CN110007019A - A kind of method that simultaneous quantitative detects three kinds of antibody contents in breast milk - Google Patents
A kind of method that simultaneous quantitative detects three kinds of antibody contents in breast milk Download PDFInfo
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Abstract
The invention discloses the methods of three kinds of antibody contents in a kind of simultaneous quantitative detection breast milk, breast milk is digested using trypsase, select the specific polypeptide in the polypeptide that antibody enzymatic hydrolysis generates in breast milk as marker, the artificial synthesized specific polypeptide is as standard substance, the specific polypeptide of isotope labelling is designed and synthesized as internal standard, quantitative detection is carried out to specific polypeptide using isotopic dilution liquid chromatography tandem mass spectrometry, then convert to obtain the content of antibody in breast milk.Three kinds of antibody contents in energy simultaneous quantitative detection breast milk of the present invention, stability is good, quantifies accurate, reproducible, specific height.
Description
Technical field
The present invention relates to technical field of food detection, in particular to three kinds of antibody contents in a kind of simultaneous quantitative detection breast milk
Method.
Background technique
Antibody in breast milk plays the role of very important in the growth and development of baby.Due to neonatal immune
System is not developed also completely, therefore the antibody in breast milk can help newborn to resist the infringement of external environment bring, help baby
The raising of immunity.Antibody in 5 can be secreted in human body in total under normal conditions, be IgG, IgA, IgM, IgD, IgE respectively.Its
In, the ratio very little of the total antibody of human body shared by IgD, IgE.Under normal conditions, baby can obtain IgG, IgA, IgM from breast milk
These three important antibody.
The antibody content in people's milk accurately and is effectively measured, can help effectively to prevent neonatal correct nursing
The generation of potential disease improves the life quality of baby.Conventional method to antibody test in people's milk is surveyed with Enzyme-linked Immunosorbent Assay
Determine the antigen and antibody method that method is representative.But due to the cross reaction of people's milk mesostroma interference and antibody, the method is surveyed
As a result stability and accuracy is frequently subjected to influence.Therefore, it is badly in need of a set of new method now, it can be accurately to mother
Main antibody substance in cream carries out quantitative analysis.
Summary of the invention
The purpose of the present invention is to provide the methods of three kinds of antibody contents in a kind of simultaneous quantitative detection breast milk.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of method that simultaneous quantitative detects three kinds of antibody contents in breast milk, includes the following steps:
(1) sample pretreatment
Human milk samples mix the mixed mark of artificial synthesized isotope labelling specific polypeptide, and must pre-process after trypsin digestion
Liquid；Isotope labelling specific polypeptide is mixed to be designated as isotope labelling IgA specific polypeptide, isotope labelling IgG specific polypeptide
And the mixture of isotope labelling IgM specific polypeptide；Each isotope labelling is special in the mixed mark of isotope labelling specific polypeptide
The concentration of property polypeptide is 20 μ g/mL；
IgA specific polypeptide sequence are as follows: SAVQGPPER；
IgG specific polypeptide sequence are as follows: VVSVLTVLHQDWLNGK；
IgM specific polypeptide sequence are as follows: QIQVSWLR；
Isotope labelling IgA specific polypeptide sequence are as follows: SAV*QGPPER；
Isotope labelling IgG specific polypeptide sequence are as follows: V*VSVLTVLHQDWLNGK；
Isotope labelling IgM specific polypeptide sequence are as follows: QIQV*SWLR
V* represents isotope labelling valine；
(2) standard curve making
