CN105884881A - Antibodies, method and kit used for detecting and determining luteoloside - Google Patents
Antibodies, method and kit used for detecting and determining luteoloside Download PDFInfo
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- CN105884881A CN105884881A CN201410521403.0A CN201410521403A CN105884881A CN 105884881 A CN105884881 A CN 105884881A CN 201410521403 A CN201410521403 A CN 201410521403A CN 105884881 A CN105884881 A CN 105884881A
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- luteoloside
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- immunogen
- conjugates
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Abstract
The invention provides hapten comprising luteoloside or its derivative, and the invention provides an immunogen comprising the aforementioned hapten coupled to an antigenicity-conferring carrier material, a conjugate comprising the aforementioned hapten coupled to a labelling agent, as well as, antibodies raised against the aforementioned immunogen and capable of binding with at least one structural epitope of intact luteoloside. The invention further provides a method and a kit for detecting, or determining the quantity of luteoloside in a sample, as well as, use of the aforementioned conjugate with the aforementioned antibodies for detecting, or determining the quantity of luteoloside. The antibodies have specificity for luteoloside, and the method and the kit can be used for detecting the existence of luteoloside and determining the luteoloside content.
Description
Technical field
Antibody that the present invention relates to small haptens and preparation method and application, particularly relates to antibody of luteoloside and derivant thereof and preparation method and application.
Background technology
The present invention relates to for detecting the method with quantitative luteoloside and test kit, and hapten, immunogen, conjugate (conjugate) and the antibody wherein used.
Wherein " detect " and refer to whether qualitative analysis material exists.
Wherein " measure " and refer to material is carried out quantitative analysis.
Luteoloside, is the representative substances of natural flavone, is primarily present in the Dracocephalum moldabium leaf of labiate, the dry aerial parts of Herba Schizonepetae；Leguminous plant narrow leaf Japanese pagodatree leaf, Herba Kummerowiae Striatae herb；Feverfew globe artichoke leaf；Caprifoliaceae plant Radix Ophiopogonis；Rosaceous plant Japan Radix Agrimoniae stem and leaf.Luteoloside content in Flos Lonicerae is higher, up to 0.09%.
Molecular formula: C21H20O11Molecular weight: 448.37
Luteoloside belongs to flavone compound, fusing point 254-256 DEG C, yellow powder；It is slightly soluble in water, methanol, ethanol, dissolves in hot water, hot methanol and ethanol, insoluble in chloroform, ether, benzene, solvent that petroleum ether isopolarity is little.
Research shows, luteoloside has stronger bactericidal antiphlogistic, effect of antipyretic-antalgic, shows preferably effect at antiviral and anti-tumor aspect.
Establish the qualitative and quantitative detection of multiple luteoloside at present and measure analysis method, such as thin layer chromatography, HPLC method, HPLC-MS method etc..The method of most common of which is high performance liquid chromatography.But the biological sample (including blood plasma, urine, saliva etc.) containing luteoloside is containing the substantial amounts of protein affecting assay and endogenous material；Luteoloside may be in bonding state simultaneously, it is necessary to through processing, get rid of endogenous impurity and the interference of metabolite, makes the luteoloside of bonding state could measure after dissociating；Also needing to concentrate to meet the requirement of instrument detection sensitivity, processing routine is complicated, wastes time and energy simultaneously；Additionally, due to luteoloside enter internal after, the distribution in tissue is extremely trace, even across enrichment, carries out assay and is still extremely difficult.It is distributed in target organ and tissue finally, for luteoloside, and cell and the research of Subcellular Localization, need by the method such as immunohistochemistry and westblot, but owing to lacking the antibody of luteoloside, hinder research in this respect.Therefore, it is necessary to develop a kind of for examining side and measuring the method for luteoloside in biological sample, to illustrating the mechanism of action of relevant medical material or compound recipe, and its distribution in vivo and metabolism research, will have and particularly be worth.
