CN1912620A - ELISA detection method of glycinin and B2 conglycinic content - Google Patents
ELISA detection method of glycinin and B2 conglycinic content Download PDFInfo
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- CN1912620A CN1912620A CN 200610020036 CN200610020036A CN1912620A CN 1912620 A CN1912620 A CN 1912620A CN 200610020036 CN200610020036 CN 200610020036 CN 200610020036 A CN200610020036 A CN 200610020036A CN 1912620 A CN1912620 A CN 1912620A
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Abstract
An ELISA method for detecting content of glycinin and conglycinin includes using purified glycinin and conglycinin as antigen separately to obtain antibodies resisting glycinin and conglycinin, applying them separately in indirect suppressed enzyme linked immunification to detect content of glycinin and conglycinin in soybean and its related products.
Description
Technical field
The present invention relates to a kind of method that detects glycinin and soybean companion globulin content in soybean, dregs of beans and the Related product, specifically, the method that relates to glycinin and soybean companion globulin content in a kind of ELISA (enzyme-linked immunosorbent assay) detection by quantitative soybean, dregs of beans and the Related product.
Background technology
Dregs of beans is one of most important protein raw materials of feedstuff industry, but glycinin in the bean-dregs feed and soybean companion globulin have stronger antigenicity, are antigen proteins main in the dregs of beans.Its anti-oxidant action mainly comprises: owing to incomplete antigen protein is not directly absorbed as complete macro-molecular protein, rather than amino acid or polypeptide, reduced the utilization of feed protein; The needs of keeping have been improved owing to activated immune system; Increase the secretion of endogenous protein, cause the increase of fecal nitrogen; Allergic reaction appears in more sensitive animal, causes diarrhoea, and production performance descends even be dead.Aspect domestic animal, mainly concentrate on piglet and calf to containing soybean companion's globulin and the anaphylactoid research of globulin feed antigen.Because the raising of production level, the early stage feed supplement of calf, the weaned piglet time shifts to an earlier date significantly, and the raising of protein level and the widespread use of soybean and converted products thereof in the wean daily ration make piglet and calf wean back malabsorption and clinical diarrhoea become the great difficult problem in the production.
In recent years, antigen protein is more and more paid attention in the research of feed and animal nutrition, this is because the production needs that people require growth of animal to get satisfies food service industry sooner, the nutrient that requires the more acquisition of animal capable can digest, easily absorb thus, and ANFs is a kind of latency that limits nutritional utilization.In the soybean overwhelming majority ANFs composition, structure and character after deliberation more clearly, abroad, the method for qualitative and quantitative detection of various ANFs is comparative maturity; At home, except glycinin and soybean companion globulin, most ANFs have had the qualitative and method for quantitatively determining of comparative maturity in the soybean.But as most important two kinds of ANFs, the qualitative and quantitative detection of glycinin and soybean companion globulin becomes the focus of internal feed and animal nutrition research.
At present domesticly mainly do not carry out qualitative detection glycinin and soybean companion globulin, also not effectively, convenient, quantitative detecting method accurately with SDS-PAGE.
Summary of the invention
The objective of the invention is: overcome the shortcoming that SDS-PAGE detects, a kind of ELISA detection method that detects glycinin and soybean companion globulin content is provided, specifically will solve following problem:
1) detection of glycinin and soybean companion globulin content in the dregs of beans raw material;
2) detection of glycinin and soybean companion globulin content in the fermented bean dregs;
3) detection of glycinin and soybean companion globulin content in other bean-dregs feeds.
The object of the present invention is achieved like this: glycinin and companion's globulin in order to method purifying such as isoelectric precipitation and affinity chromatographys obtain antibody for the antigen immune White Rabbit, detect glycinin and soybean companion globulin content in soybean, dregs of beans and the Related product used as indirect inhibition ELISA.
Its concrete steps of the present invention are as follows:
1) defatted soy flour extracts the solution that obtains containing glycinin and soybean companion globulin in buffer A,
2) pH value of solution is transferred to 6.4 and obtain precipitating 1 and supernatant.Supernatant pH transfers to 4.8, obtains precipitating 2,
3) will precipitate 1 and be dissolved in the buffer B, cross solvent resistant column, obtain the glycinin of purifying,
4) will precipitate 2 and be dissolved in the buffer B, saltouing obtains precipitating 3,
5) will precipitate 3 and be dissolved in the buffer B, cross solvent resistant column, obtain the soybean companion globulin of purifying,
6) with glycinin and soybean companion globulin immune animal, obtain antiserum,
7) antibody purification of saltouing detects antibody titer,
8) detect glycinin and soybean companion globulin content in soybean, dregs of beans and the Related product with indirect inhibitory enzyme linked immunosorbent assay.
