CN103558176A - Method for quantitatively detecting coagulant activity of bean phytohemagglutinin - Google Patents
Method for quantitatively detecting coagulant activity of bean phytohemagglutinin Download PDFInfo
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- CN103558176A CN103558176A CN201310532286.3A CN201310532286A CN103558176A CN 103558176 A CN103558176 A CN 103558176A CN 201310532286 A CN201310532286 A CN 201310532286A CN 103558176 A CN103558176 A CN 103558176A
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- bean plant
- blood coagulation
- coagulation activity
- agglutinin
- bean
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- 244000046052 Phaseolus vulgaris Species 0.000 title claims abstract description 64
- 235000010627 Phaseolus vulgaris Nutrition 0.000 title claims abstract description 44
- 230000000694 effects Effects 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 30
- 108010047620 Phytohemagglutinins Proteins 0.000 title abstract 5
- 239000000701 coagulant Substances 0.000 title abstract 5
- 230000001885 phytohemagglutinin Effects 0.000 title abstract 5
- 239000006285 cell suspension Substances 0.000 claims abstract description 23
- 239000003726 plant lectin Substances 0.000 claims description 38
- 108010089814 Plant Lectins Proteins 0.000 claims description 32
- 230000023555 blood coagulation Effects 0.000 claims description 27
- 210000003743 erythrocyte Anatomy 0.000 claims description 18
- 238000002965 ELISA Methods 0.000 claims description 13
- 238000010790 dilution Methods 0.000 claims description 13
- 239000012895 dilution Substances 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 9
- 210000004369 blood Anatomy 0.000 claims description 9
- 239000008280 blood Substances 0.000 claims description 9
- 230000031700 light absorption Effects 0.000 claims description 9
- 238000002386 leaching Methods 0.000 claims description 8
- 239000002504 physiological saline solution Substances 0.000 claims description 7
- 210000004027 cell Anatomy 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 239000003146 anticoagulant agent Substances 0.000 claims description 3
- 229940127219 anticoagulant drug Drugs 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 239000004570 mortar (masonry) Substances 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 6
- 238000011002 quantification Methods 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract 1
- 101710186708 Agglutinin Proteins 0.000 description 5
- 101710146024 Horcolin Proteins 0.000 description 5
- 101710189395 Lectin Proteins 0.000 description 5
- 101710179758 Mannose-specific lectin Proteins 0.000 description 5
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 5
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 5
- 239000000910 agglutinin Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108090001090 Lectins Proteins 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
- 208000005374 Poisoning Diseases 0.000 description 2
- 230000004520 agglutination Effects 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 238000011179 visual inspection Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010016952 Food poisoning Diseases 0.000 description 1
- 208000019331 Foodborne disease Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for quantitatively detecting the coagulant activity of bean phytohemagglutinin and belongs to the technical field of plant component detection. According to the method, a red cell suspension for detection is subjected to cell quantification; meanwhile, a coagulant activity concentration standard curve is drawn by using the bean phytohemagglutinin with the known concentration as a standard substance, and the coagulant activity concentration of the bean phytohemagglutinin is further calculated, so that the coagulant activity of the bean phytohemagglutinin is quantified.
Description
Technical field
The invention belongs to plant component detection technique field, be specifically related to a kind of bean plant agglutinin blood coagulation activity quantitative detecting method.
Background technology
The phytolectin containing in Kidney bean is one of noxious material causing Bean Food Poisoning Analysis, and fresh bean beanpod especially, usually because cooking the improper generation that causes poisoning.Kidney bean is poisoning is multifactorial, wherein most important factor is to form many " bridges " that intersect because the agglutinin in Kidney bean can be combined in erythrocyte surface with the special glycosyl identification of erythrocyte surface selectivity, make red blood cell generation aggegation, and then cause the W-response of body.In different Bean Varieties, the content of phytolectin is not quite similar, and therefore can detect phytolectin activity in Kidney bean by red cell agglutination method, to instructing people to select Bean Varieties plantation and use that phytolectin activity is lower that foundation is provided.