The mixed each 10 μ L of mark of the artificial synthesized specific polypeptide of gradient concentration is taken, it is special to be separately added into artificial synthesized isotope labelling
Specific polypeptide 10 μ L of mixed mark and 980 μ L water；Pass through the triple level four bars mass spectrums inspections of high performance liquid chromatography series connection upper after 0.22 μm of filter membrane
It surveys, while obtaining the peak area ratio of the special peptide of IgA antibody and the special peptide of isotope and corresponding IgA antibody standard solution concentration
Standard curve, the special peptide of IgG antibody and the special peptide of isotope peak area ratio and corresponding IgG antibody standard solution concentration
Standard curve, the special peptide of IgM antibody and the special peptide of isotope peak area ratio and corresponding IgM antibody standard solution concentration
Standard curve；
(3) chromatographic tandem mass spectral analysis
Pretreatment fluid is by the triple level four bars Mass Spectrometer Methods of high performance liquid chromatography series connection upper after 0.22 μm of filter membrane, obtained result generation
Enter standard curve, to obtain the dense of IgA specific polypeptide in human milk samples, IgG specific polypeptide and IgM specific polypeptide
Degree；In human milk samples in IgA antibody concentration=sample IgA specific polypeptide molar concentration × IgA antibody molal weight × mother
Milk sample product extension rate (50)/2,
In human milk samples in IgG antibody concentration=sample IgG specific polypeptide molar concentration × IgG antibody molal weight × mother
Milk sample product extension rate (50)/2,
In human milk samples in IgM antibody concentration=sample IgM specific polypeptide molar concentration × IgM antibody molal weight × mother
Milk sample product extension rate (50)/2.
The present invention first uses enzyme to digest human milk samples, selects specific enzymolysis polypeptide as marker, artificial to close
At the specific polypeptide as standard substance, quantitative detection is carried out using isotopic dilution liquid chromatography tandem mass spectrometry, is base
In the detection method of specific polypeptide, primary structure (polypeptide) stability is good, as a result stable；And peptide chain has genetic code special
Point, specificity are very high.
In step (1), human milk samples mix the mixed mark of artificial synthesized isotope labelling specific polypeptide, and through trypsase
Pretreatment fluid is obtained after enzymatic hydrolysis and obtains concrete operations are as follows: takes 20 μ L of human milk samples, 890 μ L of ammonium bicarbonate soln is added, artificial close is added
At isotope labelling specific polypeptide 10 μ L of mixed mark, 10 μ L of dithiothreitol (DTT) is added, 50 DEG C are reacted 30 minutes, and iodo is added
30 dark place μ L of acetamide is reacted 30 minutes, 20 μ L of trypsase is added, 37 DEG C are reacted 2 hours, and 10% first is added after completion of the reaction
20 μ l of acid terminate reaction, obtain pretreatment fluid.
Ammonium bicarbonate soln concentration is 0.1mol/L, and dithiothreitol (DTT) concentration is 100mmol/L, and iodoacetamido amine concentration is
100mmol/L, trypsinase concentration 250ug/mL.
In step (2), the artificial synthesized specific polypeptide of gradient concentration mixes the setting of target gradient concentration are as follows: 1ng/ml,
10ng/ml, 25ng/ml, 75ng/ml, 100ng/ml, 250ng/ml, 400ng/ml, 650ng/m, 1000ng/ml, 1500ng/
ml。
In step (3), chromatographic condition is as follows: chromatographic column is C18 chromatographic column, and column temperature is 40 DEG C；Mobile phase A is volume basis
Than the acetonitrile solution of the formic acid for 0.1%, Mobile phase B is the aqueous formic acid that percent by volume is 0.1%, gradient elution, stream
Speed is 0.3mL/min；
Wherein, gradient elution are as follows: the percent by volume of mobile phase A rises to 40% by 3% time-consuming 10min, accordingly Mobile phase B
Percent by volume drop to 60% by 97% time-consuming 10min, be then changed to 100% mobile phase A rinse 2min, be finally changed to flow
Dynamic phase A percent by volume 3% and Mobile phase B percent by volume 97% retain 3min.
