CN103698520A - Kit for quantitatively detecting alpha-glucan by monoclonal antibody immunonephelometry - Google Patents

Kit for quantitatively detecting alpha-glucan by monoclonal antibody immunonephelometry Download PDF

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CN103698520A
CN103698520A CN201310625649.8A CN201310625649A CN103698520A CN 103698520 A CN103698520 A CN 103698520A CN 201310625649 A CN201310625649 A CN 201310625649A CN 103698520 A CN103698520 A CN 103698520A
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sample
antibody
alpha
concentration
glucans
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梁达奉
曾练强
柳颖
蚁细苗
马步
林荣珍
黄曾慰
李雨虹
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GUANGXI STATE FARMS SUGAR INDUSTRIAL GROUP Co Ltd
Guangzhou Sugarcane Industry Research Institute
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GUANGXI STATE FARMS SUGAR INDUSTRIAL GROUP Co Ltd
Guangzhou Sugarcane Industry Research Institute
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Abstract

The invention discloses a kit for quantitatively detecting alpha-glucan by monoclonal antibody immunonephelometry. The kit comprises alpha-glucan antibody freeze-drying powder, an antibody diluent, a sample diluent and a standard substance, wherein the alpha-glucan antibody freeze-drying powder is freeze-drying powder of IgM type monoclonal antibody purified in mouse ascites where a hybridoma cell strain D9 with a preservation number of CCTCCC2010107 (China Center for Type Culture Collection C2010107) is injected. The quantitative detection kit provided by the invention has the characteristics of simpliness and convenience in operation, good repeatability, high sensitivity and simple test conditions, is suitable for detection of an alpha-glucan immune complex separated out from a determination reaction system, and is suitable for sugaring processes of cane juice, raw sugar, white granulated sugar, brown granulated sugar, syrup, molasses and the like.