Red cell agglutination method is the phytolectin detection method being most widely used at present.The animal erythrocytes such as that the method is utilized is natural, enzyme rabbit that process, that glutaraldehyde is fixing, sheep, people, be mixed with certain density red cell suspension, agglutinin solution is added to equal-volume red cell suspension after doubling dilution, after standing a period of time, utilize visual inspection and colourimetry to carry out activity under certain condition and judge.
The method of existing mensuration activity of lectin is mainly divided into two large classes: a class is to observe the qualitative analysis of hemagglutination effect, as V-arrangement ELISA Plate method, flat band method, test tube method etc.; Another kind of is with spectrophotometric, to take into account the semi-quantitative analysis method of the absorbance under the specific wavelength that microplate reader measures.There is following shortcoming in these methods: (1) can only carry out qualitative or semiquantitative determination; (2) preparation standard red cell suspension method differs, and multiplex percent by volume represents, poor reproducibility; (3) determine that to read the randomness of terminal time larger, what have has a visual inspection result, and subjectivity is large, and resultant error is also large, and degree of accuracy is low; (4) assay method is varied, and the result recording varies, and cannot compare.
Summary of the invention
A kind of method that provides high-throughput quantification to detect bean plant agglutinin blood coagulation activity for addressing the above problem is provided.
The technical solution adopted in the present invention is:
An agglutinin blood coagulation activity quantitative detecting method, said method comprising the steps of:
(1) to detecting, with red cell suspension, to carry out cell quantitative;
(2) with the bean plant gel element of concentration known, as standard items, draw blood coagulation activity concentration standard curve;
(3) calculate bean plant agglutinin blood coagulation activity concentration.
Further, described step (1) is specially:
(A) gather fresh rabbit blood and be placed in 5%～10% sodium citrate anticoagulant, mix with 4 times of volume A Shi (Alsever ' s) liquid that to be placed on 4 ℃ of Refrigerator stores standby;
(B) the fresh rabbit blood and the physiological saline that step (A) are obtained slowly mix in the ratio of 5:8, centrifugal 10 minutes of 800～1000rpm, collecting precipitation;
(C) repeat above-mentioned steps Washed Red Blood Cells, to supernatant fluid color be close to transparent after, by erythrocyte volume, be made into 20% red cell suspension.
(D) get the red cell suspension that step (C) obtains and after normal saline dilution, count red blood cell, and to adjust red blood cell concentration be 5 * 10
7individual/ml-5 * 10
8individual/ml, 4 ℃ of Refrigerator stores are standby.
Preferably, the fresh rabbit blood obtaining in described step (A) must not save backup over 14 days.
Further, described step (2) is specially:
(a) in ELISA Plate, by six dilutabilitys of bean plant agglutinin standard items doubling dilution of concentration known;
(b) to each hole of above-mentioned ELISA Plate, add quantitative red cell suspension, mix, and standing 10 minutes, at microplate reader 450nm place, read light absorption value, draw bean plant agglutinin blood coagulation activity concentration standard curve.
Further, in described step (a), the concentration of bean plant agglutinin standard items is 1mg/ml, and six dilutabilitys of doubling dilution are 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml and 0.03125mg/ml.
Further, described step (3) is specially:
(I) cleaned fresh bean plant after drying and weighed, and is placed in mortar, and adding mass volume ratio is the physiological saline of 1:1, in grinding on ice;
The bean plant that (II) obtains step (I) at 4 ℃, 8000rpm, centrifugal 10min, gets supernatant;
The supernatant that (III) obtains step (II), after standing 5 minutes, is got supernatant again, obtains bean plant agglutinin leaching liquor;
(IV) in ELISA Plate, three dilutabilitys of bean plant agglutinin leaching liquor doubling dilution that step (III) is obtained;
The red cell suspension that (V) adds step (1) to obtain to each hole of above-mentioned ELISA Plate, mix, and standing 10 minutes, at microplate reader 450nm place, read light absorption value, in the formula of the bean plant agglutinin blood coagulation activity concentration standard curve that this value substitution step (2) is obtained, calculate bean plant agglutinin blood coagulation activity concentration.
Preferably, described bean plant is Kidney bean beanpod or beans.
Preferably, described ELISA Plate is 96 holes.
The present invention has the following advantages:
This detection method step is simple, utilizes the method can carry out to bean plant the agglutinin blood coagulation activity detection of high-throughput quantification; It is quantitative that the red cell suspension of preparing has carried out cell, can guarantee the relatively consistent of measurement result between each batch; Detected result is more accurate simultaneously, and degree of error is little, and the result determining can compare mutually.