Summary of the invention
The hapten luteoloside of the present invention can provide the structural epitope determined, but itself does not has immunogenicity, therefore have to be coupled on the antigenic carrier mass of suitable imparting, the immunogen so formed just can induce immunne response after being expelled in host animal body.Therefore, the present invention provides the immunogen of a kind of following structure:
Luteoloside (luteoloside)
Wherein R is the alkyl bridge (connecting the bridge of hapten and carrier mass) of 0 to 6 carbon, preferably R=0, aldehyde radical after i.e. P1 directly aoxidizes with luteoloside is combined, or R is that C1-C6 is substituted or unsubstituted, straight or branched, saturated or undersaturated alkylidene, more preferably R is that C1-C4 is unsubstituted, straight chain, saturated alkylidene.P1 is to confer to antigenic carrier mass.Carrier mass is selected from protein, the polypeptide of protein sheet synthesis or semisynthetic polypeptide.Wherein protein, protein fragments are selected from albumin, serum albumin, globulin, eyepiece albumen and lipoprotein, it is preferably bovine serum albumin, ovalbumin, bovine gamma globulin(BGG), thyroxine-binding globulin, keyhole limpet hemocyanin (keyhole limpet haemocyanin, KLH), more preferably from keyhole limpet hemocyanin or bovine serum albumin (BSA).Synthesis polypeptide or semi-synthetic polypeptide are the synthesis polyamino acid with sufficient amount of available amino, preferably poly-D-lysine.Synthesis or natural polymer are the polymeric materials with reactive functional group base, particularly can be attached to hapten and produce immunogenic carbohydrate, yeast or polysaccharide.
The haptenic preparation present invention describes two adjacent hydroxyls in the glycosidic structure of hapten luteoloside and is oxidized to aldehyde radical thus the coupled action that occurs with carrier mass through sodium periodate, produces immunogen.Conjugate is successfully prepared and the most respectively through mass spectrum (MS) and proton nmr spectra and carbon spectrum (1HNMR and 13CNMR) confirmation.
The hapten of the immunogenic synthesis present invention and the connection of protein carrier can use oneself any connected mode of knowing of this area.Such as periodates oxidizing process etc..When using periodate oxidation method to prepare immunogen, it is that the hapten luteoloside of 10 milligrams is dissolved in 10ml hot water, add the sodium periodate solution 0.5 milliliter containing 8 milligrams, react one hour under room temperature, centrifugal, take in the bovine serum albumin carbonate buffer solution that supernatant joins 10-20mg/mL, stirring reaction 6 hours, loading bag filter, respectively with the PBS buffer solution dialysis 3-5d of distilled water and 0.01mol/L, subpackage is stored in the refrigerator of-20 DEG C.
The analysis method of synthetic immunogen is in order to confirm to be combined with suitable hapten on carrier mass, before immunity, use UV spectrophotography or matrix auxiliary UV laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) that each immunogen is estimated.Luteoloside immunogen reactant is compared (200nm-400nm) with the absworption peak of product, can determine whether coupling, and calculates conjugate and haptenic ratio according to the molar absorption coefficient of reactant and product.
Voyager STR biology light splitting degree method of measuring station laser desorption mass spectrometry is used to carry out MALDI-TOF mass spectrum with delay extraction combination.Each aliquot to be analyzed is diluted in the trifluoroacetic acid aqueous solution of 0.1% sample solution making 1mg/ml.Sinapinic acid matrix and bovine serum albumin is used to analyze aliquot (1uL) as external standard.
For preferred carrier mass, bovine serum albumin or KLH, preferably hapten and the combination ratio of protein are for 6-15: 1.
Second aspect, needs during immune detection luteoloside and labelled reagent are carried out coupling.Therefore, the present invention provides the conjugates of a kind of following structure；
Wherein R is the alkyl of 0 to 6 carbon, and P2 is detectable labelled reagent, and labelled reagent is peroxidase selected from enzyme, luminescent substance, radioactive substance or their mixture, more preferably labelled reagent.Most preferably labelled reagent is horseradish peroxidase (HRP).Luminescent substance is selected from bioluminescence material, chemiluminescent substance or fluorescent material.