Description

Monoclonal antibody immunity is than the kit of turbid quantitative detection alpha-glucans
Technical field
The present invention relates to a kind of macromolecule immue quantitative detection reagent box, particularly monoclonal antibody immunity is than the kit of turbid quantitative detection alpha-glucans.
Background technology
Alpha-glucans is a kind of polysaccharide being comprised of glucose monomer completely, molecular formula ((C 6h 10o 5) n), molecular weight generally several ten thousand between millions of, be generally white indefiniteness powder solid, be glue-like, viscosity increases with the increase of molecular weight and concentration, odorless, tasteless is soluble in water, dissolubility declines with the increase of molecular weight.Can stable existence in normal temperature, neutral solution, meet strong acid and decompose, in alkalescence is dissolved, its end group is easily oxidized, variable color or decomposition gradually while being heated.
From sugarcane juice to purified product, sugar refining technology process exists the harmful effect that glucosan causes from start to finish, glucosan can cause sugar part loss, obviously increase the viscosity of liquid glucose, reduce the filterableness of liquid glucose, while making crystallization of sucrose, generate the crystal of abnormal morphology, increase refining cost, also have a strong impact on sugared quality, affect the applicability of product simultaneously.Therefore, the Real-Time Monitoring of glucosan becomes an important technology of sugar refinery production control.
The general available Haze/ICUMSA method of the mensuration of alpha-glucans, Robert ' s copper method, Potical Activity Ltd.DASA method, enzymatic isolation method etc. in sugar industry.But these methods have larger limitation, as shortcomings such as accuracy is not high, complex operation, inapplicable all sugar products.
It is high that immunological detection has specificity, easy to operate, and the advantages such as credible result are the main development directions of the quantitative test of current alpha-glucans.Yet the antibody that lacks excellent performance in prior art, does not have the kit of actual available quantitative detection alpha-glucans yet.
Summary of the invention
The object of the present invention is to provide a kind of monoclonal antibody immunity than the kit of turbid quantitative detection alpha-glucans.
The technical solution used in the present invention is:
A kind of kit of quantitative detection alpha-glucans, comprise alpha-glucans antibody freeze dried powder, antibody diluent, sample diluting liquid and standard items, alpha-glucans antibody freeze dried powder is the freeze-dried powder of the IgM type monoclonal antibody of purifying from inject the mouse ascites that deposit number is CCTCC C2010107 hybridoma cell strain D9.
As a further improvement on the present invention, sample diluting liquid is that pH is 6.5～7.8, concentration is the PBS solution of 0.01～0.02mol/L, is added with mass concentration for 0.1～5% reaction promoter in PBS solution, and reaction promoter is non-ionics or cationic surfactant.
As a further improvement on the present invention, the non-ionics using in above-mentioned detection kit is selected from PEG2000, PEG4000, PEG6000, tween.Cationic surfactant is selected from DTAC, dodecyl benzyl dimethyl ammonium chloride, hexadecyltrimethylammonium chloride, OTAC.
As a further improvement on the present invention, the antibody diluent using in above-mentioned detection kit for pH be 6.5～7.8, the PBS solution that concentration is 0.01～0.02mol/L.
A method for quantitative detection alpha-glucans, comprises the steps:
1) in testing tube, add 1.0mL antibody-solutions, put into turbidimeter standing, after 2min, draw reading N mark 0; In above-mentioned antibody-solutions, adding 10 μ L concentration is C 0alpha-glucans standard solution, mix, avoid producing bubble, reading after 2min, records its turbidity data N mark 1;
2), by sample dissolution, in dilute sample, alpha-glucans content does not filter higher than 500mg/L and with 0.45 μ m filtrator;
3) in testing tube, add 1.0mL antibody-solutions, put into turbidimeter standing, after 2min, draw reading N sample 0; The sample solution that adds 10 μ L to filter in above-mentioned antibody-solutions adds wherein, mixes, and avoids producing bubble, and reading after 2min records its turbidity data N sample 1;
4) the alpha-glucans concentration in the sample solution calculating, computing method are as follows:
Δ NTU mark=N mark 1-N mark 0;
Conversion coefficient CF=C 0/ Δ NTU mark;
Δ NTU sample=N sample 1-N sample 0;
Concentration=Δ NTU of alpha-glucans in sample solution sample* CF;
Or drawing standard concentration curve in advance, according to normal concentration opisometer, calculate afterwards the concentration of alpha-glucans in sample solution;
The reagent using in above-mentioned testing process as mentioned above.
As a further improvement on the present invention, the concentration of antibody-solutions is 0.2～2mg/L.
The invention has the beneficial effects as follows:
Immue quantitative detection reagent box of the present invention, have easy and simple to handle, there is reproducible, highly sensitive, the simple feature of test condition, be suitable for the alpha-glucans immune complex of separating out in assaying reaction system, the sugar refining technology processes such as sugarcane juice, raw sugar, white granulated sugar, brown granulated sugar, syrup, molasses.
Accompanying drawing explanation