In step (3), Mass Spectrometry Conditions are as follows: capillary voltage: 3.5kv, orifice potential: 35kv, desolventizing temperature: 500
DEG C, desolventizing gas flow: 900L/min, cone hole backflow airflow amount: 30L/hr collides chamber pressure: 3.0 × 10-3mbar；Low side
Resolution ratio 1:2.5V, high-end resolution ratio 1:15.0V, ion energy 1:0.5；Low side resolution ratio 2:2.8V, high-end resolution ratio 2:
15.0V, ion energy 2:1.0；Ion source temperature: 150 DEG C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, outlet electricity
Pressure: 0.5V, collision gradient: 1.0.
In step (3), when IgA specific polypeptide Mass Spectrometer Method, parent ion 470.7, daughter ion 159.1,555.3；
When IgG specific polypeptide Mass Spectrometer Method, parent ion 603.3, daughter ion 199.1,805.4；The inspection of IgM specific polypeptide mass spectrum
When survey, parent ion 515.3, daughter ion 175.1,288.2.
In step (2), the peak area ratio of the special peptide of IgA antibody and the special peptide of isotope and corresponding IgA antibody standard items
The standard curve of solution concentration is y=2.121x-0.0369, R2=0.9993；
The standard of the peak area ratio of the special peptide of IgG antibody and the special peptide of isotope and corresponding IgG antibody standard solution concentration
Curve is y=0.3814x+0.005, R2=0.9927；
The standard of the peak area ratio of the special peptide of IgM antibody and the special peptide of isotope and corresponding IgM antibody standard solution concentration
Curve is y=0.3892x+0.0068, R2=0.9972.
Kit that is a kind of while detecting three kinds of antibody contents in breast milk, including trypsase, ammonium bicarbonate soln, two sulphur
The mixed mark of threitol, iodo-acetamide, 10% formic acid and isotope labelling specific polypeptide,
Isotope labelling specific polypeptide is mixed to be designated as isotope labelling IgA specific polypeptide, isotope labelling IgG specific polypeptide
And the mixture of isotope labelling IgM specific polypeptide；Each isotope labelling is special in the mixed mark of isotope labelling specific polypeptide
The concentration of property polypeptide is 20 μ g/mL；
IgA specific polypeptide sequence are as follows: SAVQGPPER；
IgG specific polypeptide sequence are as follows: VVSVLTVLHQDWLNGK；
IgM specific polypeptide sequence are as follows: QIQVSWLR；
Isotope labelling IgA specific polypeptide sequence are as follows: SAV*QGPPER；
Isotope labelling IgG specific polypeptide sequence are as follows: V*VSVLTVLHQDWLNGK；
Isotope labelling IgM specific polypeptide sequence are as follows: QIQV*SWLR；
V* represents isotope labelling valine.
The beneficial effects of the present invention are:
1, single injected sampling detects IgA simultaneously, and tri- kinds of antibody of IgG, IgM, sample requirements are few, time saving and energy saving；
2, quantitative accurate: isotopic dilution liquid chromatography tandem mass spectrometry is the gold standard of quantitative detection, as a result accurately and reliably；
3, reproducible: using chemically synthesized polypeptide as standard substance, can to synthesize in batches, quality is stablized, using artificial
The isotope polypeptide of synthesis improves Detection accuracy as internal standard；
4, specificity is high: selecting specific polypeptide as standard substance, polypeptide has genetic code feature, can be by target polypeptides
It is distinguished well with matrix polypeptide.
Detailed description of the invention
Fig. 1 is the chromatogram of specific polypeptide of the present invention.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is unless otherwise instructed the conventional method of this field.
Embodiment
(1) sample pretreatment
20 μ L of human milk samples is taken, 890 μ L (0.1mol/L) of ammonium bicarbonate soln is added, it is special that artificial synthesized isotope labelling is added
The mixed mark 10 μ L (20 μ g/mL) of specific polypeptide, is added 10 μ L (100mmol/L) of dithiothreitol (DTT), and 50 DEG C are reacted 30 minutes, adds
30 dark place (100mmol/L) μ L of iodo-acetamide is reacted 30 minutes, is added 20 μ L of trypsase (250 μ g/mL), 37 DEG C of reactions 2
Hour, 10% formic acid, 20 μ l is added after completion of the reaction and terminates reaction, obtains pretreatment fluid.
