CN104892757B - It is a kind of detect Rhein enzyme linked immunological kit and its application - Google Patents
It is a kind of detect Rhein enzyme linked immunological kit and its application Download PDFInfo
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Abstract
The invention discloses a kind of enzyme linked immunological kit for detecting Rhein and its applications.The monoclonal antibody of anti-Rhein used in kit of the invention is to be secreted to generate by hybridoma cell strain RH1F8.Be experimentally confirmed: the monoclonal antibody specificity of anti-Rhein provided by the present invention is strong, and the cross reacting rate with aloe-emodin is 27.039%；Cross reacting rate with rheum emodin is 0.887%；Cross reacting rate with Chrysophanol is 0.114%；Cross reacting rate with Physcion is 0.310%；Cross reacting rate with Sennoside A is 0.005%；With Sennoside B and ponticin no cross reaction.The indirect competitive enzyme-linked immunosorbent kit being prepared using the monoclonal antibody can be used for Rhein in qualitative or quantitative test sample, and linear detection range has the advantages that quick, sensitive in 0.02~0.11ng/mL.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit for detecting Rhein and its applications, belong to field of biotechnology.
Background technique
Rhein (Rhein) be parts of generic medicinal plants rheum officinale (Rhein Radix et Rhizoma) main active it
One, there are a variety of pharmacological activity such as anti-inflammatory, antitumor, protection renal.Currently, Rhein detection method has thin-layered chromatography, height
Effect liquid phase chromatogram method and capillary electrophoresis etc..
Enzyme-linked immuno-sorbent assay has the spies such as easy to operate, high sensitivity, flux are high, detection is quick, low in cost
Point, Chinese medicine in terms of have been obtained for being widely applied, but the immunologic detection method of Rhein yet there are no
Report, in addition sold rheum officinale quality is irregular in the market, so it is immune to establish a kind of easy, quickly, high-throughput Rhein
Detection method has very important significance.
Summary of the invention
It is an object of the present invention to provide a kind of monoclonal antibodies of anti-Rhein.
The monoclonal antibody of anti-Rhein provided by the invention is thin for the hybridoma of CGMCC No.10582 by deposit number
Born of the same parents' strain generates.
It is a further object to provide the hybridoma cell strains of the monoclonal antibody of one plant of anti-Rhein of secretion
RH1F8。
The deposit number of the hybridoma cell strain RH1F8 of the monoclonal antibody of the anti-Rhein of secretion provided by the invention is
CGMCC No.10582。
It is the hybridoma cell strain RH1F8, deposit number CGMCC of CGMCC No.10582 that the present invention, which protects deposit number,
The classification naming of the hybridoma cell strain RH1F8 of No.10582 be mouse hybridoma cell system, the hybridoma cell strain in
Be preserved on April 15th, 2015 China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address:
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number CGMCC
No.10582。
It is a still further object of the present invention to provide said monoclonal antibody or the purposes of above-mentioned hybridoma cell strain RH1F8.
The present invention provides said monoclonal antibodies or above-mentioned hybridoma cell strain RH1F8 in preparation detection or auxiliary detection
The reagent of Rhein Content or the application in kit in sample to be tested.
The present invention also provides said monoclonal antibody or above-mentioned hybridoma cell strain RH1F8 detect or assist detection to
Application in sample in Rhein Content.
The present invention also provides said monoclonal antibodies or above-mentioned hybridoma cell strain RH1F8 in preparation detection or auxiliary inspection
Survey the application in sample to be tested in the colloidal gold strip of Rhein Content.
Final object of the present invention is to provide a kind of enzyme for detecting or assisting Rhein Content in detection sample to be tested
Linked immunoassay reagent kit.
The enzyme linked immunological kit of Rhein Content includes only in detection provided by the invention or auxiliary detection sample to be tested
The said monoclonal antibody of vertical packaging.
The enzyme linked immunological kit of Rhein Content can also contain in detection provided by the invention or auxiliary detection sample to be tested
There are the Rhein standard items of respectively independent packaging and/or the marker enzyme for marking the monoclonal antibody；
The Rhein standard items can be Rhein or a series of rheum officinale for the various concentrations being configured to by the Rhein
Acid solution；A series of concentration of the rheum officinale acid solution of various concentrations is concretely: 0.5ng/mL, 0.25ng/mL,
0.125ng/mL, 0.0625ng/mL, 0.03125ng/mL, 0.015625ng/mL and 0.0078125ng/mL, the Rhein
The solvent of solution concretely PBS buffer solution；The solvent of the PBS buffer solution is water, solute Na2HPO4、KH2PO4With
NaCl, solute Na2HPO4、KH2PO4With concentration of the NaCl in the PBS buffer solution be respectively 0.02M, 0.0015M and
0.14M, pH value 7.5；
The marker enzyme concretely horseradish peroxidase.