Fig. 1 alpha-glucans content immunoturbidimetry range of linearity.
Embodiment
Below in conjunction with embodiment, further illustrate the present invention.Each component content in following examples, except indicating clear and definite unit, is shared mass percent; The preparation method of the monoclonal antibody of using is ascites method, and concrete operations are as follows:
1) adopt the healthy and strong Balb/c mouse that grows up, before injection hybridoma 1 week, lumbar injection 0.5mL sterilized liquid paraffin in advance;
2) every mouse lumbar injection is containing 1～5 * 10 6hybridoma D9(deposit number is CCTCC C2010107) the salt solution of 0.5mL; When mouse produces ascites, abdomen puncture is emitted ascites, and centrifugal ascites is removed cell, the centrifugal 15min of 3000rpm;
3) aseptic storage or add 0.01% Sodium azide to be placed in-20 ℃ of refrigerators frozen standby;
4) antibody purification:
The purifying of antibody is taked affinity chromatography, and operation steps is as follows:
(1) with Buffer A(0.05mol/L Boric ocid, 4.0mol/L NaCL, PH9.0) balance pillar;
(2) add a small amount of ascites, with Buffer A, rinse pillar;
(3) use again Buffer B(0.05mol/L Sodium phosphate, 0.05mol/L Sodium citrate, 0.3mol/L NaCl, pH3.0) wash-out;
(4) collect the 1.0mol/L Tris-HCL that albumen adds 5% volume, pH8.0 regulates pH value;
(5) collection albumen carries out SDS-PAGE and carries out purity testing;
5) freeze-drying of antibody: pre-freeze is to below-20 ℃, in low vacuum freeze-drying 48 hours under 100Pa, obtains alpha-glucans antibody freeze dried powder.
Embodiment 1
Each reagent composed as follows:
1, sample diluting liquid
Phosphate (damping fluid) 100mmol/L(pH7.2)
PEG6000(surfactant) 5%
KCl(electrolyte) 10%
2, antibody diluent
Phosphate (damping fluid) 100mmol/L(pH7.2)
KCl(electrolyte) 5%
3, standard items
Phosphate (damping fluid) 100mmol/L(pH7.2)
PEG6000(surfactant) 5%
KCl(electrolyte) 10%
Glucosan T-2000500mg/L
4, antibody freeze dried powder
Glucosan antibody 20mg
What this example was selected is high concentration single-point normative reference product, and it is 500mg/L that glucosan T-2000 adds concentration, then uses 0.45 μ m membrane filtration, 2-8 ℃ of preservation.During use, with sample diluting liquid, be diluted to the standard items of variable concentrations gradient, drawing standard curve.Get antibody diluent 10mL and mix and shake up with antibody freeze dried powder, after standing 5min, as single agents, use.
Embodiment 2
Each reagent composed as follows:
1, sample diluting liquid
Phosphate (damping fluid) 50mmol/L(pH7.4)
DTAC 0.5%
NaCl(electrolyte) 10%
2, antibody diluent
Phosphate (damping fluid) 50mmol/L(pH7.4)
NaCl(electrolyte) 5%
3, standard items
Phosphate (damping fluid) 50mmol/L(pH7.4)
DTAC 0.5%
NaCl(electrolyte) 10%
Glucosan T-2000500mg/L
4, antibody freeze dried powder
Glucosan antibody 20mg
What this example was selected is high concentration single-point normative reference product, and it is 500mg/L that glucosan T-2000 adds concentration, then uses 0.45 μ m membrane filtration, 2-8 ℃ of preservation.During use, with sample diluting liquid, be diluted to the standard items of variable concentrations gradient, drawing standard curve.Get antibody diluent 10mL and mix and shake up with antibody freeze dried powder, after standing 5min, as single agents, use.
Embodiment 3
Each reagent composed as follows:
1, sample diluting liquid
Phosphate (damping fluid) 100mmol/L(pH6.8)
Tween-80(surfactant) 5%
KCl(electrolyte) 10%
2, antibody diluent
Phosphate (damping fluid) 100mmol/L(pH6.8)
KCl(electrolyte) 5%
3, standard items
Phosphate (damping fluid) 100mmol/L(pH6.8)
Tween-80(surfactant) 5%
KCl(electrolyte) 10%
Glucosan T-2000500mg/L
4, antibody freeze dried powder
Glucosan antibody 20mg
What this example was selected is high concentration single-point normative reference product, and it is 500mg/L that glucosan T-2000 adds concentration, then uses 0.45 μ m membrane filtration, 2-8 ℃ of preservation.During use, with sample diluting liquid, be diluted to the standard items of variable concentrations gradient, drawing standard curve.Get antibody diluent 10mL and mix and shake up with antibody freeze dried powder, after standing 5min, as single agents, use.
Detection method:
For the purpose of convenient, in following test experience, the antibody concentration of institute is 1mg/L, and those skilled in the art can use the antibody of other concentration as required, as long as the concentration of antibody is enough to react with glucosan the precipitation of generation q.s.Preferably, the concentration of antibody is at 0.2～2mg/L.
The composition of the kit below using in experiment is identical with the formula of embodiment 3.
1, Specification Curve of Increasing:
According to the requirement of technology path, calculate dextran standard water cut.Take the glucosan of undried, make it contain glucosan 0.100g without moisture in beaker, after fully dissolving, be surely dissolved in the volumetric flask of 100mL, use PBS liquid that pH is 6.8 to be mixed with the glucosan standard solution of 1.0mg/mL.Configuration concentration is 800mg/L, 600mg/L, 500mg/L, 400mg/L, 300mg/L, 200mg/L, 100mg/L, 50mg/L, 25mg/L, 12mg/L solution (this solution needs fresh preparation).
In testing tube, add 1.0mL antibody-solutions, put into turbidimeter standing, after 2min, draw reading N mark 0; In above-mentioned antibody-solutions, adding 10 μ L concentration is C 0alpha-glucans standard solution, mix, avoid producing bubble, reading after 2min, records its turbidity data N mark 1;
Δ NTU mark=N mark 1-N mark 0;