Isotope labelling specific polypeptide is mixed to be designated as isotope labelling IgA specific polypeptide, isotope labelling IgG specificity
The mixture of polypeptide and isotope labelling IgM specific polypeptide,
IgA specific polypeptide sequence are as follows: SAVQGPPER (SEQ ID No.1)；
IgG specific polypeptide sequence are as follows: VVSVLTVLHQDWLNGK (SEQ ID No.2)；
IgM specific polypeptide sequence are as follows: QIQVSWLR (SEQ ID No.3)；
Isotope labelling IgA specific polypeptide sequence are as follows: SAV*QGPPER；
Isotope labelling IgG specific polypeptide sequence are as follows: V*VSVLTVLHQDWLNGK；
Isotope labelling IgM specific polypeptide sequence are as follows: QIQV*SWLR；
V* represents isotope labelling valine.
(2) standard curve making
The mixed each 10 μ L of mark of the artificial synthesized specific polypeptide of gradient concentration is taken, it is special to be separately added into artificial synthesized isotope labelling
Specific polypeptide 10 μ L of mixed mark and 980 μ L water；Pass through the triple level four bars mass spectrums inspections of high performance liquid chromatography series connection upper after 0.22 μm of filter membrane
It surveys, while obtaining the peak area ratio of the special peptide of IgA antibody and the special peptide of isotope and corresponding IgA antibody standard solution concentration
Standard curve, the special peptide of IgG antibody and the special peptide of isotope peak area ratio and corresponding IgG antibody standard solution concentration
Standard curve, the special peptide of IgM antibody and the special peptide of isotope peak area ratio and corresponding IgM antibody standard solution concentration
Standard curve；The typical chromatogram of each specific polypeptide is shown in Fig. 1.
The mixed mark gradient concentration setting of artificial synthesized specific polypeptide are as follows: 1ng/ml, 10ng/ml, 25ng/ml, 75ng/
Ml, 100ng/ml, 250ng/ml, 400ng/ml, 650ng/m, 1000ng/ml, 1500ng/ml.
The mixed mixture for being designated as IgA specific polypeptide, IgG specific polypeptide and IgM specific polypeptide of specific polypeptide,
IgA specific polypeptide sequence are as follows: SAVQGPPER (SEQ ID No.1)；
IgG specific polypeptide sequence are as follows: VVSVLTVLHQDWLNGK (SEQ ID No.2)；
IgM specific polypeptide sequence are as follows: QIQVSWLR (SEQ ID No.3).
The peak area ratio of the special peptide of IgA antibody and the special peptide of isotope and corresponding IgA antibody standard solution concentration
Standard curve is y=2.121x-0.0369, R2=0.9993；
The standard of the peak area ratio of the special peptide of IgG antibody and the special peptide of isotope and corresponding IgG antibody standard solution concentration
Curve is y=0.3814x+0.005, R2=0.9927；
The standard of the peak area ratio of the special peptide of IgM antibody and the special peptide of isotope and corresponding IgM antibody standard solution concentration
Curve is y=0.3892x+0.0068, R2=0.9972.
(3) chromatographic tandem mass spectral analysis
Pretreatment fluid is by the triple level four bars Mass Spectrometer Methods of high performance liquid chromatography series connection upper after 0.22 μm of filter membrane, obtained result generation
Enter standard curve, to obtain the dense of IgA specific polypeptide in human milk samples, IgG specific polypeptide and IgM specific polypeptide
Degree；In human milk samples in IgA antibody concentration=sample IgA specific polypeptide molar concentration × IgA antibody molal weight × mother
Milk sample product extension rate (50)/2,
In human milk samples in IgG antibody concentration=sample IgG specific polypeptide molar concentration × IgG antibody molal weight × mother
Milk sample product extension rate (50)/2,
In human milk samples in IgM antibody concentration=sample IgM specific polypeptide molar concentration × IgM antibody molal weight × mother
Milk sample product extension rate (50)/2.