The enzyme linked immunological kit of Rhein Content can also contain in detection provided by the invention or auxiliary detection sample to be tested
There are the coating buffer and/or cleaning solution and/or substrate buffer solution and/or stop buffer and/or envelope of respectively independent packaging
Close liquid；
The solvent of the coating buffer is water, solute Na2CO3And NaHCO3；Solute Na2CO3And NaHCO3Described
The concentration being coated in buffer is respectively 0.01M and 0.04M；The pH value of the coating buffer is 9.6；
The solvent of the cleaning solution is water, solute Na2HPO4、KH2PO4, NaCl and Tween-20；Solute Na2HPO4、
KH2PO4, the concentration of NaCl and Tween-20 in the cleaning solution be respectively 0.02M, 0.0015M, 0.14M and 0.1% (volume
Percentage composition)；The pH value of the cleaning solution is 7.5；
The solvent of the substrate buffer solution is water, and solute is trisodium citrate and Na2HPO4；Solute trisodium citrate and
Na2HPO4Concentration in the substrate buffer solution is respectively 0.01M and 0.03M；The pH value of the substrate buffer solution is 5.5；
The stop buffer is the aqueous sulfuric acid of 2M；
The solvent of the confining liquid is water, solute Na2HPO4、KH2PO4, NaCl and skim milk；Solute Na2HPO4、
KH2PO4, the concentration of NaCl and skim milk in the confining liquid be respectively 0.02M, 0.0015M, 0.14M and 3% (quality hundred
Divide content)；The pH value of the confining liquid is 7.5.
In above-mentioned enzyme linked immunological kit, the sample is rheum officinale；The rheum officinale is specially that sorrel, Tang's Gu are especially big
Huang, Rheum officinale, Rumex madaio or North China rheum officinale.
Application of the above-mentioned enzyme linked immunological kit in detecting or assisting detection sample to be tested in Rhein Content also belongs to
Protection scope of the present invention.
In above-mentioned application, the sample is rheum officinale；The rheum officinale be specially sorrel, Rheum tanguticum Maxim, Rheum officinale,
Rumex madaio or North China rheum officinale.
The present invention provides a kind of enzyme linked immunological kits for detecting Rhein.Anti- Rhein provided by the present invention
Monoclonal antibody is to pass through indirect competition with Rhein-OVA (conjugate of Rhein and oralbumin) for envelope antigen
ELISA method screens hybridoma RH1F8.It is experimentally confirmed: anti-Rhein provided by the present invention
Monoclonal antibody specificity is strong, and the cross reacting rate with aloe-emodin is 27.039%；Cross reacting rate with rheum emodin is
0.887%；Cross reacting rate with Chrysophanol is 0.114%；Cross reacting rate with Physcion is 0.310%；With kind
The cross reacting rate for rushing down glycosides A is 0.005%；With Sennoside B and ponticin no cross reaction.It is prepared using the monoclonal antibody
Obtained indirect competitive enzyme-linked immunosorbent kit can be used for Rhein in qualitative or quantitative test sample, and linear detection range exists
0.02~0.11ng/mL has the advantages that quick, sensitive.
Detailed description of the invention
Fig. 1 is Rhein chemical structure.
Fig. 2 is the suppression curve of Rhein in indirect competitive ELISA.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Freund's complete adjuvant is the product of Sigma company, catalog number F5881.
Incomplete Freund's adjuvant is the product of Sigma company, catalog number F5506.
Be coated with buffer: solvent is water, solute Na2CO3And NaHCO3；Solute Na2CO3And NaHCO3In coating buffer
In concentration be respectively 0.01M and 0.04M；PH value is 9.6.
Cleaning solution: solvent is water, solute Na2HPO4、KH2PO4, NaCl and Tween-20；Solute Na2HPO4、KH2PO4、
The concentration of NaCl and Tween-20 in cleaning solution is respectively 0.02M, 0.0015M, 0.14M and 0.1% (volumn concentration)；
PH value is 7.5.