Accompanying drawing explanation
Fig. 1 is that in embodiment, Kidney bean phytolectin standard items are measured curve map.
Embodiment
Below in conjunction with drawings and embodiments, the present invention will be further described in detail.
(1) preparation standard red cell suspension and bioassay standard curve:
1. with heart blood collection method, gather fresh rabbit blood 2ml, be placed in 8% sodium citrate anticoagulant of 300ul, then mix and be placed on 4 ℃ of Refrigerator stores standby (in 2 weeks, red blood cell is normal, surpasses 2 weeks, and haemolysis appears in red blood cell) with 8ml Alsever ' s liquid.
2. take the fresh rabbit blood 2.5ml that Alsever ' s liquid is preserved, slowly mix in the ratio of 5:8 with physiological saline, centrifugal 10 minutes of 800-1000rpm, collects erythroprecipitin.
3. repeat after above-mentioned steps Washed Red Blood Cells five times, supernatant fluid color is completely transparent, then is made into 20% red cell suspension according to the volume of erythroprecipitin.
4. get the above-mentioned red cell suspension of 100ul with after 10 times of normal saline dilutions, get the red cell suspension 100ul after dilution, then dilute 10 times, then get the red cell suspension counting erythrocyte number of 15ul after dilute twice, adjusting red blood cell concentration is 5 * 10
7individual/ml-5 * 10
8individual/ml.
5. in 96 hole ELISA Plate, add the 1mg/ml bean plant agglutinin standard items of 100ul, and do 6 dilutabilitys of doubling dilution, drip variable concentrations (5 * 10
7individual/ml, 1 * 10
8individual/ml, 2 * 10
8individual/ml, 4 * 10
8individual/ml) 10ul red cell suspension, observes blood coagulation situation, and measures the light absorption value at 450nm place.
With reference to Fig. 1, result shows that red blood cell concentration is 2 * 10
8individual/during ml, blood coagulation reaction velocity is moderate, and the linearly dependent coefficient of the typical curve of making according to the light absorption value at 450nm place is greater than 0.99.
(2) preparation of bean plant agglutinin leaching liquor
1. fresh Kidney bean beanpod or beans are weighed after cleaning and drying, and are placed in mortar, and adding mass volume ratio is the physiological saline of 1:1, in grinding on ice.
2. at 4 ℃, the centrifugal 10min of 8000r/min, gets supernatant.
3. after standing 5 minutes, again get supernatant, be bean plant agglutinin leaching liquor.
(3) bean plant activity of lectin concentration determination
1. in 96 hole ELISA Plate, add the Kidney bean beanpod leaching liquor of 58 kinds of different cultivars respectively
100ul。
2. in each hole, add 2 * 10
8the red cell suspension 10ul of individual/ml, mixes, and standing 10 minutes, at microplate reader 450nm place, read light absorption value, the formula of this value substitution typical curve is carried out to the calculating of bean plant agglutinin blood coagulation activity concentration, formula is as shown in Figure 1.
Higher for agglutinin content, exceed 15 kinds of Kidney bean beanpod leaching liquors of standard curve determination scope, with physiological saline, carry out 3 concentration gradients of doubling dilution, every hole 100ul, then add 2 * 10 in each hole
8the red cell suspension 10ul of individual/ml, mixes, and standing 10 minutes, at microplate reader 450nm place, read light absorption value, get the formula that light absorption value is positioned at the numerical value substitution typical curve of typical curve and carry out the calculating of bean plant agglutinin blood coagulation activity concentration.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (8)
1. a bean plant agglutinin blood coagulation activity quantitative detecting method, is characterized in that, said method comprising the steps of:
(1) to detecting, with red cell suspension, to carry out cell quantitative;
(2) with the bean plant gel element of concentration known, as standard items, draw blood coagulation activity concentration standard curve;
(3) calculate bean plant agglutinin blood coagulation activity concentration.