Wherein target polypeptides (special peptide) and daughter ion information are shown in Table 1, and liquid phase chromatogram condition and Mass Spectrometry Conditions are as follows:
Liquid phase chromatogram condition
Chromatographic column is C18 chromatographic column, and column temperature is 40 DEG C；Mobile phase A is the acetonitrile solution for the formic acid that percent by volume is 0.1%,
Mobile phase B is the aqueous formic acid that percent by volume is 0.1%, gradient elution, flow velocity 0.3mL/min；
Wherein, gradient elution are as follows: the percent by volume of mobile phase A rises to 40% by 3% time-consuming 10min, accordingly Mobile phase B
Percent by volume drop to 60% by 97% time-consuming 10min, be then changed to 100% mobile phase A rinse 2min, be finally changed to flow
Dynamic phase A percent by volume 3% and Mobile phase B percent by volume 97% retain 3min.
Mass Spectrometry Conditions
Capillary voltage: 3.5kv, orifice potential: 35kv, desolventizing temperature: 500 DEG C, desolventizing gas flow: 900L/min, cone
Hole blowback throughput: 30L/hr collides chamber pressure: 3.0 × 10-3mbar；Low side resolution ratio 1:2.5V, high-end resolution ratio 1:
15.0V, ion energy 1:0.5；Low side resolution ratio 2:2.8V, high-end resolution ratio 2:15.0V, ion energy 2:1.0；Ion source temperature
Degree: 150 DEG C, extractor voltage: 3.0V, entrance lens voltage: 0.5V, exit potential: 0.5V, collision gradient: 1.0；
Table 1: mass ions information
# is quota ion.
Methodology validation
By the way that the mixed polypeptide of high, medium and low three kinds of various concentrations is added in people's milk sample, to its method rate of recovery, precision into
Row test.Experimental method are as follows: the mixed mark solution of configuration, concentration is respectively 50ug/mL, and 20ug/mL, 5ug/mL. take people's milk sample
Ammonium bicarbonate soln 970ul (0.1mol/L) is added in 20ul, adds the mixing inner mark solution 10uL prepared, is filtered by 0.22um
Upper machine analysis, daily 5 parallel sample are analyzed 4 days altogether after film.
Sensitivity is calculated public by the way that low concentration internal standard polypeptide is added, obtain signal-to-noise ratio and calculates its detection limit and quantitative limit
Formula is detection limit=3* signal-to-noise ratio/sample introduction concentration, quantitative limit=10* signal-to-noise ratio/sample introduction concentration (the results are shown in Table 2).
Table 2: methodology validation table
Actual sample detection
Multiple human milk samples are detected using method of the invention, wherein typical testing result is as follows:
Kit that is a kind of while detecting three kinds of antibody contents in breast milk, including trypsase, ammonium bicarbonate soln, two sulphur
The mixed mark of threitol, iodo-acetamide, 10% formic acid and isotope labelling specific polypeptide,
Isotope labelling specific polypeptide is mixed to be designated as isotope labelling IgA specific polypeptide, isotope labelling IgG specific polypeptide
And the mixture of isotope labelling IgM specific polypeptide；Each isotope labelling is special in the mixed mark of isotope labelling specific polypeptide
The concentration of property polypeptide is 20 μ g/mL；
IgA specific polypeptide sequence are as follows: SAVQGPPER；
IgG specific polypeptide sequence are as follows: VVSVLTVLHQDWLNGK；
IgM specific polypeptide sequence are as follows: QIQVSWLR；
Isotope labelling IgA specific polypeptide sequence are as follows: SAV*QGPPER；
Isotope labelling IgG specific polypeptide sequence are as follows: V*VSVLTVLHQDWLNGK；
Isotope labelling IgM specific polypeptide sequence are as follows: QIQV*SWLR；
V* represents isotope labelling valine.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form
Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.