Sample diluting liquid: solvent is water, solute Na2HPO4、KH2PO4With NaCl, Tween-20 and gelatin；Solute
Na2HPO4、KH2PO4It is respectively 0.02M, 0.0015M and 0.14M with concentration of the NaCl in the sample diluting liquid；It is described to spit
Volumn concentration of the temperature -20 in the sample diluting liquid is 0.1%；Quality of the gelatin in the sample diluting liquid
Percentage composition is 0.5%.
Substrate buffer solution: solvent is water, and solute is trisodium citrate and Na2HPO4；Solute trisodium citrate and Na2HPO4
Concentration in substrate buffer solution is respectively 0.01M and 0.03M；PH value is 5.5.
Stop buffer: concentration is the aqueous sulfuric acid of 2M.
Confining liquid: solvent is water, solute Na2HPO4、KH2PO4, NaCl and skim milk；Solute Na2HPO4、KH2PO4、
The concentration of NaCl and skim milk in confining liquid is respectively 0.02M, 0.0015M, 0.14M and 3% (mass percentage)；pH
Value is 7.5.
PBS buffer solution: solvent is water, solute Na2HPO4、KH2PO4And NaCl, solute Na2HPO4、KH2PO4Exist with NaCl
Concentration in PBS buffer solution is respectively 0.02M, 0.0015M and 0.14M；PH value is 7.5.
Bal b/C small white mouse is purchased from Military Medical Science Institute's Experimental Animal Center.
SP2/0 myeloma cell is purchased from China Veterinery Drug Inspection Office.
Embodiment 1, the acquisition of hybridoma cell strain and the preparation of anti-Rhein monoclonal antibody
One, the synthesis of Rhein-BSA immunogene and Rhein-OVA coating antigen
1, Rhein standard items (are purchased from National Institute for Food and Drugs Control, catalogue number is 110757, chemistry knot
Structure formula is shown in Fig. 1) 5.2mg, it is dissolved in 3mL n,N-Dimethylformamide (purchased from Sigma company, catalogue number is D4551)
In, magnetic agitation under room temperature obtains Rhein standard solution.
2, n-hydroxysuccinimide is added into the Rhein standard solution in step 1 (to be purchased from Sigma company, produce
Product catalog number is 56480) 4.2mg, is protected from light after being stirred to react 30min；7.5mg dicyclohexylcarbodiimide is added (to be purchased from
Sigma company, catalogue number is D3128), 4 DEG C are stirred to react 4h, are centrifuged (3000g, 5min), take supernatant.
3, by 20mg BSA (bovine serum albumin(BSA) is purchased from Sigma company, and catalogue number is A4737) and 20mg
OVA (chicken ovalbumin is purchased from Sigma company, and catalogue number is A5503) is dissolved separately in 2mL PBS buffer solution,
Centrifugation, takes supernatant (BSA protein solution and OVA protein solution), and supernatant is divided into two equal portions respectively.
4, the supernatant of step 2 is added dropwise to respectively in the BSA protein solution and OVA protein solution of step 3,
After 4 DEG C are stirred to react 12h, it is packed into bag filter, PBS dialyses 3 days, and Rhein-BSA immunogen solution is respectively obtained after dialysis
(4mg/mL) and Rhein-OVA are coated with original solution (4mg/mL).
Two, animal immune
1, using the Bal b/C small white mouse of 8-10 week old as experimental animal.
2, fundamental immunity: the Rhein-BSA of 1mg/mL is added to isometric Freund after sterilizing filter filters and is helped completely
Emulsification is sufficiently stirred with magnetic stirring apparatus in agent, until instilling indiffusion in water.Abdominal cavity and back are used with the immunogene emulsified
Subcutaneous multi-point injection Bal b/C small white mouse, injection dosage are that every mouse injects 0.1mg antigen.
3, booster immunization: after fundamental immunity 2 weeks, taking the Rhein-BSA of 1mL 1mg/mL, and 1mL Freund is added and not exclusively helps
Emulsification is sufficiently stirred with magnetic stirring apparatus in agent, until instilling indiffusion in water.The immunogene emulsified is used into abdominal cavity and back
Subcutaneous multi-point injection Bal b/C mouse, injection dosage are that every mouse injects 0.1mg antigen.
