CN101315384A - Method for detecting immunoglobulinlg content in cow's milk - Google Patents
Method for detecting immunoglobulinlg content in cow's milk Download PDFInfo
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- CN101315384A CN101315384A CNA2008100819008A CN200810081900A CN101315384A CN 101315384 A CN101315384 A CN 101315384A CN A2008100819008 A CNA2008100819008 A CN A2008100819008A CN 200810081900 A CN200810081900 A CN 200810081900A CN 101315384 A CN101315384 A CN 101315384A
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Abstract
The invention relates to a method for detecting components in milk, in particular to a detection method for detecting the content of milk immunoglobulin (IgG), and belongs to the detection technology field. The detection method for detecting the content of the milk immunoglobulin (IgG) through a capillary electrophoresis instrument of the invention comprises the steps of sample pretreatment and sample loading detection, reading calibration, and report editing, etc. In the invention, the electrophoresis of a sample runs on the capillary electrophoresis instrument, and the absorption value of the sample is detected by a capillary chromatographic system and expressed in the form of mapping, thereby the content of the milk immunoglobulin (IgG) can be accurately detected, so as to provide technology support for product development, quality control and component analysis.
Description
Technical field
The present invention relates to the method for a kind of analyzing and testing Ruzhong albumen, the method for immunoglobulin (Ig) (IgG) content in particularly a kind of detection cow's milk.Belong to the detection technique field.
Background technology
Cow's milk is nutriment indispensable in the daily life, and it contains rich nutrient substances, as casein, lactalbumin, complement, fat and immunoglobulin (Ig) etc.The immunoglobulin (Ig) that contains in the cow's milk is mainly IgG, IgA and IgM.Wherein, IgG content is the highest, is about 80% of immunoglobulin (Ig) total amount.Cow divides endocrine Ruzhong immunoglobulin content just of first day puerperium the highest, studies show that the mean concentration of its IgG can reach 59～80mg/ml.Yet 5-7 days first Ruzhong IgG concentration is flat near normal whey, is about 0.7～1.0mg/ml.Immunoglobulin (Ig) has multiple bioactive functions, mainly be pathogen such as identification and the bacterium that resists an invasion and virus, in the immunocompetence of enhancing body, prevent bacterium and poisoning intrusion, complement activation, neutralize a toxin, aspect such as kill tumor cell bringing into play important effect.
At present, domestic national standard and the industry standard that newborn immunoglobulin (Ig) (IgG) detection method of content is not arranged as yet.The method that detects newborn immunoglobulin (Ig) (IgG) has in the serum and experiment, the experiment of immunofluorescence antibody, agar diffusion experiment, complement fixation test, electron microscopic observation, methods such as ELISA, PCR.Owing to consider human and material resources and efficient, the detection method of domestic scientific research institutions newborn immunoglobulin (Ig) (IgG) commonly used is ELISA.The ELISA method is highly sensitive, and minimum detectable level is low, is suitable for the detection of newborn immunoglobulin (Ig) (IgG), but its detection time is longer, is unsuitable for being applicable in the middle of the practical application.In view of the relative merits of this method, this patent has been invented the method that will use capillary electrophoresis apparatus fast detecting breast immunoglobulin (Ig) (IgG) content.Capillary electrophoresis apparatus is that a class comprises that the liquid phase differential of electrophoresis, chromatogram and intersection content thereof is from technology.It can accurately detect the functional component of mingling composition, microbiotic, interpolation, lactalbumin, immunoglobulin (Ig), enzyme, sugar, adjuvant and other micromolecular content in the milk fast.This method is easy and simple to handle, accurate, and detecting out as a result, the time only is about 1h.
At present, the application prospect of capillary electrophoresis apparatus is very extensive.Capillary electrophoresis method has all been approved as FDA, U.S.P, CBER, CDER etc. by authoritative institution of many international officials.The CE that approves impurity in Le Woka-bass front yard hydrochloride in authoritative institution's European Pharmacopoeia measures; Also approve the capillary electrophoresis apparatus mensuration of hematopoietin (EPO) etc. in the European Pharmacopoeia.Capillary electrophoresis apparatus has become a conventional scientific research instrument of various fields such as chemistry, agricultural, medical science, bioengineering, pharmacy.