The hapten of the synthesis present invention of conjugates and the connection of protein carrier can use oneself any connected mode of knowing of this area.Such as periodates oxidizing process etc..When using periodate oxidation method to prepare immunogen, it is that the hapten luteoloside of 10 milligrams is dissolved in 10ml hot water, add the sodium periodate solution 0.5 milliliter containing 8 milligrams, react one hour under room temperature, centrifugal, take in the bovine serum albumin carbonate buffer solution that supernatant joins 10-20mg/mL, stirring reaction 6 hours, loading bag filter, respectively with the PBS buffer solution dialysis 3-5d of distilled water and 0.01mol/L, subpackage is stored in the refrigerator of-20 DEG C.
On the other hand, the present invention relates to the antibody that the immunogen of first aspect present invention is produced, these antibody can at least the epi-position in a luteoloside structure be combined, preferably combine with complete luteoloside structure epi-position, this antibody is polyclonal, or monoclonal, preferably monoclonal antibody, and there is the specific antibody to luteoloside.
This antibody is fixed on during immune detection on support substrate.Preferably, the method farther includes to be fixed to described serum antibody support on substrate, preferably solid support, most preferably polystyrene solid support.
The present invention further provides a kind of method preparing antibody, the method comprises the immunogen immune animal by repeating to give the luteoloside according to the present invention, the preferably young animal of ridge, most preferably mammal, and collects the step of the serum obtained from immune animal.
On the other hand, present invention resides in the method detecting or measuring luteoloside in sample, the method includes that the conjugates by the present invention or its mixture contact sample with antibody or its mixture of the present invention；The quantity of the conjugate that detection or mensuration combine；And from standard curve infer luteoloside sample existence or quantity.
On the other hand, invention further describes and how to be used for developing the universaling analysis method that can be used to detect and measure the existence of luteoloside and corresponding test kit by the antibody produced for this immunogen.This test kit includes the conjugates by the present invention or its mixture and the antibody of the present invention or its mixture, and this test kit can at random include applying described conjugate and described antibody detect in the sample or measure the guidance of luteoloside simultaneously.Preferably, sample is solution, such as biofluid.It is highly preferred that sample is serum or urine.In the method and test kit of the present invention, the most different first-selected cross-linking agent (immunogenic and conjugates).
On the other hand, the present invention includes that the conjugates using the present invention or its mixture and the antibody of the present invention or its mixture detect in test sample is such as biofluid or measure luteoloside.
In order to produce polyclonal antiserum, immunogen is mixed with Freund adjuvant, and mixture is injected in host animal body, such as rabbit, sheep, Mus, guinea pig or horse.Inject (reinforcement) further and take Serum samples evaluation antibody titer.When reaching optimal titre, subsequently host animal blood-letting is obtained the specific antiserum of proper volume.The antibody purification level required is depending on the application of plan.For many purposes, do not need purification, but, in other cases, such as antibody is fixing on a solid support, and purification step can remove undesired material and remove nonspecific combination.Specific antibody of the present invention needs purification as reagent content of the luteoloside in mensuration or biological fluid in immunity test.
The monoclonal antibody of the present invention of preparing of monoclonal antibody refers to the antibody of the antibody population available from substantially homology, and the antibody individuality i.e. forming this colony is the most identical, except there may be the most possible spontaneous mutation.
The antibody of the present invention can be prepared by oneself the various technology known of those skilled in that art.Such as, complete antigen of the present invention, animal can be applied to induce the generation of monoclonal antibody.For monoclonal antibody, available hybridoma technology is prepared or can use recombinant DNA method to prepare.The myeloma cell line of the optional muroid of myeloma cell, including derived from the myeloma cell line of MOPC-21 and MPC-11 mouse tumor and SP-2, N20 or X63-Ag8-653 cell, and human myeloma and mouse-human heteromyeloma's cell line.