Three, cell fusion and cloning
Booster immunization is primary every 10 days, immune 7th day latter every time since third time booster immunization, from mouse orbit
Blood sampling measures antibody titer, the serum diluting multiple that the definition of potency is OD value when being 1.After potency is greater than 1:8000, selection
The optimal mouse (potency 1:80000) of serum titer, extracting spleen cell are thin in 9:1 (quantitative proportion) ratio and SP2/0 myeloma
Born of the same parents' fusion；Using the hybridoma cell strain of limiting dilution assay screening secrete monoclonal antibody；It is anti-using indirect competitive ELISA measurement
Body potency, with the high monoclonal cell strain of Rhein standard solution screening secretory antibody potency, selection can identify rheum officinale acidity scale
The monoclonal antibody of quasi- product solution, finally screening obtains the monoclonal hybridoma strain of the anti-Rhein antibody of stably excreting, orders
The classification naming of entitled RH1F8, the hybridoma cell strain are mouse hybridoma cell system, and the hybridoma cell strain is in 2015
It was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Beijing on April 15
The institute 3 of Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number CGMCC
No.10582。
The method of above-mentioned indirect competitive ELISA measurement antibody titer is as follows:
1) it is coated with: the Rhein-OVA solution of 100 μ L 250ng/mL being added in 96 hole elisa Plates, 37 DEG C of coatings 3 are small
When, it is washed 4 times with cleaning solution.
2) close: 1h is closed in 150 hole μ L/ of confining liquid in 37 DEG C of wet box, abandons confining liquid, is washed 3 times.
3) product are loaded: inhibiting hole that 50 μ L of pre-configured standard items (100ng/mL) is added, 50 μ L samples are added in blank well
Product dilution.
4) add antibody: the monoclonal antibody that the increment cultivation of different extension rates obtains being added in ELISA Plate, is set 37 in wet box
30min under the conditions of DEG C, board-washing 4 times.
5) add ELIAS secondary antibody: sheep anti mouse ELIAS secondary antibody IgG-HRP (is purchased from Jackson company, cat. no is
79556) (0.1mg/mL) dilutes 1000 times, and every hole adds 100 μ L, sets in wet box 30min under the conditions of 37 DEG C, and board-washing 4 times.
6) it develops the color: taking 20mg o-phenylenediamine (OPD) to be dissolved in 10mL substrate dilution, add 4 μ L 30%H2O2, substrate is molten
Liquid is added in ELISA Plate, every 100 μ L of hole.It is protected from light colour developing 10min.With the OD value for measuring each hole at microplate reader 492nm, partial results
It is shown in Table 1.In table 1, the hybridoma that 2G2,2G12 and 3G2 are respectively represented in the tissue culture plate difference hole being added in screening process is thin
Born of the same parents, inhibiting rate (%)=[(blank well OD value-inhibition hole OD value)/blank well OD value] × 100%.
The result of table 1, indirect competitive ELISA method screening hybridoma
Light absorption value is bigger, illustrates that antibody is higher to the affinity of antigen；Discrimination is higher, illustrates that the specificity of antibody is got over
It is good.The discrimination highest that can be seen that 2G12 from the data of table 1, up to 87.2%, while with the affinity of coating antigen also compared with
Height, the hybridoma for choosing 2G12 continue to screen, and finally obtain the hybridoma that secretory antibody specificity is good, affinity is high
Cell strain RH1F8.
Four, cell cryopreservation and recovery
Hybridoma cell strain RH1F8CGMCC No.10582 is made 1 × 10 with frozen stock solution6The cell suspension of a/mL,
It is saved for a long time in liquid nitrogen.When recovery, cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds is immediately placed in and melts, moved into after centrifugation removal frozen stock solution
Cultivate culture in glassware.
Five, the preparation and purification of monoclonal antibody
1, increment cultivation
The preparation method of cell culture medium: calf serum is added into DMEM culture medium and (is purchased from GIBCOBRL, catalogue
Number be 26170-043) and sodium bicarbonate, final concentration of 20% (mass percentage) of calf serum, the end of sodium bicarbonate it is dense
Degree is 0.2% (mass percentage), pH 7.4.
Hybridoma cell strain RH1F8CGMCC No.10582 is placed in above-mentioned cell culture medium, 37 DEG C are cultivated 2 days, are used
In preparing monoclonal antibody solution.