At present, domestic capillary electrophoresis apparatus detection technique is applied in the field of dairy products gradually, in fields such as product development and control of quality problem.But the method for the capillary electrophoresis technique newborn immunoglobulin (Ig) of still untapped detection (IgG).The present invention has successfully remedied the shortcoming in the field of dairy products, can accurately measure the content of newborn immunoglobulin (Ig) (IgG), thereby help in the middle of product development and the quality control.
Summary of the invention
Purpose of the present invention: newborn immunoglobulin (Ig) (IgG) content detecting method in a kind of cow's milk.
The newborn immunoglobulin (Ig) of a kind of detection of the present invention (IgG) content detecting method, wherein, comprise the steps: 1) newborn immunoglobulin (Ig) (IgG) is dissolved in sample buffer, prepare the standard solution of a plurality of different contents breast immunoglobulin (Ig)s (IgG), and obtain the typical curve between newborn immunoglobulin (Ig) (IgG) content and the testing result after a plurality of solution are detected respectively; 2) sample is detected, and obtain testing result; 3), obtain the content of newborn immunoglobulin (Ig) (IgG) in the sample with described testing result and the comparison of described typical curve; Wherein, the equipment of test sample is the high voltage capillary electrophoresis analyser.
Above-mentioned steps 1) method of the formulation of typical curve is preferably as follows in: prepare newborn immunoglobulin (Ig) (IgG) standard solution, dissolving newborn immunoglobulin (Ig) (IgG) standard substance is mixed with and is respectively 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml, 0.05mg/ml in sample buffer, prepare standard solution, shake up the back and use the high voltage capillary electrophoresis analysis, obtain testing result, promptly obtain the relation curve of newborn immunoglobulin (Ig) (IgG) content and the average correction factor; Wherein, described testing result is calculated as follows: go out the average correction factor by calculated by peak area.
The above-mentioned standard solution or the disposal route of detected sample are: standard solution or detected sample are packed in the centrifuge tube, centrifugal 5-15min under the 2000-6000r/min centrifugal condition, with sample and damping fluid according to 1: the ratio of 4-20 adds frozen pipe (or other containers), and shake up, accurately extracting 10-200ul handles in the sample adding sample hose, be positioned over supersonic wave cleaning machine 1～10min, use capillary electrophoresis apparatus to measure.
Above-mentioned sample buffer is: the damping fluid that protein is disperseed is preferably as follows damping fluid:
Add 40m mol/L 3-morpholino propane sulfonic acid in the 160m mol/L trimethyl aminomethane buffer solution, the 60mmol/L disodium ethylene diamine tetraacetate, 7mol/L urea, the beta-mercaptoethanol of interpolation 5ul/ml when handling sample, methyl hydroxyethylcellulose 0.05%, pH value are 8.5
The above-mentioned step that shakes up adopts eddy mixer.
The use detected parameters that above-mentioned high voltage capillary electrophoresis is analyzed is as follows: the temperature that Capillary Electrophoresis detects is controlled between 15～40 ℃, capillary column diameter 25～75 μ m, length 20～600mm, DAD (or ultraviolet light) detecting device, pressure sample introduction (or electrokinetic injection), pressure is 0～1psi, sample injection time is 1s～20s, and separation temperature is 15～40 ℃, and operating voltage is 10～30kV.
Above-mentioned capillary column is a quartz capillary column, comprises coating and coating not.
Electrophoretic is a zone electrophoresis during detection of above-mentioned high voltage capillary electrophoresis analysis, and its electrophoretic buffer is pH2.0～9.0, and principal ingredient is the inorganic salts damping fluid, and is preferred: phosphate, boric acid or citric acid.
The disposal route of above-mentioned testing result is as follows: testing result is the collection of illustrative plates of sample separation, the utilization capillary electrophoresis apparatus with analysis software, the area at each peak of integration calculates sample solution concentration, takes advantage of extension rate again, promptly gets actual concentrations.
Content 20mg～the 350mg/L of newborn immunoglobulin (Ig) (IgG) in the normal breast, its molecular weight is 12000.Capillary electrophoresis apparatus detects least concentration can reach 10
-5～10
-8Mol/L.So capillary electrophoresis apparatus can detect its content.The method of using the capillary electrophoresis apparatus exploitation to detect newborn immunoglobulin (Ig) (IgG) content comprise properties of samples understanding, clastotype, set up buffer system, optimize buffer system and change step such as clastotype.The method of developing can be applied in product development, quality control and the analyzing and testing field.