SEQUENCE LISTING
<110>Lv Chengnongke detection technique Co., Ltd
<120>a kind of method that simultaneous quantitative detects three kinds of antibody contents in breast milk
<130> 2019.3.29
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 1
Ser Ala Val Gln Gly Pro Pro Glu Arg
1 5
<210> 2
<211> 16
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 2
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
1 5 10 15
<210> 3
<211> 8
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 3
Gln Ile Gln Val Ser Trp Leu Arg
1 5
Claims (9)
1. a kind of method of three kinds of antibody contents in simultaneous quantitative detection breast milk, which comprises the steps of:
(1) sample pretreatment
Human milk samples mix the mixed mark of artificial synthesized isotope labelling specific polypeptide, and must pre-process after trypsin digestion
Liquid；
Isotope labelling specific polypeptide is mixed to be designated as isotope labelling IgA specific polypeptide, isotope labelling IgG specific polypeptide
And the mixture of isotope labelling IgM specific polypeptide；Each isotope labelling is special in the mixed mark of isotope labelling specific polypeptide
The concentration of property polypeptide is 20 μ g/mL；
IgA specific polypeptide sequence are as follows: SAVQGPPER；
IgG specific polypeptide sequence are as follows: VVSVLTVLHQDWLNGK；
IgM specific polypeptide sequence are as follows: QIQVSWLR；
Isotope labelling IgA specific polypeptide sequence are as follows: SAV*QGPPER；
Isotope labelling IgG specific polypeptide sequence are as follows: V*VSVLTVLHQDWLNGK；
Isotope labelling IgM specific polypeptide sequence are as follows: QIQV*SWLR；
V* represents isotope labelling valine；
(2) standard curve making
The mixed each 10 μ L of mark of the artificial synthesized specific polypeptide of gradient concentration is taken, it is special to be separately added into artificial synthesized isotope labelling
Specific polypeptide 10 μ L of mixed mark and 980 μ L water；Pass through the triple level four bars mass spectrums inspections of high performance liquid chromatography series connection upper after 0.22 μm of filter membrane
It surveys, while obtaining the peak area ratio of the special peptide of IgA antibody and the special peptide of isotope and corresponding IgA antibody standard solution concentration
Standard curve, the special peptide of IgG antibody and the special peptide of isotope peak area ratio and corresponding IgG antibody standard solution concentration
Standard curve, the special peptide of IgM antibody and the special peptide of isotope peak area ratio and corresponding IgM antibody standard solution concentration
Standard curve；
(3) chromatographic tandem mass spectral analysis
Pretreatment fluid is by the triple level four bars Mass Spectrometer Methods of high performance liquid chromatography series connection upper after 0.22 μm of filter membrane, obtained result generation
Enter standard curve, to obtain the dense of IgA specific polypeptide in human milk samples, IgG specific polypeptide and IgM specific polypeptide
Degree；In human milk samples in IgA antibody concentration=sample IgA specific polypeptide molar concentration × IgA antibody molal weight × mother
Milk sample product extension rate/2,
In human milk samples in IgG antibody concentration=sample IgG specific polypeptide molar concentration × IgG antibody molal weight × mother
Milk sample product extension rate/2
In human milk samples in IgM antibody concentration=sample IgM specific polypeptide molar concentration × IgM antibody molal weight × mother
Milk sample product extension rate/2.
2. according to the method described in claim 1, it is characterized by: human milk samples mix artificial synthesized same position in step (1)
The mixed mark of element label specific polypeptide, and obtain pretreatment fluid after trypsin digestion and obtain concrete operations are as follows: 20 μ L of human milk samples is taken,
890 μ L of ammonium bicarbonate soln is added, artificial synthesized isotope labelling specific polypeptide 10 μ L of mixed mark are added, two sulphur threoses are added
10 μ L of alcohol, 50 DEG C are reacted 30 minutes, are added 30 dark place μ L of iodo-acetamide and are reacted 30 minutes, are added 20 μ L of trypsase, and 37
DEG C reaction 2 hours, be added after completion of the reaction 10% formic acid, 20 μ l terminate reaction, obtain pretreatment fluid.