2, prepared by ascites
Bal b/c mouse peritoneal injection sterilizing paraffin oil (0.2mL/ is only).Above-mentioned hybridoma cell strain is injected intraperitoneally after 7 days
RH1F8CGMCC No.10582(106A/only).Ascites is acquired after 7 days, is purified with saturated ammonium sulfate method to ascites, is obtained
The monoclonal antibody of anti-Rhein, -20 DEG C of ascites preservations after purification.
Specific step is as follows for above-mentioned saturated ammonium sulfate purifying ascites method:
1) ascites is mixed with isometric PBS solution at room temperature.Isometric saturation sulfuric acid is added dropwise under stirring
Ammonium (even if ammonium sulfate concentrations reach 50%), after 4 DEG C of standing 2h, 4 DEG C of 3000g are centrifuged 15min, abandon supernatant, take precipitating.It is described
The solvent of PBS buffer solution is water, solute Na2HPO4、KH2PO4And NaCl, solute Na2HPO4、KH2PO4With NaCl in the PBS
Concentration in buffer is respectively 0.02M, 0.0015M and 0.14M, pH value 7.5.The solvent of the saturated ammonium sulfate solution is
Water, solute are (NH4)2SO4, concentration 4.10M, pH 7.2.
2) PBS solution of 2 times of volumes of former ascites volume is added into gained precipitating, sufficiently dissolves, half PBS body is added dropwise
Long-pending saturated ammonium sulfate (i.e. saturated ammonium sulfate concentration reaches 33%), after 4 DEG C of standing 2h, 4 DEG C of 3000g are centrifuged 15min, in abandoning
Clearly, precipitating is taken.
3) the PBS solution dissolution of the former ascites half volume of precipitating, 4 DEG C are dialysed 2 days, and every 6h replaces a dialyzate.Thoroughly
After the completion of analysis, freeze-drying, -40 DEG C of preservations.The dialyzate is PBS buffer solution.
Six, the identification of monoclonal antibody
Using monoclonal antibody type detection kit (being purchased from Sigma, product number ISO2-1KT) in step 5
2 obtained monoclonal antibody solutions in the hypotype of monoclonal antibody detected.
The result shows that: the hypotype of the monoclonal antibody of 2 in step 5 obtained anti-Rheins is IgG1 type.
Embodiment 2 utilizes the cross reaction of Indirect Competitive ELISA method measurement monoclonal antibody
Main dissociated anthraquinone constituents in rhubarb medicinal material are Rhein, rheum emodin, Physcion, Chrysophanol, aloe
5 kinds of rheum emodin, and furthermore also contain in rheum officinale in " Chinese Pharmacopoeia " also with this 5 kinds of ingredients as the index ingredient of rhubarb medicinal material
There are the flavones ingredients such as Sennoside A, Sennoside B；Ponticin is the main feature ingredient for rhubarb medicinal material authenticity.Cause
Monoclonal antibody and Rhein, rheum emodin, Physcion, Chrysophanol, aloe-emodin, sennoside are determined in this this experiment
A, the cross reaction of Sennoside B and ponticin.Specific step is as follows:
1, it is coated with: taking 96 hole elisa Plates, be coated with using Rhein-OVA solution, 100 holes μ L/；Rhein-OVA is molten
The concentration of liquid is 62.5ng/mL；37 DEG C are coated with 3 hours, are washed 4 times with cleaning solution.
2, product are loaded: being added following compound solutions of 50 μ L in every hole respectively: rheum officinale acid solution, rheum emodin solution,
Physcion solution, rheum officinale phenol solution, aloe-emodin solution, Sennoside A solution, Sennoside B solution and ponticin are molten
Liquid, the solvent of above compound solution are sample diluting liquid.Wherein, rheum officinale acid solutions be respectively as follows: 0.5ng/mL,
0.25ng/mL, 0.125ng/mL, 0.0625ng/mL, 0.03125ng/mL, 0.015625ng/mL, 0.0078125ng/mL and
0.00390625ng/mL；Aloe-emodin solution concentration be respectively as follows: 2ng/mL, 1ng/mL, 0.5ng/mL, 0.25ng/mL,
0.125ng/mL, 0.0625ng/mL, 0.03125ng/mL and 0.015625ng/mL；Rheum emodin solution concentration is respectively as follows: 20ng/
ML, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.3125ng/mL and 0.15625ng/mL；Greatly
Yellow phenol solution concentration be respectively as follows: 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL,
0.3125ng/mL and 0.15625ng/mL；Physcion solution concentration is respectively as follows: 1000ng/mL, 500ng/mL, 125ng/
ML, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL, 7.8125ng/mL and 3.90625ng/mL；Sennoside A solution concentration
Respectively 40000ng/mL, 20000ng/mL, 10000ng/mL, 5000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL
And 312.5ng/mL；Sennoside B solution concentration be respectively 40000ng/mL, 20000ng/mL, 10000ng/mL, 5000ng/mL,
2500ng/mL, 1250ng/mL, 625ng/mL and 312.5ng/mL；Ponticin solution concentration be respectively 40000ng/mL,
20000ng/mL, 10000ng/mL, 5000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL and 312.5ng/mL；Control
Hole is the sample diluting liquid of 50 μ L.