Growth of Hybridoma Cell is analyzed in culture medium therein the generation with detection with required specific monoclonal antibody, as by external binding analysis, such as Enzyme Linked Immunoadsorbent Assay (ELISA) or radioimmunoassay, RIA (RIA).The position of the cell expressing antibody can be detected with FACS.Then, hybridoma clone can be formed sub-clone by limiting dilution procedures, and be grown by standard method.The culture medium being suitable for used to reach this purpose includes, such as, DMEM or RPMI-1640 culture medium.Additionally, hybridoma can grow as ascites tumor in animal body.
The monoclonal antibody secreted by sub-clone is from culture medium, ascites or serum, suitably being separated by conventional immunoglobulin purification technique, these purifying process are Protein A-agarose method (protein A-Sepharose), hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatograph etc..
In one preferred scheme of the present invention, prepared by the method that luteoloside monoclonal antibody uses Balb/C mouse ascites to produce monoclonal antibody.By about 106-107Hybridoma is inoculated in the mouse peritoneal of sensitization, and in 2-4 week, visible abdominal part substantially swells.Extraction ascites, is slightly proposed IgG through saturated sulphuric acid by the sedimentation method, then by the antibody that slightly carries through affinity column (Protein G-Sephrose) purification.
Detailed description of the invention
Embodiment 1
The hapten luteoloside of 5 milligrams is dissolved in 1mlCBS, add the sodium periodate solution 0.3 milliliter containing 4 milligrams, react one hour under room temperature, join in the bovine serum albumin carbonate buffer solution of 10-20mg/mL, stirring reaction 6 hours, loading bag filter, respectively with the PBS buffer solution dialysis 3-5d of distilled water and 0.01mol/L, subpackage is stored in the refrigerator of-20 DEG C.
Embodiment 2
The hapten luteoloside of 5 milligrams is dissolved in 1mlCBS, add the sodium periodate solution 0.3 milliliter containing 4 milligrams, react one hour under room temperature, centrifugal, take in the ovalbumin carbonate buffer solution that supernatant joins 10-20mg/mL, stirring reaction 6 hours, load bag filter, respectively with the PBS buffer solution dialysis 3-5d of distilled water and 0.01mol/L, subpackage is stored in the refrigerator of-20 DEG C.
Embodiment 3
Preparation method, with embodiment 1, simply changes bovine serum albumin (BSA) into KLH, prepares luteoloside-KLH complete antigen 120mg.
Embodiment 4
Three rabbits of immunogen (luteoloside-BSA, luteoloside-KLH) the most each immunity.From the beginning of booster immunization second time, within the 8th day after each immunity, after suitably dilution, measure titer with indirect ELISA in rabbit ear edge vein exploitating blood, serum.When the 4th immunity, rabbit obtains the antibody of high-titer, purified make lyophilized powder after titer be respectively 30000: 1.
Embodiment 5
First by luteoloside antigen and Freund adjuvant emulsifying, with PBS, complete antigen luteoloside-KLH is configured to the solution of 1mg/ml, then complete antigen solution is mixed with Freund adjuvant equal-volume, form uniform emulsion with the concussion of high speed oscillator, this emulsion is used for the immunity of animal.The mice selecting 7 week old carries out injecting immune.Take the healthy Balb/C mice 6 of 7 week old, at the 0th day, every lumbar injection complete Freund's adjuvant emulsifying antigen 1 00uL.14th day, then to every mouse peritoneal injection incomplete Freund's adjuvant emulsifying antigen 1 00uL.21st day, to pluck the blood sampling of eyeball of mouse method, detect antibody titers from serum (titre) by ELISA method.28th day, lumbar injection incomplete Freund's adjuvant emulsifying antigen 1 00uL again.In cell fusion reaction the last week, with 100ug/100ul antigen normal saline booster immunization again.Through measuring after blood sampling, gained antiserum titre is 1: 30000.Cell mixing operation checks after 3 days that cell merges situation, adds HT ' complete medium after 8 days, and every hole adds 1ml.Between 12 days, it is possible to observe the clone of hybrid cell.When cloning diameter and growing to about 1mm, the coated elisa plate of employment luteoloside-BSA filters out the hybridoma of anti-luteoloside.The positive colony limiting dilution assay screened carries out cloning, screens the specific hybrid oncocyte of anti-luteoloside with PF-BSA coated ELISA method ELISA Plate.After five time clonings, obtain a strain and can secrete the anti-luteoloside monoclonal cell of specific antibodies.