3. according to the method described in claim 2, it is characterized by: ammonium bicarbonate soln concentration is 0.1mol/L, two sulphur threoses
Determining alcohol is 100mmol/L, and iodoacetamido amine concentration is 100mmol/L, trypsinase concentration 250ug/mL.
4. according to the method described in claim 1, it is characterized by: in step (2), the artificial synthesized specificity of gradient concentration
Polypeptide mixes the setting of target gradient concentration are as follows: 1ng/ml, 10ng/ml, 25ng/ml, 75ng/ml, 100ng/ml, 250ng/ml,
400ng/ml, 650ng/m, 1000ng/ml, 1500ng/ml.
5. according to the method described in claim 1, it is characterized by: chromatographic condition is as follows in step (3): chromatographic column is C18 color
Column is composed, column temperature is 40 DEG C；Mobile phase A is the acetonitrile solution for the formic acid that percent by volume is 0.1%, and Mobile phase B is volume basis
Than the aqueous formic acid for 0.1%, gradient elution, flow velocity 0.3mL/min；
Wherein, gradient elution are as follows: the percent by volume of mobile phase A rises to 40% by 3% time-consuming 10min, accordingly Mobile phase B
Percent by volume drop to 60% by 97% time-consuming 10min, be then changed to 100% mobile phase A rinse 2min, be finally changed to flow
Dynamic phase A percent by volume 3% and Mobile phase B percent by volume 97% retain 3min.
6. according to the method described in claim 1, it is characterized by: Mass Spectrometry Conditions are as follows: capillary voltage in step (3):
3.5kv, orifice potential: 35kv, desolventizing temperature: 500 DEG C, desolventizing gas flow: 900L/min, cone hole backflow airflow amount:
30L/hr collides chamber pressure: 3.0 × 10-3mbar；Low side resolution ratio 1:2.5V, high-end resolution ratio 1:15.0V, ion energy 1:
0.5；Low side resolution ratio 2:2.8V, high-end resolution ratio 2:15.0V, ion energy 2:1.0；Ion source temperature: 150 DEG C, extractor
Voltage: 3.0V, entrance lens voltage: 0.5V, exit potential: 0.5V, collision gradient: 1.0.
It is female when IgA specific polypeptide Mass Spectrometer Method 7. according to the method described in claim 2, it is characterized by: in step (3)
Ion is 470.7, daughter ion 159.1,555.3；When IgG specific polypeptide Mass Spectrometer Method, parent ion 603.3, daughter ion
It is 199.1,805.4；When IgM specific polypeptide Mass Spectrometer Method, parent ion 515.3, daughter ion 175.1,288.2.
8. according to the method described in claim 1, it is characterized by: the special peptide of IgA antibody and isotope are special in step (2)
The peak area ratio of peptide is y=2.121x-0.0369, R with the standard curve of corresponding IgA antibody standard solution concentration2=
0.9993；
The standard of the peak area ratio of the special peptide of IgG antibody and the special peptide of isotope and corresponding IgG antibody standard solution concentration
Curve is y=0.3814x+0.005, R2=0.9927；
The standard of the peak area ratio of the special peptide of IgM antibody and the special peptide of isotope and corresponding IgM antibody standard solution concentration
Curve is y=0.3892x+0.0068, R2=0.9972.
