazine hydrochloride
1) with following proportional quantities, 25mg Dextran T 500s are weighed up, in the PBS for being dissolved in 0.5ml 0.1M, pH7.2；
2) 0.5ml 0.1M NaIO are added4, lucifuge reaction 2 ± 0.2 hours at room temperature；
3) desalination is carried out with 0.1M, pH7.2 PBS with PD-10 desalting columns, collects the eluate containing glucan, plus 0.6g hydrochloric acid Hydrazine, uses 2M NaHCO3PH to 9.0 is adjusted, reaction is gently agitated at room temperature 2 ± 0.2 hours；
4) add 5mg trimethylamine boranes compound (TMAB) again, react at room temperature 2 ± 0.2 hours；
5) desalination is carried out with 0.1M, pH7.2 PBS with PD-10 desalting columns, collects the liquid of first peak, it is standby in 2~8 DEG C of preservations With；
(2) preparation and purifying of polymer enzyme marker (Poly-HRP)
1) with following proportional quantities, claim 25mg horseradish peroxidases (HRP), be dissolved in 1ml distilled waters, then add 0.15ml0.1M NaIO4, react at room temperature 15 ± 2min；
2) desalination is carried out with 0.1M, pH7.2 PBS with PD-10 desalting columns, collects HRP eluate, and collect with step (1) Liquid mixed, react at room temperature 2 ± 0.2 hours；
3) 8mg gauge is added by every milliliter of reaction solution, TMAB is added, reacted at room temperature 2 ± 0.2 hours；
4) Glycine crystals are added, it is 0.2M to make its ultimate density, room temperature reaction crosses Superdex200 after 2 ± 0.2 hours Post, is eluted with 0.1M, pH7.2 PBS, collects the liquid of first peak, is corrected to 3ml with pipe concentration is concentrated by ultrafiltration, is Purified Poly-HRP, is saved backup in 2~4 DEG C；
(3) sulfydryl modification c-hepatitis antibody
Using DMF be solvent compound concentration as 1mg/ml N- succinimides S- acetylthio-acetates salt (SATA) solution, by such as Lower proportional quantities, take 200ul and add 1ml it is to be coupled using 0.1M PBS be solvent prepare concentration as the third of 5mg/ml, pH7.2 Liver antibody, reacts at room temperature 1 ± 0.2 hour, that is, obtains sulfydryl modification c-hepatitis antibody；
(4) sulfydryl modification c-hepatitis antibody and the glue connection of polymer enzyme marker and purifying
1) using DMF be solvent compound concentration as 1mg/ml N- [ε-maleimide acetyl group oxygen] thiosuccimide ester (EMCS) solution, equal volume amounts add polymer enzyme marker (Poly-HRP) made from step (2), room temperature reaction 2 ± 0.2 Hour, that is, obtain the poly-HRP of EMCS activation；
2) by sulfydryl modification c-hepatitis antibody made from step (3), the 0.5M of equal volume amounts hydroxylamine hydrochloride, room temperature reaction are added After 15 ± 2min, desalination is carried out with 0.1M, pH7.2 PBS with PD-10 desalting columns, the liquid of first peak is collected, and by isometric Amount is added to step 1) in the poly-HRP that have activated of EMCS that obtain, room temperature reaction 2 ± 0.2 hours；
3) it is 2% by every milliliter of load responsive fluid addition 300ul percent weight in volume concentration prepared by solvent of distilled water 2-MEA, reacts at room temperature 15 ± 2min；
4) again by step 3) every milliliter of load responsive fluid add 150ul using DMF be the concentration prepared of solvent as 80mg/ml N- ethyls Superdex200 posts are crossed after maleimide (NEM) solution, 15 ± 2min of room temperature reaction, are washed with 0.1M, pH7.2 PBS It is de-, the liquid of first peak is collected, 8ml is corrected to pipe concentration is concentrated by ultrafiltration, that is, purified c-hepatitis antibody polymer enzyme is made Label.
5. c-hepatitis antibody polymer enzyme marker described in claim 1 is preparing the cAg enzyme linked immunological of HCV Application in detection kit or chemiluminescence detection kit.
6. application according to claim 5, it is characterised in that the application method of the c-hepatitis antibody polymer enzyme marker It is：Polymer described in the supporting claim 2 that it is used is added by matrix of the c-hepatitis antibody polymer enzyme marker for preparing Enzyme stabilizers, then carry out 1 with dilution described in the supporting claim 3 that it is used:It is used for hepatitis C after 2000 ± 200 dilutions The cAg enzyme linked immunosorbent detection or chemiluminescence detection of virus.
CN201710467575.8A 2017-06-20 2017-06-20 A kind of c-hepatitis antibody polymer enzyme marker and its preparation and application Active CN107037207B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710467575.8A CN107037207B (en) 2017-06-20 2017-06-20 A kind of c-hepatitis antibody polymer enzyme marker and its preparation and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710467575.8A CN107037207B (en) 2017-06-20 2017-06-20 A kind of c-hepatitis antibody polymer enzyme marker and its preparation and application