The present invention mainly uses capillary electrophoresis apparatus can accurately measure the content of newborn immunoglobulin (Ig) (IgG), thereby is applied in product development, quality control and the analyzing and testing field.
The present invention uses the high voltage capillary electrophoresis instrument to detect the content of newborn immunoglobulin (Ig) (IgG) on the basis in the principle of traditional electrophoresis and chromatogram, reaches the content that accurately detects newborn immunoglobulin (Ig) (IgG) in the dairy products.By test, finally determine the optimal parameter condition of detection method.Specific as follows:
Detect newborn immunoglobulin (Ig) (IgG) content detecting method, comprise sample pre-treatments, sample detection, also comprise steps such as calibration reading.The temperature that Capillary Electrophoresis detects is controlled between 15～40 ℃, the quartz capillary column of capillary column diameter 50～75 μ m, length 20～600mm, DAD (or ultraviolet light) detecting device, the pressure sample introduction, pressure is 0.5psi, sample injection time is 5s～20s, and separation temperature is 20～40 ℃, and operating voltage is 15-25kV.
The formulation of calibration curve: prepare newborn immunoglobulin (Ig) (IgG) concentration of standard solution and be respectively 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml, 0.051mg/ml.Shake up the back and use the high voltage capillary electrophoresis analysis, calculate the average correction factor by peak height.
The detection step is:
The raw material milk sample is packed in the centrifuge tube of 15ml into centrifugal 5min under the 5000r/min centrifugal condition.Accurately extract sample 100ul and insert frozen pipe.Extract the 400ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.Accurately extract 40ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 3min, use capillary electrophoresis apparatus to measure.
The quantitative reading of sample: the collection of illustrative plates of Fen Liing per sample, the utilization capillary electrophoresis apparatus with analysis software, each peak height of integration (or peak area) calculates sample solution concentration, takes advantage of extension rate again, promptly gets actual concentrations.
Beneficial effect
The existing regression equation related coefficient of the inventive method＞0.98, its range of linearity are at 0.01mg/ml～2mg/ml, and the minimum line that detects is 0.15ng, and concentration limit is 0.005mg/ml.Degree of accuracy reaches more than 90%, and the pillar recovery reaches more than 90%, and sample recovery rate reaches more than 70%.Can effectively detect the content of the newborn immunoglobulin (Ig) (IgG) in the sample.
Embodiment
Immunoglobulin (Ig) (IgG) content detecting method in 1 one kinds of cow's milk of embodiment
The Capillary Electrophoresis temperature is controlled at 25 ℃, and using diameter is 50 μ m, the length quartz capillary column as 20mm, and DAD detecting device, sample introduction pressure are 0.5psi, and sample injection time is 10s, and operating voltage is 15kV.
The formulation of calibration curve: utilize newborn immunoglobulin (Ig) (IgG) standard items compound concentration to be respectively the standard solution of 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml.Shake up the back and use the high voltage capillary electrophoresis analysis, peak area behind the integration is respectively 189614,87015,44216,20897,13210, utilize software to draw typical curve, the linear regression related coefficient is 0.9973, the minimum line that detects is 0.009ug, the minimum content that detects is 0.01mg/ml, and the standard model recovery reaches 90%.
Handle the sample buffer collocation method: add 40m mol/L 3-morpholino propane sulfonic acid in the 160m mol/L trimethyl aminomethane buffer solution, the 60mmol/L disodium ethylene diamine tetraacetate, 6mol/L urea, add the beta-mercaptoethanol of 5ul/ml when handling sample, methyl hydroxyethylcellulose 0.05% uses sodium hydroxide solution that the pH value is transferred to 8.5.
The collocation method of electrophoretic buffer: 0.32mol/L citric acid, 20m mol/L trisodium citrate, 7mol/L urea, hydroxypropyl methylcellulose 0.05%.