Buffer A of the present invention is the Tris-HCl damping fluid of 0.03-0.06mol/L, contains the 10mmol/L beta-mercaptoethanol, pH8.0.
Buffer B of the present invention is phosphate buffer (2.6mmol/L KH
2PO
4, 32.5mmol/L K
2HPO
4), contain the NaCl of 0.5mol/L, 10mmol/L beta-mercaptoethanol, pH8.0.
Gel filtration medium of the present invention is Sepharose 6B.
Immune animal of the present invention is healthy male new zealand white rabbit.
Immunization method of the present invention is the mixed immunity method.
Enzyme-linked immunoassay method of the present invention is indirect inhibitory enzyme linked immunosorbent assay.
Describe in further detail below glycinin of the present invention and soybean companion globulin content the ELISA detection method set up process and testing process is as follows:
1) with defatted soy flour and 0.03-0.06mol/L, the Tris-HCl buffer A of pH 8.0 is mixed, material-water ratio 1: 10-20, and 28-37 ℃ was extracted 1-2 hour.Collect supernatant 1.1pH transfers to 6.4,10 with supernatant, the centrifugal 20min of 000rpm.Obtain precipitating 1 and supernatant 2.
2) will precipitate the 1 usefulness phosphate buffer B (NaCl that contains 0.5mol/L, the 10mmol/L beta-mercaptoethanol, pH 8.0) dissolving, centrifugal collection supernatant is crossed Sepharose 6B, with phosphate buffer B (the same) wash-out, collect the solution of the corresponding absorption peak of stream, i.e. the glycinin of purifying (11S albumen).
3) glycinin (11S albumen) of purifying is packed into molecular cut off 8,000-12,000 bag filter is to PBS damping fluid dialysed overnight.Collect dislysate, concentrate, the aliquot packing, freeze drying is preserved.
4) pH with supernatant 2 modulates 4.8,10, the centrifugal 20min collecting precipitation 2 of 000rpm.To precipitate 2 usefulness with phosphate buffer damping fluid (the same) wash-out, dissolve, cross Sepharose6B.With phosphate buffer B (the same) wash-out, collect the solution of the corresponding absorption peak of stream, i.e. the soybean of purifying companion globulin (7S albumen).
5) soybean of purifying companion's globulin (7S albumen) is packed into molecular cut off 8,000-12,000 bag filter is to PBS damping fluid dialysed overnight.Collect dislysate, concentrate, the aliquot packing, freeze drying is preserved.
6) get two kinds of albumen of above-mentioned purifying and be made into the solution of 1-5mg/ml with the PBS damping fluid, respectively with the Freund's complete adjuvant emulsification of equivalent, the healthy male new zealand white rabbit of initial immunity, every White Rabbit dosage 1-1.5ml, immune position be that the both feet back is slapped subcutaneous.With the emulsion booster immunization of above two kinds of protein solutions and incomplete Freund, immunizing dose is a 1-1.5ml/ White Rabbit after two weeks, and immune position is two backs leg lymphonodi popliteis.Countercurrent electrophoresis or indirect ELISA detect antibody titer after the week again.Antiserum is collected in the suitable rear neck artery bloodletting of tiring.
7) with the Chinese People's Anti-Japanese Military and Political College legumin of collection and the antiserum salting out method purifying of the beans companion of Chinese People's Anti-Japanese Military and Political College globulin, detect the back packing in a small amount of tiring once more, a part of freeze drying preservation, another part mixes-20 ℃ of preservations in back with the aseptic glycerine of equivalent.