Publications (2)

Publication Number Publication Date
CN107037207A true CN107037207A (en) 2017-08-11
CN107037207B CN107037207B (en) 2018-08-24

Family

ID=59541532

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710467575.8A Active CN107037207B (en) 2017-06-20 2017-06-20 A kind of c-hepatitis antibody polymer enzyme marker and its preparation and application

Country Status (1)

Country Link
CN (1) CN107037207B (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109358199A (en) * 2018-10-18 2019-02-19 郑州标源生物科技有限公司 A kind of preparation method of RBP ELISA quality-control product
CN112305222A (en) * 2020-11-05 2021-02-02 东莞市医本生物科技有限公司 Small polymer enzyme-antibody fragment, preparation and application thereof
CN112924665A (en) * 2021-02-19 2021-06-08 山东莱博生物科技有限公司 Antibody horseradish peroxidase marker and preparation and application thereof
CN113358863A (en) * 2021-06-10 2021-09-07 武汉原谷生物科技有限责任公司 Method for preparing polymerase label
CN113495157A (en) * 2020-03-20 2021-10-12 郑州达诺生物技术有限公司 Enzyme conjugate diluent, total thyroxine quantitative detection kit and use method thereof
CN114295824A (en) * 2021-12-14 2022-04-08 东方伊诺(苏州)医疗科技有限公司 Xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody, and preparation method and kit thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5543332A (en) * 1991-07-04 1996-08-06 Immunodex K/S Water-soluble, polymer-based reagents and conjugates comprising moieties derived from divinyl sulfone
CN1743846A (en) * 2004-08-30 2006-03-08 北京源德生物医学工程有限公司 Enzyme tag stabilizing agent
CN101178402A (en) * 2007-12-06 2008-05-14 武汉赛欣生物技术有限公司 Preparation method of polyase marker antibody
CN101561432A (en) * 2009-05-27 2009-10-21 福建省洪诚生物药业有限公司 Dilution being capable of maintaining high stability of enzyme marker solution
CN104569428A (en) * 2014-12-30 2015-04-29 浙江普康生物技术股份有限公司 ELISA (enzyme-linked immunosorbent assay) kit for EV (enterovirus) 71 inactivated vaccine antigen

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5543332A (en) * 1991-07-04 1996-08-06 Immunodex K/S Water-soluble, polymer-based reagents and conjugates comprising moieties derived from divinyl sulfone
CN1743846A (en) * 2004-08-30 2006-03-08 北京源德生物医学工程有限公司 Enzyme tag stabilizing agent
CN101178402A (en) * 2007-12-06 2008-05-14 武汉赛欣生物技术有限公司 Preparation method of polyase marker antibody
CN101561432A (en) * 2009-05-27 2009-10-21 福建省洪诚生物药业有限公司 Dilution being capable of maintaining high stability of enzyme marker solution
CN104569428A (en) * 2014-12-30 2015-04-29 浙江普康生物技术股份有限公司 ELISA (enzyme-linked immunosorbent assay) kit for EV (enterovirus) 71 inactivated vaccine antigen