The concentration of each concentration dextran standard of take is horizontal ordinate, and Δ NTU is ordinate mapping (Fig. 1): analyzing known is that the following linear relationship of 500mg/L is good in concentration, and equation of linear regression is y=0.0759x+0.7437, coefficient R 2=0.9967; And concentration is the above departs from linear of 500mg/L, reason may be antibody quantity not sufficient.It is typical curve that the good part of the following linear relationship of 0.5mg/mL is used in this experiment.The results are shown in following table:
Figure BDA0000424917880000061
2, detect lower limit:
Take distilled water as blank sample, and replication 5,10,20mg/L standard items 6 times, compare and whether have significant difference with blank with t inspection statistics determination data, and the Cmin that defines significant difference is 10mg/L.The results are shown in following table:
Figure BDA0000424917880000062
Figure BDA0000424917880000071
T value is greater than 0.05 for difference is not remarkable, is less than 0.05 for significant difference, is less than 0.01 for difference is extremely remarkable, from the above results, only has 5mg/L and 0mg/L standard items difference not remarkable, can not offer an explanation all the other equal significant difference heteropole levels of signifiance.Therefore under the detection of this kit of deducibility, be limited to 10mg/L, at least can offer an explanation the difference of 5-10mg/L concentration.
3, repeatability:
Replication 500mg/L glucosan mark product and a raw sugar sample each 8 times, calculate relative standard deviation (RSD) value and be respectively 3.0% and 3.7%.The results are shown in following table:
500mg/L standard items ΔNTU Raw sugar sample mg/kg
1 42.7 1 356.2
2 45.5 2 333.0
3 41.2 3 353.7
4 44.3 4 353.7
5 44.0 5 373.2
6 43.1 6 366.0
7 43.3 7 374.1
8 42.5 8 359.4
RSD 3.0% RSD 3.7%
From above, can obtain, the RSD value of this kit measurement standard items and sample is all less than 5%, can meet testing requirement.
4, mark-on reclaims:
The raw sugar, sugarcane juice, the molasses sample that add a certain amount of glucosan mark product, calculation sample recovery of standard addition.Computing formula: the recovery=(measured value-former state beta-dextran content)/glucosan addition * 100%.Measurement result sees the following form:
Figure BDA0000424917880000081
From above, can obtain, the recovery of this method is fabulous, and deviation, in 2%, illustrates that the detection accuracy of this method is good.
5, alpha-glucans assay in sugar cube sample
1) in testing tube, add 1.0mL antibody-solutions, put into turbidimeter standing, after 2min, draw reading N mark 0;
2) with liquid-transfering gun, draw 10 μ L500mg/L dextran standards and add wherein, evenly mix, do not produce bubble, start timer, 2min reading just in time, record data N mark 1; Calculate Δ NTU mark(sample turbidity value-blank turbidity value)=N mark 1-N mark 0, conversion coefficient CF=500/ Δ NTU mark;
3) precision takes 5.000g raw sugar, white granulated sugar, brown granulated sugar, purified sugar sample respectively, with dilution, dissolves, and constant volume, in 10mL volumetric flask, makes need testing solution with 0.45 μ m membrane filtration;
4) in testing tube, add 1.0mL antibody-solutions, put into turbidimeter standing, after 2min, draw reading N sample 0;
5) with liquid-transfering gun, draw 10 μ L need testing solutions and add wherein, evenly mix, do not produce bubble, start timer, 2min reading just in time, record data N sample 1; Calculate Δ NTU(sample turbidity value-blank turbidity value)=N sample 1-N sample 0.
Beta-dextran content (mg/mL)=Δ NTU sample* CF * extension rate.Measurement result sees the following form:
Figure BDA0000424917880000082
The test that this kit reclaims by examination criteria product and sample mark-on, method of proof is reliable, accurately.This example is applied to kit the mensuration of beta-dextran content in the solid samples such as raw sugar, white granulated sugar, brown granulated sugar, purified sugar, has quick, accurate, easy feature, and the detection time of a sample is 5～10min approximately.
6, alpha-glucans assay in liquid sugar sample
Take respectively 1g sugarcane juice and molasses, with 0.45 μ m membrane filtration, make need testing solution, measure solution hammer degree Bx.Measure and computing method same " Specification Curve of Increasing ", measurement result sees the following form:
Sample Beta-dextran content mg/L
Sugarcane juice 1 1070.2
Sugarcane juice 2 551.0
Molasses 1 2533.7
Molasses 2 591.5
This example is applied to kit the mensuration of beta-dextran content in the various fluid samples such as sugarcane juice, molasses, has quick, accurate, easy feature, about 5-10min detection time of a sample.
The invention discloses a kind of kit of quantitative detection alpha-glucans, comprise alpha-glucans antibody freeze dried powder, antibody diluent, sample diluting liquid and standard items, alpha-glucans antibody freeze dried powder is the freeze-dried powder of the IgM type monoclonal antibody of purifying from inject the mouse ascites that deposit number is CCTCCC2010107 hybridoma cell strain D9.Immue quantitative detection reagent box of the present invention, have easy and simple to handle, there is reproducible, highly sensitive, the simple feature of test condition, be suitable for the alpha-glucans immune complex of separating out in assaying reaction system, the sugar refining technology processes such as sugarcane juice, raw sugar, white granulated sugar, brown granulated sugar, syrup, molasses.