3, add antibody: the monoclonal antibody of 2 obtained anti-Rheins in the step of 50 embodiment 1 μ L five is added in every hole
Dilution (250ng/mL)；Three multiple holes are arranged in every kind of dilution；Set in wet box 30min under the conditions of 37 DEG C, board-washing 4 times.
4, add ELIAS secondary antibody: by sheep anti mouse ELIAS secondary antibody IgG-HRP, (0.1mg/mL is purchased from Jackson company, commodity mesh
79556) to dilute 1000 times, every hole adds 100 μ L for record number, sets in wet box 30min under the conditions of 37 DEG C, and board-washing 4 times.
5, it develops the color: taking 20mg o-phenylenediamine (OPD) to be dissolved in 10mL substrate dilution, add 4 μ L 30%H2O2, substrate is molten
Liquid is added in ELISA Plate, every 100 μ L of hole.It is protected from light colour developing 15min.
6, terminate: 50 μ L terminate liquids are added in every hole, with the OD value for measuring each hole at microplate reader 492nm.
Using compound solution concentration as abscissa, OD/OD0(light absorption value when OD value is indicated containing compound, OD0It indicates not
Light absorption value when containing compound) it is ordinate, Rhein, rheum emodin, rheum emodin are calculated separately with 8 software of OriginPro
Methyl ether, Chrysophanol, aloe-emodin, Sennoside A, Sennoside B and ponticin IC50It is anti-that intersection is calculated according to the following formula in value
It should rate: cross reacting rate (%)=(IC50(Rhein)/IC50(rheum officinale acid-like substance)) × 100%.
The results are shown in Table 2: the monoclonal antibody of anti-Rhein is 27.039% to the cross reacting rate of aloe-emodin；
Cross reacting rate to rheum emodin is 0.887%；Cross reacting rate to Chrysophanol is 0.114%；Friendship to Physcion
Pitching reactivity is 0.310%；Cross reacting rate to Sennoside A is 0.005%；It is anti-without intersecting to Sennoside B and ponticin
It answers, illustrates that monoclonal antibody specificity of the invention is strong.
The cross reacting rate of table 2, the monoclonal antibody of anti-Rhein and Rhein and its structurally similar compounds
The sensitivity technique of embodiment 3, monoclonal antibody
Standard curve is established by indirect competitive ELISA experiment, and determines that detection sensitivity is 0.04ng/mL, it is linear to examine
Range is surveyed in 0.02~0.11ng/mL.The specific method is as follows:
1, it is coated with: taking 96 hole elisa Plates, be coated with using Rhein-OVA solution, 100 holes μ L/；Rhein-OVA is molten
The concentration of liquid is 62.5ng/mL；37 DEG C are coated with 3 hours, are washed 4 times with cleaning solution.
2, add standard solution and sample solution: 50 μ L Rhein standard solutions and testing sample solution is added in every hole,
Rhein standard solution concentration is successively are as follows: 0.5ng/mL, 0.25ng/mL, 0.125ng/mL, 0.0625ng/mL,
0.03125ng/mL, 0.015625ng/mL, 0.0078125ng/mL and 0.00390625ng/mL；Control wells are the sample of 50 μ L
Dilution.
3, add antibody: the monoclonal antibody of 2 obtained anti-Rheins in the step of 50 embodiment 1 μ L five is added in every hole
Dilution (is diluted to 500ng/mL with sample diluting liquid)；Set in wet box 30min under the conditions of 37 DEG C, board-washing 4 times.