8) suppress the foundation of ELISA typical curve indirectly.To detect glycinin is example, its method is: with the glycinin optium concentration coated elisa plate of indirect ELISA detection, 4 ℃ of bags are spent the night, wash plate next day and glycinin is done 0 (reference) respectively, 50,500,1,000,5,000,10, the serial dilution of 000ng/ml is got 50 μ l and is added successively in the ELISA Plate hole, 6 repetitions of each gradient, add Chinese People's Anti-Japanese Military and Political College's legumin antibody 50 μ l that press the optimum dilution degree dilution then in each hole respectively, mixing is hatched 30-60min for 28-37 ℃, wash plate, every hole added 1: 10, and each 100 μ l of 000 enzyme mark goat anti-rabbit igg were hatched 1 hour, wash plate then, every hole adds tmb substrate 100 μ l, and behind 37 ℃ of colour developing 10-15min, every hole adds the sulfuric acid stop buffer of 50 μ l 2mol/L.Send into then and under the 450nm wavelength, detect every hole OD value in the microplate reader.Inhibiting rate (%)=(reference opening OD value-detection hole OD value) * 100/ reference opening OD value wherein.With the Detection of antigen of above gradient dilution to the OD value set up typical curve: be the longitudinal axis with the inhibiting rate, the logarithm of antigen concentration is a transverse axis.The typical curve method for building up of soybean companion globulin is the same.
The detection by quantitative of glycinin and soybean companion globulin.By method 8) set up typical curve, the testing protein solution dilution behind the suitable concn also by method 8) detect the OD value after doing 6 repetitions.OD value according to testing protein is found the actual concentrations that the corresponding concentration logarithm value converses protein solution again in typical curve.
Superiority of the present invention is that this method detects accurately, process is simple, highly sensitive, can satisfy the needs of glycinin and soybean companion globulin content in relevant enterprise unit batch detection soybean, dregs of beans and the Related product.
Description of drawings
Fig. 1 suppresses ELISA typical curve legend indirectly.
Wherein, inhibiting rate is the longitudinal axis, and the logarithm of antigen concentration is a transverse axis.
Embodiment
By the ELISA detection method of glycinin of the present invention and soybean companion globulin content, concrete implementation and operation step is as follows:
1) with defatted soy flour and 0.03mol/L, the Tris-HCl damping fluid of pH 8.0 mixes, material-water ratio 1: 20, and 37 ℃ were extracted 1 hour.Collect supernatant.PH transfers to 6.4,10 with supernatant, the centrifugal 20min of 000rpm.Obtain precipitating 1.
2) will precipitate with the phosphate buffer damping fluid (NaCl that contains 0.5mol/L, the 10mmol/L beta-mercaptoethanol, pH 8.0) dissolving, centrifugal collection supernatant is crossed Sepharose 6B, with phosphate buffer damping fluid (the same) wash-out, collect the solution of the corresponding absorption peak of stream, i.e. the glycinin of purifying (11S albumen).
3) glycinin (11S albumen) of purifying is packed into molecular cut off 8,000-12,000 bag filter is to PBS damping fluid dialysed overnight.Collect dislysate, concentrate, the aliquot packing, freeze drying is preserved.
4) get an amount of glycinin and be made into the solution of 5mg/ml with the PBS damping fluid, with the Freund's complete adjuvant emulsification of equivalent, the healthy male new zealand white rabbit of initial immunity, every White Rabbit dosage 1ml, immune position be that the both feet back is slapped subcutaneous.With the emulsion booster immunization of above-mentioned solution and incomplete Freund, immunizing dose is 11/ White Rabbit after two weeks, and immune position is two backs leg lymphonodi popliteis.A week back countercurrent electrophoresis or detect antibody titer again.Antiserum is collected in the suitable rear neck artery bloodletting of tiring.
5) with the antiserum salting out method purifying of the Chinese People's Anti-Japanese Military and Political College legumin of collecting, indirect ELISA detects the back packing in a small amount of tiring, and a part of freeze drying is preserved ,-20 ℃ of preservations after another part mixes with the aseptic glycerine of equivalent.
6) suppress the foundation of ELISA typical curve indirectly.Its method is: wrap by concentration 4 μ l/ml coated elisa plates with glycinin the best that indirect ELISA detects, every hole 100 μ l, 4 ℃ of bags are spent the night, wash plate next day and glycinin is done 0 (reference) respectively, 50,500,1,000,5,000,10, the dilution of 000ng/ml, getting 50 μ l adds in the ELISA Plate hole successively, 6 repetitions of each gradient add Chinese People's Anti-Japanese Military and Political College's legumin antibody 50 μ l of optimum dilution degree (1: 640, the 000) dilution that indirect ELISA detects then respectively in each hole, mixing, hatch 30min for 37 ℃, wash plate, every hole added 1: 10, each 100 μ l of 000 enzyme mark goat anti-rabbit igg, hatched 1 hour, and washed plate then, every hole adds tmb substrate 100 μ l, behind 37 ℃ of colour developing 15min, every hole adds the sulfuric acid stop buffer of 50 μ l 2mol/L.Send into then and under the 450nm wavelength, detect every hole OD value in the microplate reader.Inhibiting rate (%)=(reference opening OD value-detection hole OD value) * 100/ reference opening OD value wherein.With the Detection of antigen of above gradient dilution to the OD value set up typical curve: be the longitudinal axis with the inhibiting rate, the logarithm of antigen concentration is a transverse axis.The typical curve method for building up of soybean companion globulin is the same.