The detection step is: the milk sample is added in the centrifuge tube of 15ml centrifugal 10min under the 5000r/min centrifugal condition.Accurately extract sample 100ul and insert frozen pipe (or other containers).Extract the 400ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.Accurately extract 40ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 3min, use capillary electrophoresis apparatus to measure afterwards.
The quantitative reading of sample: the collection of illustrative plates of Fen Liing per sample, the utilization capillary electrophoresis apparatus with analysis software, the peak area of integration breast immunoglobulin (Ig) (IgG) is 9851, utilize analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation and be 0.1435mg/ml, take advantage of extension rate 5 again, promptly getting actual concentrations is 0.72mg/ml, so newborn immunoglobulin (Ig) (IgG) concentration that this milk sample contains is 0.72g/L.
Newborn immunoglobulin (Ig) (IgG) content detecting method in 2 one kinds of cow's milk of embodiment
The Capillary Electrophoresis temperature is controlled at 30 ℃, and using diameter is 75 μ m, the length quartz capillary column as 600mm, and DAD detecting device, sample introduction pressure are 0.5psi, and sample injection time is 5s, and operating voltage is 25kV.
The formulation of calibration curve: utilize newborn immunoglobulin (Ig) (IgG) standard items compound concentration to be respectively the standard solution of 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml.Shake up the back and use the high voltage capillary electrophoresis analysis, peak area behind the integration is respectively, 153413,76214,37502,18305,8923 utilize software to draw typical curve, the linear regression related coefficient is 0.999, the minimum line that detects is 0.01ug, the minimum content that detects is 0.01mg/ml, and the standard model recovery reaches 92%.
Handle the sample buffer collocation method: add 40m mol/L 3-morpholino propane sulfonic acid in the 160m mol/L trimethyl aminomethane buffer solution, the 60mmol/L ethylenediamine tetraacetic acid, 6mol/L urea, the beta-mercaptoethanol of interpolation 5ul/ml when handling sample, methyl hydroxyethylcellulose 0.4%.Using 0.1M NaOH to transfer to the pH value is 8.5.
The collocation method of electrophoretic buffer: 0.32mol/L citric acid, 20m mol/L trisodium citrate, 6mol/L urea, hydroxypropyl methylcellulose 0.4%.
The detection step is: the milk sample is added in the centrifuge tube of 15ml centrifugal 5min under the 5000r/min centrifugal condition.Accurately extract sample 100ul and insert frozen pipe (or other containers).Extract the 400ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.Accurately extract 40ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 3min, use capillary electrophoresis apparatus to measure afterwards.
The quantitative reading of sample: the collection of illustrative plates of Fen Liing per sample, the utilization capillary electrophoresis apparatus with analysis software, the peak area of integration breast immunoglobulin (Ig) (IgG) is 4738, utilize analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation and be 0.0614mg/ml, take advantage of extension rate 5 again, promptly getting actual concentrations is 0.307mg/ml, so newborn immunoglobulin (Ig) (IgG) concentration that this milk sample contains is 0.307g/L.
Claims (9)
1. one kind is detected newborn immunoglobulin (Ig) (IgG) content detecting method, it is characterized in that: comprise the steps: 1) newborn immunoglobulin (Ig) (IgG) is dissolved in sample buffer, prepare the standard solution of a plurality of different contents breast immunoglobulin (Ig)s (IgG), and obtain the typical curve between newborn immunoglobulin (Ig) (IgG) content and the testing result after a plurality of solution are detected respectively; 2) sample is detected, and obtain testing result; 3), obtain the content of newborn immunoglobulin (Ig) (IgG) in the sample with described testing result and the comparison of described typical curve; Wherein, the equipment of test sample is the high voltage capillary electrophoresis analyser.
2. detection method according to claim 1, it is characterized in that: the method for the formulation of typical curve is preferably as follows in the described step 1): prepare newborn immunoglobulin (Ig) (IgG) standard solution, dissolving newborn immunoglobulin (Ig) (IgG) standard substance is mixed with and is respectively 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml, 0.05mg/ml in sample buffer, prepare standard solution, shake up the back and use the high voltage capillary electrophoresis analysis, obtain testing result, promptly obtain the relation curve of newborn immunoglobulin (Ig) (IgG) content and the average correction factor; Wherein, described testing result is calculated as follows: go out the average correction factor by calculated by peak area.