Claims (7)

1. a kit that quantitatively detects alpha-glucans, comprise alpha-glucans antibody freeze dried powder, antibody diluent, sample diluting liquid and standard items, it is characterized in that: alpha-glucans antibody freeze dried powder is the freeze-dried powder of the IgM type monoclonal antibody of purifying from inject the mouse ascites that deposit number is CCTCC C2010107 hybridoma cell strain D9.
2. kit according to claim 1, it is characterized in that: described sample diluting liquid is that pH is 6.5～7.8, concentration is the PBS solution of 0.01～0.02mol/L, in PBS solution, be added with mass concentration for 0.1～5% reaction promoter, reaction promoter is non-ionics or cationic surfactant.
3. kit according to claim 2, is characterized in that: described non-ionics is selected from PEG2000, PEG4000, PEG6000, tween.
4. kit according to claim 2, is characterized in that: described cationic surfactant is selected from DTAC, dodecyl benzyl dimethyl ammonium chloride, hexadecyltrimethylammonium chloride, OTAC.
5. according to the kit described in claim 1～4 any one, it is characterized in that: described antibody diluent is that pH is 6.5～7.8, the PBS solution that concentration is 0.01～0.02mol/L.
6. quantitatively detect a method for alpha-glucans, comprise the steps:
1) in testing tube, add 1.0mL antibody-solutions, put into turbidimeter standing, after 2min, draw reading N mark 0; In above-mentioned antibody-solutions, adding 10 μ L concentration is C 0alpha-glucans standard solution, mix, avoid producing bubble, reading after 2 min, records its turbidity data N mark 1;
2), by sample dissolution, in dilute sample, alpha-glucans content does not filter higher than 500 mg/L and with 0.45 μ m filtrator;
3) in testing tube, add 1.0mL antibody-solutions, put into turbidimeter standing, after 2min, draw reading N sample 0; The sample solution that adds 10 μ L to filter in above-mentioned antibody-solutions adds wherein, mixes, and avoids producing bubble, and reading after 2 min records its turbidity data N sample 1;
4) the alpha-glucans concentration in the sample solution calculating, computing method are as follows:
Δ NTU mark=N mark 1-N mark 0;
Conversion coefficient CF=C 0/ Δ NTU mark;
Δ NTU sample=N sample 1-N sample 0;
Concentration=Δ NTU of alpha-glucans in sample solution sample* CF;
Or drawing standard concentration curve in advance, according to normal concentration opisometer, calculate afterwards the concentration of alpha-glucans in sample solution;
The reagent using in above-mentioned testing process is as described in claim 1～5 any one.
7. method according to claim 6, is characterized in that: the concentration of antibody-solutions is 0.2～2mg/L.
CN201310625649.8A 2013-11-28 2013-11-28 Kit for quantitatively detecting alpha-glucan by monoclonal antibody immunonephelometry Pending CN103698520A (en)