7) glycinin detection by quantitative.By method 8) set up typical curve, the testing protein solution dilution behind the suitable concn also by method 8) detect the OD value after doing 6 repetitions.OD value according to testing protein is found the actual concentrations that the corresponding concentration logarithm value converses protein solution again in typical curve.
Claims (7)
1, the ELISA detection method of glycinin and soybean companion globulin content, be that its uses isoelectric precipitation, saltouts and gel permeation chromatography purifying glycinin and soybean companion globulin obtain antibody as antigen, it is used separately as indirect inhibitory enzyme linked immunosorbent assay detects glycinin and soybean companion globulin content in soybean, dregs of beans and the Related product, its concrete steps are as follows:
1) defatted soy flour extracts the solution that obtains containing glycinin and soybean companion globulin in buffer A,
2) pH value of solution is transferred to 6.4 and obtain precipitating 1 and supernatant.Supernatant pH transfers to 4.8, obtains precipitating 2,
3) will precipitate 1 and be dissolved in the buffer B, cross solvent resistant column, obtain the glycinin of purifying,
4) will precipitate 2 and be dissolved in the buffer B, saltouing obtains precipitating 3,
5) will precipitate 3 and be dissolved in the buffer B, cross solvent resistant column, obtain the soybean companion globulin of purifying,
6) with glycinin and soybean companion globulin immune animal, obtain antiserum,
7) antibody purification of saltouing detects antibody titer,
8) detect glycinin and soybean companion globulin content in soybean, dregs of beans and the Related product with indirect inhibitory enzyme linked immunosorbent assay.
2, by the ELISA detection method of described glycinin of claim 1 and soybean companion globulin content, it is characterized in that: buffer A is the Tris-HCl damping fluid of 0.03-0.06mol/L, contains the 10mmol/L beta-mercaptoethanol, pH8.0.
3, by the ELISA detection method of described glycinin of claim 1 and soybean companion globulin content, it is characterized in that: buffer B is a phosphate buffer, contains the NaCl of 0.5mol/L, 10mmol/L beta-mercaptoethanol, pH8.0.
4, by the ELISA detection method of described glycinin of claim 1 and soybean companion globulin content, it is characterized in that: gel filtration medium is Sepharose 6B.
5, by the ELISA detection method of described glycinin of claim 1 and soybean companion globulin content, it is characterized in that: immune animal is healthy male new zealand white rabbit.
6, by the ELISA detection method of described glycinin of claim 1 and soybean companion globulin content, it is characterized in that: immunization method is the mixed immunity method.
7, by the ELISA detection method of described glycinin of claim 1 and soybean companion globulin content, it is characterized in that: enzyme-linked immunoassay method is indirect inhibitory enzyme linked immunosorbent assay.
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| CN 200610020036 CN1912620A (en) | 2006-08-24 | 2006-08-24 | ELISA detection method of glycinin and B2 conglycinic content |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102879585A (en) * | 2012-09-28 | 2013-01-16 | 安徽农业大学 | Indirect ELISA (enzyme linked immunosorbent assay) method for detecting soybean antigenic protein serum antibody |
| CN105004863A (en) * | 2015-07-08 | 2015-10-28 | 河南省农业科学院 | Colloidal gold immunochromatography test paper for rapidly detecting soybean sensitizing protein beta-conglycinin, and production method thereof |
-
2006
- 2006-08-24 CN CN 200610020036 patent/CN1912620A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102879585A (en) * | 2012-09-28 | 2013-01-16 | 安徽农业大学 | Indirect ELISA (enzyme linked immunosorbent assay) method for detecting soybean antigenic protein serum antibody |
| CN105004863A (en) * | 2015-07-08 | 2015-10-28 | 河南省农业科学院 | Colloidal gold immunochromatography test paper for rapidly detecting soybean sensitizing protein beta-conglycinin, and production method thereof |
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