3. detection method as claimed in claim 1 or 2, it is characterized in that: standard solution or detected sample are packed in the centrifuge tube, centrifugal 5-15min under the 2000-6000r/min centrifugal condition, with sample and damping fluid according to 1: the ratio of 4-20 adds frozen pipe (or other containers), and shake up, accurately extract 10-200ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 1～10min, use capillary electrophoresis apparatus to measure.
4. detection method as claimed in claim 3, it is characterized in that: described sample buffer is: the damping fluid that protein is disperseed, be preferably as follows damping fluid: add 40m mol/L 3-morpholino propane sulfonic acid in the 160m mol/L trimethyl aminomethane buffer solution, the 60mmol/L disodium ethylene diamine tetraacetate, 7mol/L urea, add the beta-mercaptoethanol of 5ul/ml when handling sample, methyl hydroxyethylcellulose 0.05%, the pH value is 8.5.
5. detection method as claimed in claim 2 is characterized in that: the described step that shakes up adopts eddy mixer.
6. detection method as claimed in claim 1 or 2, it is characterized in that: the use detected parameters that described high voltage capillary electrophoresis is analyzed is as follows: the temperature that Capillary Electrophoresis detects is controlled between 15～40 ℃, capillary column diameter 25～75 μ m, length 20～600mm, DAD (or ultraviolet light) detecting device, pressure sample introduction (or electrokinetic injection), pressure are 0～1psi, and sample injection time is 1s～20s, separation temperature is 15～40 ℃, and operating voltage is 7～30kV.
7. detection method as claimed in claim 6 is characterized in that: described capillary column is a quartz capillary column, comprises coating and coating not.
8. detection method as claimed in claim 1 or 2, it is characterized in that: electrophoretic is a zone electrophoresis during detection of described high voltage capillary electrophoresis analysis, its electrophoretic buffer is pH2.0～9.0, and principal ingredient is the inorganic salts damping fluid, and is preferred: phosphate, boric acid or citric acid.
9. according to the detection method described in the claim 2, it is characterized in that: the disposal route of described testing result is as follows: testing result is the collection of illustrative plates of sample separation, the utilization capillary electrophoresis apparatus with analysis software, the area at each peak of integration, calculate sample solution concentration, take advantage of extension rate again, promptly get actual concentrations.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101782550A (en) * | 2010-03-29 | 2010-07-21 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting immune globulin IgM content in human milk |
| CN101793866A (en) * | 2010-03-29 | 2010-08-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting content of immunoglobulin IgG in human milk |
| CN101806770A (en) * | 2010-03-29 | 2010-08-18 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting immunoglobulin IgA content in human milk |
| CN108693278A (en) * | 2018-07-06 | 2018-10-23 | 漳州傲农牧业科技有限公司 | The assay method of IgG content in a kind of Swine plasma protein |
| CN113552368A (en) * | 2021-07-21 | 2021-10-26 | 苏州立禾生物医学工程有限公司 | Tumor necrosis factor alpha detection kit and detection method thereof |
| CN115398219A (en) * | 2020-04-07 | 2022-11-25 | 阿瑞斯贸易股份公司 | Capillary gel electrophoresis and its use in complex biomolecules |
-
2008
- 2008-05-20 CN CNA2008100819008A patent/CN101315384A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101782550A (en) * | 2010-03-29 | 2010-07-21 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting immune globulin IgM content in human milk |
| CN101793866A (en) * | 2010-03-29 | 2010-08-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting content of immunoglobulin IgG in human milk |
| CN101806770A (en) * | 2010-03-29 | 2010-08-18 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting immunoglobulin IgA content in human milk |
| CN108693278A (en) * | 2018-07-06 | 2018-10-23 | 漳州傲农牧业科技有限公司 | The assay method of IgG content in a kind of Swine plasma protein |
| CN115398219A (en) * | 2020-04-07 | 2022-11-25 | 阿瑞斯贸易股份公司 | Capillary gel electrophoresis and its use in complex biomolecules |
| CN113552368A (en) * | 2021-07-21 | 2021-10-26 | 苏州立禾生物医学工程有限公司 | Tumor necrosis factor alpha detection kit and detection method thereof |
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Application publication date: 20081203 |