4, add ELIAS secondary antibody: by sheep anti mouse ELIAS secondary antibody IgG-HRP, (0.1mg/mL is purchased from Jackson company, commodity mesh
79556) to dilute 1000 times, every hole adds 100 μ L for record number, sets in wet box 30min under the conditions of 37 DEG C, and board-washing 4 times.
5, it develops the color: taking 20mg o-phenylenediamine (OPD) to be dissolved in 10mL substrate dilution, add 4 μ L 30%H2O2, substrate is molten
Liquid is added in ELISA Plate, every 100 μ L of hole.It is protected from light colour developing 15min.
6, terminate: 50 μ L terminate liquids are added in every hole, with the OD value for measuring each hole at microplate reader 492nm.
7, standard curve is drawn: using the Rhein standard solution of various concentration as abscissa, B/B0(B is indicated containing big
OD value when yellow acid, B0Indicate OD value when control wells) it is ordinate, with 8 Software on Drawing standard curve of OriginPro.It is real
It tests and sets 3 repetitions, take the average value of experimental result three times, obtained standard curve is as shown in Figure 2.Calibration curve equation is y=
0.05023+(0.96399-0.05023)/(1+(x/0.046)^1.8303)(R2=0.99118).
8, by the standard curve established, B/B is defined0Corresponding Rhein standard concentration is detection when numerical value is 0.5
Sensitivity (i.e. 0.04ng/mL) defines B/B0Numerical value Rhein standard concentration range corresponding when being respectively 0.2 and 0.8
For the linear detection range (i.e. 0.02~0.11ng/mL) of the detection method.
Embodiment 4, the enzyme linked immunological kit for detecting Rhein and its application
One, the preparation of kit
The enzyme linked immunological kit of detection Rhein of the invention is by Rhein-OVA solution (coating antigen), anti-Rhein
Monoclonal antibody, sheep anti mouse ELIAS secondary antibody IgG-HRP, coating buffer, cleaning solution, standard solution, substrate buffer solution and end
Only buffer and confining liquid composition.Wherein, Rhein-OVA solution is that the step one in embodiment 1 is prepared；Anti- rheum officinale
2 in the step of monoclonal antibody of acid is embodiment 1 five are prepared.
Two, application of the monoclonal antibody of anti-Rhein in test sample on Rhein Content
Using kit described in step 1 respectively to sample to be tested: sorrel, Rheum tanguticum Maxim, Rheum officinale, soil
Rhein Content in rheum officinale, North China rheum officinale (deriving from National Institute for Food and Drugs Control) is detected, and specific steps are such as
Under:
1, sample to be tested is crushed, through methanol ultrasonic extraction, obtains extracting solution；Into extracting solution plus appropriate amount of sample dilutes
Liquid obtains testing sample solution.
2, it is coated with: taking 96 hole elisa Plates, be coated with using Rhein-OVA solution, 100 holes μ L/；Rhein-OVA is molten
The concentration of liquid is 62.5ng/mL；37 DEG C are coated with 3 hours, are washed 4 times with cleaning solution.
3, add standard solution and sample solution: 50 μ L Rhein standard solutions and testing sample solution is added in every hole,
Rhein standard solution concentration is successively are as follows: 0.5ng/mL, 0.25ng/mL, 0.125ng/mL, 0.0625ng/mL,
0.03125ng/mL, 0.015625ng/mL, 0.0078125ng/mL and 0.00390625ng/mL；Control wells are the sample of 50 μ L
Dilution.
4, add antibody: the monoclonal antibody of 2 obtained anti-Rheins in the step of 50 embodiment 1 μ L five is added in every hole
Dilution (is diluted to 500ng/mL with sample diluting liquid)；Set in wet box 30min under the conditions of 37 DEG C, board-washing 4 times.
5, add ELIAS secondary antibody: by sheep anti mouse ELIAS secondary antibody IgG-HRP, (0.1mg/mL is purchased from Jackson company, commodity mesh
79556) to dilute 1000 times, every hole adds 100 μ L for record number, sets in wet box 30min under the conditions of 37 DEG C, and board-washing 4 times.
6, it develops the color: taking 20mg o-phenylenediamine (OPD) to be dissolved in 10mL substrate dilution, add 4 μ L 30%H2O2, substrate is molten
Liquid is added in ELISA Plate, every 100 μ L of hole.It is protected from light colour developing 15min.