2. bean plant agglutinin blood coagulation activity quantitative detecting method according to claim 1, is characterized in that, described step (1) is specially:
(A) gather fresh rabbit blood and be placed in 5%～10% sodium citrate anticoagulant, mix with 4 times of volume A Shi liquid that to be placed on 4 ℃ of Refrigerator stores standby;
(B) the fresh rabbit blood and the physiological saline that step (A) are obtained slowly mix in the ratio of 5:8, centrifugal 10 minutes of 800～1000rpm, collecting precipitation;
(C) repeat above-mentioned steps Washed Red Blood Cells, to supernatant fluid color be close to transparent after, by erythrocyte volume, be made into 20% red cell suspension.
(D) get the red cell suspension that step (C) obtains and after normal saline dilution, count red blood cell, and to adjust red blood cell concentration be 5 * 10
7individual/ml-5 * 10
8individual/ml, 4 ℃ of Refrigerator stores are standby.
3. bean plant agglutinin blood coagulation activity quantitative detecting method according to claim 2, is characterized in that, the fresh rabbit blood obtaining in described step (A) must not save backup over 14 days.
4. bean plant agglutinin blood coagulation activity quantitative detecting method according to claim 1, is characterized in that, described step (2) is specially:
(a) in ELISA Plate, by six dilutabilitys of bean plant agglutinin standard items doubling dilution of concentration known;
(b) to each hole of above-mentioned ELISA Plate, add quantitative red cell suspension, mix, and standing 10 minutes, at microplate reader 450nm place, read light absorption value, draw bean plant agglutinin blood coagulation activity concentration standard curve.
5. bean plant agglutinin blood coagulation activity quantitative detecting method according to claim 4, it is characterized in that, in described step (a), the concentration of bean plant agglutinin standard items is 1mg/ml, and six dilutabilitys of doubling dilution are 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml and 0.03125mg/ml.
6. bean plant agglutinin blood coagulation activity quantitative detecting method according to claim 1, is characterized in that, described step (3) is specially:
(I) cleaned fresh bean plant after drying and weighed, and is placed in mortar, and adding mass volume ratio is the physiological saline of 1:1, in grinding on ice;
The bean plant that (II) obtains step (I) at 4 ℃, 8000rpm, centrifugal 10min, gets supernatant;
The supernatant that (III) obtains step (II), after standing 5 minutes, is got supernatant again, obtains bean plant agglutinin leaching liquor;
(IV) in ELISA Plate, three dilutabilitys of bean plant agglutinin leaching liquor doubling dilution that step (III) is obtained;
The red cell suspension that (V) adds step (1) to obtain to each hole of above-mentioned ELISA Plate, mix, and standing 10 minutes, at microplate reader 450nm place, read light absorption value, in the formula of the bean plant agglutinin blood coagulation activity concentration standard curve that this value substitution step (2) is obtained, calculate bean plant agglutinin blood coagulation activity concentration.
7. bean plant agglutinin blood coagulation activity quantitative detecting method according to claim 6, is characterized in that, described bean plant is Kidney bean beanpod or beans.
8. according to the bean plant agglutinin blood coagulation activity quantitative detecting method described in claim 4 or 6, it is characterized in that, described ELISA Plate is 96 holes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104237474A (en) * | 2014-08-20 | 2014-12-24 | 江汉大学 | Method for rapidly detecting toxicity of beans |
| CN104880541A (en) * | 2015-05-08 | 2015-09-02 | 吉林农业大学 | Micro-gel bead ELISA kit for determination of soybean agglutinin agglutination activity |
| CN111458301A (en) * | 2019-12-24 | 2020-07-28 | 江汉大学 | Kidney bean agglutinin detection kit |
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2013
- 2013-10-31 CN CN201310532286.3A patent/CN103558176B/en not_active Expired - Fee Related
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104237474A (en) * | 2014-08-20 | 2014-12-24 | 江汉大学 | Method for rapidly detecting toxicity of beans |
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| CN104880541A (en) * | 2015-05-08 | 2015-09-02 | 吉林农业大学 | Micro-gel bead ELISA kit for determination of soybean agglutinin agglutination activity |
| CN111458301A (en) * | 2019-12-24 | 2020-07-28 | 江汉大学 | Kidney bean agglutinin detection kit |
| CN111458301B (en) * | 2019-12-24 | 2023-12-19 | 江汉大学 | Bean lectin detection kit |
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