Embodiment 6
Through 4 immune mices in Example 4, measure sero-fast titre with the coated elisa plate of luteoloside-BSA.Being diluted in the 0.05M carbonate buffer solution of pH 9.6 with 10ug/ml by envelope antigen, 100ul/ hole joins 96 hole ELISA Plate in 4 DEG C overnight.Discard and close at 37 DEG C 2 hours with 1% gelatin/PBS after being coated liquid, then wash plate 3 times by PBS-Tween-20 washing liquid, add the luteoloside antiserum after dilution, be placed in 37 DEG C and hatch 1 hour.Plate is washed 3 times by PBS-Tween-20 washing liquid, add the sheep anti-mouse igg multi-resistance of the horseradish peroxidase-labeled of 1: 2000,37 DEG C hatch 1 hour after wash plate 3 times by PBS-Tween-20 washing liquid, add TMB/H202 substrate and carry out the 10min that develops the color, and by stop buffer (0.1M sulphuric acid) color development stopping.
Under 450nm optical wavelength, measure absorption value (OD), with the OD average ratio of blank control wells relatively, use the t inspection of measurement data, take the minimum dilution factor titer as this antibody of P ＜ 0.05.Through colorimetric determination, the sero-fast titer of gained luteoloside is 1: 30000.
Embodiment 7
The fusion efficiencies analysis aobvious green of the lymphocyte of CFSE green fluorescence preliminary making, by the aobvious redness of the SPZ/O cell of the anti-mouse B22O monoclonal antibody preliminary making of PE labelling, two kinds of cells merge the hybridoma formed with Two Colour Fluorescence.Measure through flow cytometer and analyze double fluorecyte ratio, be used for reflecting fusion efficiencies.In experiment, SP2/0 (myeloma) cell of PE labelling anti-mouse B22O monoclonal antibody preliminary making, CFSE green fluorescence preliminary making lymphocyte, it is hybridoma with Two Colour Fluorescence.In this experimental system, the initial fusion efficiencies of splenocyte and SP2/0 is about 50%, and it is 13% that ELISA method measures positive rate.
Claims (20)
1. the immunogen of a below formula:
Luteoloside (luteoloside)
Wherein R is the alkyl bridge (connecting the bridge of hapten and carrier mass) of 0 to 6 carbon, P1It is to confer to antigenic carrier mass.
Immunogen the most according to claim 1, R=0, i.e. P1Aldehyde radical after directly aoxidizing with luteoloside is combined.
Immunogen the most according to claim 1, R is that C1-C6 is substituted or unsubstituted, straight or branched, saturated or undersaturated
Alkylidene.
4., according to the immunogen of claim 1 or 3, R is that C1-C4 is unsubstituted, straight chain, saturated alkylidene.
Immunogen the most according to claim 1, wherein carrier mass is protein, protein fragments, synthesis or natural polypeptides or narrows
Become polypeptide, synthesis or natural polymer.
6. according to the immunogen of claim 1 or 5, wherein protein, protein fragments selected from albumin, serum albumin, globulin,
And lipoprotein.