9. a kind of kit for detecting three kinds of antibody contents in breast milk simultaneously, it is characterised in that: including trypsase, ammonium hydrogen carbonate
The mixed mark of solution, dithiothreitol (DTT), iodo-acetamide, 10% formic acid and isotope labelling specific polypeptide,
Isotope labelling specific polypeptide is mixed to be designated as isotope labelling IgA specific polypeptide, isotope labelling IgG specific polypeptide
And the mixture of isotope labelling IgM specific polypeptide；Each isotope labelling is special in the mixed mark of isotope labelling specific polypeptide
The concentration of property polypeptide is 20 μ g/mL；
IgA specific polypeptide sequence are as follows: SAVQGPPER；
IgG specific polypeptide sequence are as follows: VVSVLTVLHQDWLNGK；
IgM specific polypeptide sequence are as follows: QIQVSWLR；
Isotope labelling IgA specific polypeptide sequence are as follows: SAV*QGPPER；
Isotope labelling IgG specific polypeptide sequence are as follows: V*VSVLTVLHQDWLNGK；
Isotope labelling IgM specific polypeptide sequence are as follows: QIQV*SWLR；
V* represents isotope labelling valine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111551739A (en) * | 2020-04-27 | 2020-08-18 | 杭州璞湃科技有限公司 | Detection method and kit for immunoglobulin A and immunoglobulin A1 |
| CN112630319A (en) * | 2020-10-22 | 2021-04-09 | 北京食品科学研究院 | Method for determining pork content in meat product by LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry) internal standard method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030219838A1 (en) * | 2002-05-24 | 2003-11-27 | Johnson Richard S. | Polypeptide analyses using stable isotope labeling |
| US20120264155A1 (en) * | 2009-10-09 | 2012-10-18 | Symphogen A/S | Multiplex Quantitation of Individual Recombinant Proteins in a Mixture by Signature Peptides and Mass Spectrometry |
| CN103642895A (en) * | 2013-12-06 | 2014-03-19 | 浙江贝因美科工贸股份有限公司 | Method and kit for quantitative detection of content of human alpha-lactalbumin |
| US20160146796A1 (en) * | 2002-10-03 | 2016-05-26 | Anderson Forschung Group, Inc. | High sensitivity quantitation of peptides by mass spectrometry |
| CN109071606A (en) * | 2016-06-13 | 2018-12-21 | 国立大学法人鹿儿岛大学 | Utilize the locus specificity labelled with radioisotope antibody of IgG binding peptide |
-
2019
- 2019-03-29 CN CN201910249510.5A patent/CN110007019A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030219838A1 (en) * | 2002-05-24 | 2003-11-27 | Johnson Richard S. | Polypeptide analyses using stable isotope labeling |
| US20160146796A1 (en) * | 2002-10-03 | 2016-05-26 | Anderson Forschung Group, Inc. | High sensitivity quantitation of peptides by mass spectrometry |
| US20120264155A1 (en) * | 2009-10-09 | 2012-10-18 | Symphogen A/S | Multiplex Quantitation of Individual Recombinant Proteins in a Mixture by Signature Peptides and Mass Spectrometry |
| CN103642895A (en) * | 2013-12-06 | 2014-03-19 | 浙江贝因美科工贸股份有限公司 | Method and kit for quantitative detection of content of human alpha-lactalbumin |
| CN109071606A (en) * | 2016-06-13 | 2018-12-21 | 国立大学法人鹿儿岛大学 | Utilize the locus specificity labelled with radioisotope antibody of IgG binding peptide |
Non-Patent Citations (3)
| Title |
|---|
| LIN-ZHI CHEN 等: "Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation", 《JOURNAL OF IMMUNOLOGY RESEARCH》 * |
| 周愿 等: "基于稳定同位素标记的蛋白质组学定量方法研究进展", 《色谱》 * |
| 周长林: "《微生物学与免疫学》", 31 July 2013, 中国医药科技出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111551739A (en) * | 2020-04-27 | 2020-08-18 | 杭州璞湃科技有限公司 | Detection method and kit for immunoglobulin A and immunoglobulin A1 |
| CN112630319A (en) * | 2020-10-22 | 2021-04-09 | 北京食品科学研究院 | Method for determining pork content in meat product by LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry) internal standard method |
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