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Cited By (5)

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CN105527438A (en) * 2015-12-25 2016-04-27 广州甘蔗糖业研究所 Colloidal gold test strip for semi-quantitative detection of alpha-glucan, and detection method thereof
CN105628917A (en) * 2015-12-25 2016-06-01 广州甘蔗糖业研究所 Gold-colloid test strip for detecting alpha-glucosan and detection method
CN106405094A (en) * 2016-09-29 2017-02-15 广州华弘生物科技有限公司 Chemiluminiscence immune kit used for detecting TU M2-PK (Tumor M2-Pyruvate Kinase)
CN106483298A (en) * 2016-09-29 2017-03-08 广州华弘生物科技有限公司 For detecting the chemiluminescence immunoassay kit of Cyfra21-1 fragment
CN113201072A (en) * 2020-11-25 2021-08-03 广东省科学院生物工程研究所 Anti-dextran monoclonal antibody D24 and application thereof in determination of dextranase enzyme activity in sugar products

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105527438A (en) * 2015-12-25 2016-04-27 广州甘蔗糖业研究所 Colloidal gold test strip for semi-quantitative detection of alpha-glucan, and detection method thereof
CN105628917A (en) * 2015-12-25 2016-06-01 广州甘蔗糖业研究所 Gold-colloid test strip for detecting alpha-glucosan and detection method
CN106405094A (en) * 2016-09-29 2017-02-15 广州华弘生物科技有限公司 Chemiluminiscence immune kit used for detecting TU M2-PK (Tumor M2-Pyruvate Kinase)
CN106483298A (en) * 2016-09-29 2017-03-08 广州华弘生物科技有限公司 For detecting the chemiluminescence immunoassay kit of Cyfra21-1 fragment
CN106483298B (en) * 2016-09-29 2017-08-11 广州华弘生物科技有限公司 Chemiluminescence immunoassay kit for detecting cytokeratin 19 fragment
CN106405094B (en) * 2016-09-29 2018-03-06 广州华弘生物科技有限公司 For detecting the chemiluminescence immunoassay kit of knubble type M 2 pyruvate kinase
CN113201072A (en) * 2020-11-25 2021-08-03 广东省科学院生物工程研究所 Anti-dextran monoclonal antibody D24 and application thereof in determination of dextranase enzyme activity in sugar products
CN113201072B (en) * 2020-11-25 2022-09-13 广东省科学院生物工程研究所 Anti-dextran monoclonal antibody D24 and application thereof

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