7, terminate: 50 μ L terminate liquids are added in every hole, with the OD value for measuring each hole at microplate reader 492nm.
8, standard curve is drawn: using the Rhein standard solution of various concentration as abscissa, B/B0(B is indicated containing big
OD value when yellow acid, B0Indicate OD value when control wells) it is ordinate, with 8 Software on Drawing standard curve of OriginPro.It is real
It tests and sets 3 repetitions, take the average value of experimental result three times, obtained standard curve is as shown in Figure 2.Calibration curve equation is y=
0.05023+(0.96399-0.05023)/(1+(x/0.046)^1.8303)(R2=0.99118).
9, by the B/B of sample to be tested0It substitutes into standard curve, is calculated in sample to be tested greatly according to calibration curve equation
The content of yellow acid.
The testing result of sorrel, Rheum tanguticum Maxim, Rheum officinale, Rumex madaio, Rhein Content in the rheum officinale of North China
As shown in table 3.Wherein, the content of Rhein is 0.653 ± 0.071mg/g in sorrel；Rhein in Rheum tanguticum Maxim
Content is 3.587 ± 0.065mg/g；The content of Rhein is 1.200 ± 0.054mg/g in Rheum officinale；Rheum officinale in Rumex madaio
The content of acid is 0.708 ± 0.009mg/g；Rhein Content in the rheum officinale of North China is 0.042 ± 0.005mg/g.
Rhein Content testing result in table 3, rheum officinale sample
Claims (9)
1. the monoclonal antibody of anti-Rhein is generated by the hybridoma cell strain that deposit number is CGMCC No.10582.
2. the hybridoma cell strain RH1F8, deposit number CGMCC of the monoclonal antibody of one plant of anti-Rhein of secretion
No.10582。
3. monoclonal antibody described in claim 1 or hybridoma cell strain RH1F8 as claimed in claim 2 in preparation detection or
The reagent of Rhein Content or the application in kit in auxiliary detection sample to be tested.
4. monoclonal antibody described in claim 1 or hybridoma cell strain RH1F8 as claimed in claim 2 are being detected or are being assisted
Detect the application in sample to be tested in Rhein Content.
5. monoclonal antibody described in claim 1 or hybridoma cell strain RH1F8 as claimed in claim 2 in preparation detection or
Application in auxiliary detection sample to be tested in the colloidal gold strip of Rhein Content.
6. the enzyme linked immunological kit of Rhein Content, the power including independent packaging in a kind of detection or auxiliary detection sample to be tested
Benefit require 1 described in monoclonal antibody.
7. enzyme linked immunological kit according to claim 6, it is characterised in that: the sample is rheum officinale；The rheum officinale tool
Body is that the rheum officinale is specially sorrelRheum palmatumL., Rheum tanguticum MaximRheum tanguticum
Maxim.ex Balf., Rheum officinaleRheum offcihale Baill., Rumex madaioRheum tanguticum Maxim.ex
Or North China rheum officinale Rhei Franzenbachii Radix Balf..
8. enzyme linked immunological kit described in claim 6 or 7 is in detecting or assisting detection sample to be tested in Rhein Content
Application.
9. application according to claim 8, it is characterised in that: the sample is rheum officinale；The rheum officinale is specially that palm leaf is big
It is yellowRheum palmatumL., Rheum tanguticum MaximRheum tanguticum Maxim.ex Balf., Rheum officinaleRheum offcihale Baill., Rumex madaioRheum tanguticum Maxim.ex Balf. or North China rheum officinale Rhei
Franzenbachii Radix。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003000246A1 (en) * | 2001-06-25 | 2003-01-03 | Arakis Ltd. | The use of rhein and derivatives thereof in pain treatment |
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2015
- 2015-05-14 CN CN201510246118.7A patent/CN104892757B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003000246A1 (en) * | 2001-06-25 | 2003-01-03 | Arakis Ltd. | The use of rhein and derivatives thereof in pain treatment |
Non-Patent Citations (2)
| Title |
|---|
| 大黄酸人工抗原合成及免疫原性鉴定;张波等;《中国中药杂志》;20150430;第40卷(第8期);1463-1467 * |
| 甘草酸、柚皮苷单克隆抗体的制备及其酶联免疫分析方法的建立;张越;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20130815(第08期);E057-74 * |
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