7. according to the immunogen of claim 1 or 5, wherein protein, protein fragments selected from bovine serum albumin, ovalbumin,
Bovine gamma globulin(BGG), thyroxine-binding globulin, keyhole limpet hemocyanin (keyhole limpet haemocyanin, KLH)
Deng.
8. according to the immunogen of claim 1 or 5, wherein synthesis or natural polypeptides or semi-synthetic polypeptide be have sufficient amount of can profit
With the synthesis polyamino acid of amino.
9., according to the immunogen of claim 1 or 5, wherein synthesis or natural polymer are selected from carbohydrate, yeast or polysaccharide.
10. resisting such as the immunogenic antibody of claim 1 to 9, wherein antibody can be with at least one structure in complete luteoloside
Property epitope combine.
The antibody of 11. such as claim 10, is wherein fixed on support substrate when antigen, particularly during polystyrene solid support,
Antibody can be specific binding with it.
The 12. preparations such as method of the antibody in claim 10 or 11, the method includes such as the immune original weight of claim 1 to 9
Give animal again and make animal immune, and from immune animal, collect obtained serum antibody；And utilize can in immunology
The cell line that the cell line accepted and the immune animal monoclonal cell system producing antibody produce after merging, prepared monoclonal anti
Body.
The method of 13. such as claim 12, wherein the method also includes being fixed on by described serum antibody on support substrate.Including such as power
Profit requires the conjugate covalently bound with the label that can detect that of the hapten any one of 1 to 3.
The conjugates of 14. 1 kinds of following structures:
Luteoloside (luteoloside)
Wherein R is the alkyl of 0 to 6 carbon, and P2 is detectable labelled reagent.
15. conjugates according to claim 14, labelled reagent is selected from enzyme, luminescent substance, radioactive substance or their mixture.
16. according to the conjugates of claim 14 or 16, and labelled reagent is peroxidase.
17. conjugates according to claim 16, labelled reagent is horseradish peroxidase (HRP).
18. according to the conjugates of claims 14 or 15, and luminescent substance is bioluminescence material, chemiluminescent substance or fluorescent material.
19. for detecting and the method for luteoloside in quantitative sample, and the method includes by test sample with such as claim 14 to 18
Any one of conjugate or its mixture, contact with antibody or its mixture such as claim 10 or 11；Detection or quantitative
In conjunction with conjugate；And from calibration curve, release existence or the content of luteoloside in sample.
20. for detection and the test kit of quantitative luteoloside, and this test kit includes the coupling as any one of claim 14 to 18
Thing or its mixture, and such as the antibody of claim 10 or 11 or its mixture.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108164598A (en) * | 2016-12-07 | 2018-06-15 | 南开大学 | Conjugate of Bergenin and preparation method and application |
| CN108164599A (en) * | 2016-12-07 | 2018-06-15 | 南开大学 | Conjugate of barbaloin and preparation method and application |
| CN108164597A (en) * | 2016-12-07 | 2018-06-15 | 南开大学 | Conjugate of galuteolin and preparation method and application |
| CN110787178A (en) * | 2019-12-05 | 2020-02-14 | 上海健康医学院 | Medical application of luteoloside |
-
2014
- 2014-09-30 CN CN201410521403.0A patent/CN105884881A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108164598A (en) * | 2016-12-07 | 2018-06-15 | 南开大学 | Conjugate of Bergenin and preparation method and application |
| CN108164599A (en) * | 2016-12-07 | 2018-06-15 | 南开大学 | Conjugate of barbaloin and preparation method and application |
| CN108164597A (en) * | 2016-12-07 | 2018-06-15 | 南开大学 | Conjugate of galuteolin and preparation method and application |
| CN108164598B (en) * | 2016-12-07 | 2021-09-03 | 南开大学 | Bergenin conjugate and preparation method and application thereof |
| CN110787178A (en) * | 2019-12-05 | 2020-02-14 | 上海健康医学院 | Medical application of luteoloside |
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Application publication date: 20160824 |