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109358199A (en) * 2018-10-18 2019-02-19 郑州标源生物科技有限公司 A kind of preparation method of RBP ELISA quality-control product
CN113495157A (en) * 2020-03-20 2021-10-12 郑州达诺生物技术有限公司 Enzyme conjugate diluent, total thyroxine quantitative detection kit and use method thereof
CN112305222A (en) * 2020-11-05 2021-02-02 东莞市医本生物科技有限公司 Small polymer enzyme-antibody fragment, preparation and application thereof
CN112924665A (en) * 2021-02-19 2021-06-08 山东莱博生物科技有限公司 Antibody horseradish peroxidase marker and preparation and application thereof
CN112924665B (en) * 2021-02-19 2023-10-03 山东莱博生物科技有限公司 Antibody horseradish peroxidase marker and preparation and application thereof
CN113358863A (en) * 2021-06-10 2021-09-07 武汉原谷生物科技有限责任公司 Method for preparing polymerase label
CN114295824A (en) * 2021-12-14 2022-04-08 东方伊诺(苏州)医疗科技有限公司 Xanthan gum-based HRP-labeled mouse anti-human troponin I monoclonal antibody, and preparation method and kit thereof

Also Published As

Publication number Publication date
CN107037207B (en) 2018-08-24

Similar Documents

Publication Publication Date Title
CN107037207B (en) A kind of c-hepatitis antibody polymer enzyme marker and its preparation and application
Ozer et al. Chemical and biological sensors for viral detection
Karash et al. Rapid detection of avian influenza virus H5N1 in chicken tracheal samples using an impedance aptasensor with gold nanoparticles for signal amplification
Baeumner et al. Biosensor for dengue virus detection: sensitive, rapid, and serotype specific
Lowen et al. High temperature (30 C) blocks aerosol but not contact transmission of influenza virus
CN1969189B (en) Method of immunoassay having nonspecific reaction inhibited and reagent therefor
Shafagati et al. The use of Nanotrap particles for biodefense and emerging infectious disease diagnostics
Rettcher et al. Simple and portable magnetic immunoassay for rapid detection and sensitive quantification of plant viruses
CN101968490A (en) Indirect ELISA (Enzyme-Linked Immunosorbent Assay) method and kit for detecting haemophilus parasuis antibodies
TW201300421A (en) Reagents and methods for PRRSV detection
CN106676197A (en) Dual fluorescence RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for PDCoV (porcine Delta coronavirus) and PEDV (porcine epidemic diarrhea virus) and application thereof
Lee et al. Development and clinical evaluation of a highly accurate dengue NS1 rapid test: from the preparation of a soluble NS1 antigen to the construction of an RDT
KR101203399B1 (en) Diagnostic kit for influenza virus H1N1 using rapid immunochromatography
Ye et al. Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157: H7 detection
Duan et al. The serologic investigation and viral isolation of bluetongue virus in Shangri‐La in Southwest China
KR20170063000A (en) Method of Detecting Virus Using Virus Specific Nucleic Acid Aptamer-Nanoparticle Complex
CN101769919B (en) Immuno-chromatography detection device and detection method thereof
CN1570638A (en) SARS virus antibody detecting method, rapid diagnosis kit and preparation method
Desvoyes et al. RNA: protein interactions associated with satellites of panicum mosaic virus
CN104297494A (en) Enzyme-linked immunoassay kit for anti-hepatitis B virus X protein antibody and preparation method for kit
CN109856397A (en) A kind of colloidal-gold detecting-card and preparation method thereof of quick detection Rabbit pest virus
KR101032956B1 (en) Rapid diagnostic kit of hemorrhagic fever with renal syndrome detecting specific IgM and IgG using nucleocapsid protein derived from Soochong virus
Leakey et al. The Ability of Helicobactev pylori to Activate Neutrophils Is Determined by Factors Other Than H. pylori Neutrophil-Activating Protein
WO2007012945A1 (en) Diagnostic kit for detection of carnation mottle virus
Herrmann et al. Conjugation of enzymes to anti-poliovirus globulin: effect of enzyme molecular weight on virus neutralization capacity

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant