CN115598277A

CN115598277A - Method for quantitatively detecting immunoglobulin G

Info

Publication number: CN115598277A
Application number: CN202211597786.0A
Authority: CN
Inventors: 王象欣; 解庆刚; 张影; 崔东影; 高鹏; 陆思宇; 蒋士龙; 张永久
Original assignee: Feihe Harbin Dairy Co ltd; Heilongjiang Feihe Dairy Co Ltd
Current assignee: Feihe Harbin Dairy Co ltd; Heilongjiang Feihe Dairy Co Ltd
Priority date: 2022-12-14
Filing date: 2022-12-14
Publication date: 2023-01-13

Abstract

The invention belongs to the field of detection, and provides a quantitative detection method of immunoglobulin G, which comprises the following steps: a pretreatment step of removing at least casein from a test object to obtain a sample; a step of detecting, which is to perform quantitative detection on the sample by using a two-dimensional chromatographic apparatus and a detector, wherein the two-dimensional chromatographic apparatus comprises a one-dimensional chromatographic system and a two-dimensional chromatographic system, the one-dimensional chromatographic system comprises an immunoglobulin G specific affinity chromatographic column and a fraction collector, the two-dimensional chromatographic system comprises an antibody chromatographic column, the one-dimensional chromatographic system and the two-dimensional chromatographic system are connected or switched through a multi-way valve group, and in the step of detecting: immunoglobulin G in the sample is enriched in the fraction collector after purification in the immunoglobulin G-specific affinity chromatography column and is detected by the detector after passing through the two-dimensional chromatography system.

Description

Method for quantitatively detecting immunoglobulin G

Technical Field

The invention belongs to the field of detection, particularly relates to the field of separation and analysis detection of protein/peptide substances, and more particularly relates to a method for quantitative analysis of immunoglobulin G, in particular to quantitative analysis of immunoglobulin G content in dairy products.

Background

Immunoglobulin (Ig) is an important immune molecule, can specifically recognize and bind to invading pathogens such as bacteria and viruses, mediates immune reactions such as complement activation and immunoregulation, plays an important role in enhancing the immunity of the body, preventing invasion of bacteria and viruses and the like, and is present in serum, normal milk and colostrum.

Igs are a family of bioactive whey proteins that, unlike casein, are soluble upon acidification to about pH 4.6.

The predominant Ig in colostrum is isotype IgG (80%). This differs from serum IgG in the ratio of the two subclasses IgG1 and IgG2 (IgG 1: igG2; colostrum 90, see serum 56 44), where IgG1 is selectively transported from serum to colostrum. IgG in colostrum was high and rapidly dropped to levels comparable to those in normal milk after 4 days.

With the increasing production of IgG-containing products, igG detection methods are also receiving more attention. IgG has unique molecular structure and bioactivity, and the detection method is complicated. The existing popular IgG detection methods mainly comprise two methods of analyzing the content of IgG and the content of IgG activity in a product. Moreover, the research on IgG has mainly focused on its bioactive function and separation from other immunoglobulins, and few reports have been reported for the detection of the active content in food (dairy products), and the detection of IgG content is mainly based on an immunological detection method, ELISA.

IgG is a heat labile protein that rapidly denatures at temperatures above 75 ℃. In order to maintain their physiological functions, they must be made invariable and remain in their original state. The IgG content of cow milk is measured, and the purpose is to distinguish the cow colostrum milk from the normal milk sold in the market by using the IgG content. The ideal detection method is the ability to stabilize, accurately and distinguish between native and denatured IgG.

In the prior art, some standards such as NY/T2070-2011 mainly detect the content of immunoglobulin G in bovine colostrum and products thereof by a spectrophotometry method; standard SN/T3132-2012 determines the immunoglobulin G content in the exported bovine milk product by enzyme linked immunosorbent assay. In addition, although HPLC method using reversed phase bond and chromatographic column ZORBAX300SB-C18 is also attempted to determine the content of immunoglobulin G in bovine colostrum, the above standard or method is only applicable to bovine colostrum products and bovine milk products added with bovine colostrum components, and some methods cannot distinguish the content of active immunoglobulin.

At present, a detection method for immunoglobulin G in cow regular milk and products thereof is T/TDTIA 003-2021, and the content of the immunoglobulin G in milk is detected by Protein G affinity column purification and high performance liquid chromatography.

In addition, cited document 1 detects bovine immunoglobulin G (IgG) using an affinity Liquid Chromatography (LC) method, which uses an agarose support with coupled Protein G as a stationary phase to allow quantification of IgG in colostrum powder.

Therefore, in general, the prior art methods for measuring the content of immunoglobulin still have the disadvantages of complicated operation, long time, low accuracy, poor applicability and reliability, etc.

Cited documents:

citation 1: "Affinity liquid chromatography method for the quantification of immunoglobulin G in bone collagen powders", david E J Copetake et al, J AOAC int. 2006 Sep-Oct;89 (5): 1249-56.

Disclosure of Invention

Problems to be solved by the invention

Through long-term research, igG and foreign proteins flow out together by adopting the analytical method only using the POROS Protein G immunoaffinity column in the citation document 1, the result accuracy is poor, and IgG adsorption capacities of POROS Protein G immunoaffinity columns in batches are inconsistent, and the recovery rate is poor. In addition, the POROS Protein G immunoaffinity column is easy to damage, the third sample leaks, the sieve plate is separated from the filler, the sieve plate needs to be replaced and rebalanced, and the conventional operation difficulty is high.

In addition, although the standard T/TDSIIA 003-2021 adopts a Protein G filler solid-phase extraction mode, the problems are avoided, the solid-phase extraction consumes much time, has high cost, the recovery rate is not easy to guarantee, the adsorption capacity of a Protein G filler solid-phase extraction column between batches cannot be determined, and the phenomenon is more serious because a pretreatment sample is turbid; the solid phase extraction effluent is subjected to on-machine analysis by adopting a large-aperture C4 chromatographic column, the effluent time is 2min, and the sensitivity of a low-content sample is low.

Therefore, the invention aims to provide a rapid and convenient method for quantitatively detecting immunoglobulin G in dairy products (especially liquid milk and milk powder).

In addition, the quantitative analysis method for detecting the immunoglobulin G provided by the invention can also be an automatic detection method, and provides a possibility for reliably and quantitatively detecting the content of the immunoglobulin G in the production process of the dairy product on line.

Means for solving the problems

The following technical problems can be solved by implementing the following technical scheme:

[1] a method for the quantitative detection of immunoglobulin G, wherein the method comprises:

a pretreatment step of removing at least casein from the test object to obtain a sample containing immunoglobulin;

a step of detecting, by using a two-dimensional chromatography device and a detector, the immunoglobulin G-containing sample by quantitative detection,

the two-dimensional chromatography apparatus comprises a one-dimensional chromatography system and a two-dimensional chromatography system, wherein,

the one-dimensional chromatographic system comprises an immunoglobulin G specificity affinity chromatographic column and a fraction collector, the two-dimensional chromatographic system comprises an antibody chromatographic column, the one-dimensional chromatographic system and the two-dimensional chromatographic system are connected or switched through a multi-way valve bank,

and, in the step of detecting:

the igg in the igg-containing sample is enriched in the fraction collector after purification in the igg-specific affinity chromatography column and then detected by the detector after passing through the two-dimensional chromatography system.

[2] The method of [1], wherein the method further comprises determining the retention time of immunoglobulin G on the immunoglobulin G-specific affinity chromatography column using the detector.

[3] The method according to [1] or [2], wherein the detection object is selected from liquid milk or powdered milk; in the pretreatment step, the object to be detected is further subjected to degreasing and/or desugarization treatment.

[4] The method according to any one of [1] to [3], wherein the immunoglobulin G-specific affinity chromatography column is selected from Protein G affinity chromatography columns.

[5] The method according to any one of [1] to [4], wherein the antibody chromatography column in the two-dimensional chromatography system is selected from a monoclonal antibody chromatography column.

[6] The method according to any one of [1] to [5], wherein the multi-way valve block is a six-way valve.

[7] The method according to any one of [1] to [6], wherein the detector comprises one or more ultraviolet light detectors.

[8] The method of any one of [1] to [7], wherein the two-dimensional chromatography device comprises one or more pumps to provide mobile phase to the one-dimensional chromatography system and the two-dimensional chromatography system.

[9] Further, the present invention also provides a method for the online quantitative detection of immunoglobulin G, which comprises the method according to any one of the above [1] to [8].

[10] Moreover, the present invention also provides a method for producing a dairy product, comprising using the method for online quantitative determination according to [9] above to online detect whether the immunoglobulin G content in the dairy product meets a preset standard, and adjusting or not adjusting the method for producing a dairy product according to the result of the method for online quantitative determination.

ADVANTAGEOUS EFFECTS OF INVENTION

Through the implementation of the technical scheme, the invention can obtain the following technical effects:

1) According to the invention, the sample containing the immunoglobulin G is detected by the two-dimensional chromatographic device, and the immunoglobulin G is purified, collected and detected by one-time computer processing, so that the detection efficiency is greatly improved, the detection result also has good accuracy and reproducibility, in some specific embodiments, the variation coefficient of 100 detection results of the same sample is lower than 0.8%, and the reliability of the detection method is greatly improved.

2) The purification, collection and chromatographic detection of the immunoglobulin G are finished by one-time on-machine processing, so that the problems of low total recovery rate, long time consumption and the like in the method for performing the on-machine detection of the chromatography independently by solid-phase extraction in the prior art can be solved.

3) The detection method of the invention can be used as a method for detecting the immunoglobulin G on line (and full-automatically) due to extremely high detection efficiency, and can realize real-time monitoring of the product quality of the production line of the dairy products containing the immunoglobulin G.

Drawings

FIG. 1: a diagram of the operating state of an affinity chromatography column in a one-dimensional chromatography column in some embodiments of the invention;

FIG. 2: a diagram of the operating state of a fraction collector in a one-dimensional chromatography system in some embodiments of the invention;

FIG. 3: a two-dimensional chromatographic column operating state diagram in some embodiments of the invention;

FIG. 4: shows the DAD profile of the separation assay using affinity chromatography columns only;

FIG. 5: shows the DAD profile when a C4 column is used in place of the distillate collection loop of the present invention;

FIG. 6: showing the DAD profile when a standard solution was tested using the detection method of the present invention;

FIG. 7: shows direct analysis using an affinity column in a one-dimensional chromatography system.

Detailed Description

The following describes embodiments of the present invention, but the present invention is not limited to these embodiments. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims, and embodiments and examples obtained by appropriately combining the technical means disclosed in the respective embodiments and examples are also included in the technical scope of the present invention.

< definition >

In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end points of numerical values a and B.

In the present specification, the numerical ranges indicated by "above" or "below" mean the numerical ranges including the numbers.

In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In this specification, the use of "optional" or "optional/optionally" means that certain substances, components, performance steps, application conditions, and the like are used or not used.

In the present specification, the term "normal temperature" as used herein generally means a temperature of 23. + -. 3 ℃ unless otherwise specified.

In the present specification, the unit names used are all international standard unit names, and the "%" used means weight or mass% content, if not specifically stated.

In the present specification, the term "parts" means parts by weight.

In the present specification, the term "substantially" means that the deviation from the standard is 1% or less, preferably 0.5% or less.

In the present specification, "column" and "chromatography column" have the same meaning.

In the present specification, the term "near" used in the description of the wavelength means that the systematic error introduced by the detection device is included.

In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

Unless defined otherwise, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention mainly provides a quantitative analysis and detection method of immunoglobulin G contained in dairy products, in particular liquid dairy products and powdered milk products, and the invention is mainly completed based on the following findings:

aiming at the problems that the prior art adopts an immunological method which is expensive and inconvenient to popularize and use, and the prior art adopts a chromatographic detection technology after solid phase extraction which has the problems of poor recovery rate, long time consumption, poor reliability and the like in the solid phase extraction step, the invention thinks that the detection efficiency of the whole detection can be improved by using a two-dimensional chromatographic device, thereby saving trouble and labor. And, set up the fraction collector in front of two-dimentional chromatographic system of the two-dimentional chromatographic apparatus, can solve the problem that how much the miscellaneous peak is difficult to quantitative analysis when only using one-dimentional chromatogram effectively, and avoid producing solvent effect and bad problem of peak type because of the change of mobile phase, stationary phase when using one-dimentional chromatogram and two-dimentional chromatogram jointly. Meanwhile, the invention considers that the combination of a fraction collector (instead of a reversed-phase chromatographic trapping column) and a two-dimensional chromatographic system improves the separation effect, shortens the peak time and greatly improves the detection efficiency.

In addition, by further using an automatic control device, the invention can provide the automatic detection or on-line detection function which is not existed in the past, and has good accuracy and reliability (reproducibility) and improved detection efficiency.

Based on the above findings, the present invention provides a method for quantitatively detecting immunoglobulin G by a two-dimensional chromatography device. The quantitative measurement method of the present invention mainly comprises: a pretreatment step and a detection step. Removing at least casein from the test object by the pretreatment step to obtain a sample containing immunoglobulin; in the step of detecting, the immunoglobulin G-containing sample is quantitatively detected using a two-dimensional chromatography apparatus and a detector, and the immunoglobulin G in the immunoglobulin G-containing sample is enriched in the fraction collector after being purified in the immunoglobulin G-specific affinity chromatography column, and then is quantitatively analyzed by being detected by the detector after passing through the two-dimensional chromatography system.

(detection target)

The detection target of the present invention is not particularly limited in principle, and may be various immunoglobulin G-containing products, particularly dairy products.

For the source of the immunoglobulin G of the present invention, it may be derived from human milk or animal milk in general. As the animal milk, cow's milk, goat's milk, horse's milk, camel's milk, etc. may be mentioned.

In some preferred embodiments of the present invention, the detection object may be liquid milk, such as liquid cow milk, goat milk, etc., or may be a powdered milk product. Further, as the liquid milk, liquid milk subjected to or not subjected to sterilization treatment may be used; for milk powder products, it may include infant formula milk powder, adult milk powder, middle aged and elderly people milk powder, protein powder, or milk powder as a complementary food, etc. In some more preferred embodiments, the test object may include liquid milk of human milk, cow milk, or goat milk, which may or may not be sterilized, or infant formula.

(step of pretreatment)

In the quantitative analysis and detection method of the present invention, before the quantitative detection of the present invention, appropriate pretreatment may be performed depending on the detection target to reduce the influence on the column and remove the foreign proteins or other adverse components that may affect the quantitative detection.

In some specific embodiments, the pretreatment step includes a step of removing casein, for example, in cow milk, the content of casein may be about 80% of the total protein content, and the presence of a large amount of casein may cause turbidity of the test object, which is not favorable for subsequent quantitative detection. It is therefore advantageous to remove the casein in order to obtain as clear a test sample as possible.

As the method for removing casein, there is in principle no particular limitation, and specific examples include removing casein in a solid form by a precipitation method at an isoelectric point (obtaining a whey fraction). The method for adjusting the isoelectric point is not particularly limited, and casein may be precipitated by forming a solution (microemulsion) of the treatment object and then adjusting the pH of the solution to 4.3 to 4.6 using an acidic component. As the acidic component, an organic acid such as acetic acid and the like is generally used. In some specific embodiments, 80% by mass or more, preferably 90% by mass or more, or substantially all of the casein in the object to be detected is removed by the step of removing casein.

In some other specific embodiments of the present invention, the pretreatment step may further comprise one or more of a step of removing fat, a step of removing lactose, and a step of concentrating or diluting, in addition to the step of removing casein, according to any other requirement. Through the pretreatment steps, the interference on the quantitative detection of the subsequent two-dimensional chromatographic device can be reduced.

With respect to the step of removing fat, there is in principle no particular limitation, and for example, a step of removing fat in the detection object by centrifugation can be cited. In some specific embodiments, at least 80% by mass, preferably at least 90% by mass, or substantially all of the fat in the test object is removed by the fat removal step.

With respect to the step of removing lactose, there is in principle no particular limitation, and a method including separation by (ultrafiltration) membrane treatment can be exemplified. In some specific embodiments, 80% by mass or more, preferably 90% by mass or more, or substantially all of the lactose in the test object is removed by the lactose removal step.

The concentration or dilution step is mainly required for subsequent detection, so that the sample detected on the computer has a proper substance content. The specific dry matter content can be determined according to the specific standards or requirements of the measuring instrument.

One-dimensional chromatographic system

The one-dimensional chromatographic system comprises one or more immunoglobulin G specific affinity chromatographic columns and a fraction collector. In some preferred embodiments, the one-dimensional chromatography system comprises an immunoglobulin G-specific affinity chromatography column and fraction collector in series.

The immunoglobulin G-specific affinity column is not particularly limited in principle, and a column having affinity for immunoglobulin G, which is known in the art, such as a gel having an immunoglobulin-binding protein-coupled agarose or glycosaminoglycan, a porous resin having an immunoglobulin-binding protein coupling (a crosslinked methacrylic resin, etc.), and the like, may be used. In some particular embodiments of the invention may be an affinity chromatography column with Protein G coupling.

In addition, in some preferred embodiments of the present invention, the affinity chromatography column is characterized by no diffusion, no micropores, no dead volume, in order to facilitate rapid mass transfer between the mobile phase and the stationary phase, from the viewpoint of improving the recovery rate of immunoglobulin G. Specific examples of the affinity chromatography column include Bio-Monolith Protein G chromatography column available from Agilent, and Pierce Protein G chromatography column available from Thermo Scientific.

The immunoglobulin G fraction collector according to the present invention can be used for enriching the immunoglobulin G fraction retained in the specific affinity column. The present invention recognizes that the use of a fraction collector in the one-dimensional chromatography system allows the detection result to be suitably peaked and also allows the detection to be unexpectedly shortened.

As the fraction collector usable in the present invention, there may be a commonly used fraction collecting ring or the like. In some preferred embodiments, the fraction collector has an automatic control element to assist automatic collection of the fraction. The capacity of the fraction collector ring is in principle not particularly critical, and in some specific embodiments the internal capacity may be in the range of 0.5 to 5ml, such as 1ml, 1.5ml, 2ml, etc.

Furthermore, in a preferred embodiment, the fraction to be finally determined is collected into the fraction collector at an appropriate time by previously measuring the retention time of immunoglobulin G from the affinity chromatography column.

Two-dimensional chromatographic system

The two-dimensional chromatography system of the invention comprises one or more antibody chromatography columns, and in some preferred embodiments, the two-dimensional chromatography system of the invention comprises one antibody chromatography column.

The antibody column is selected from a monoclonal antibody chromatographic column bonded with phenyl as a functional group on a high-purity integrated silica gel matrix, and is used for separating natural IgG for the first time. Natural IgG, because it is affected by processing, has a severe peak broadening when analyzed using a conventional chromatographic column, unlike monoclonal antibody IgG. The monolithic column with phenyl as the functional group has obviously better retention of natural IgG than the conventional chromatographic column with C4 as the functional group. Typically, monoclonal antibody chromatography columns may be used with the present invention.

Further, in some preferred embodiments of the present invention, the pore size of the antibody column in the two-dimensional chromatography system is 40 nm or more, preferably 55 to 70 nm, and the antibody column is controlled to have a suitable pore size to obtain a suitable retention time and to improve the recovery rate of the immunoglobulin G detection.

Pump and mobile phase supply

The two-dimensional chromatography apparatus of the invention also has one or more liquid phase pumps to provide mobile phase to the one-dimensional chromatography system and the two-dimensional chromatography system during operation.

In some embodiments of the invention, there may be one or more liquid pumps, and preferably two mutually independent liquid pumps may be provided to supply mobile phase to the one-dimensional chromatography system and the two-dimensional chromatography system, respectively.

For one-dimensional chromatography systems, the liquid pump (1D) provides a mobile phase (a) capable of enhancing the binding of immunoglobulin G to the affinity chromatography column, mainly during the working phase of the affinity chromatography column, such mobile phase (a) may be an aqueous solution containing a hydrogen phosphate salt or a hydrate thereof, typically, the hydrogen phosphate salt may be monohydrogen phosphate, dihydrogen phosphate, or the like, such as a potassium salt thereof, or the like. In addition, as for the concentration of the mobile phase (A), the concentration of the hydrogen phosphate thereof may be typically 45 to 55 mM, for example, 50mM, and the pH of the mobile phase (A) may be 6.5 to 7.5, preferably 6.8 to 7.2, from the viewpoint of promoting the affinity of the affinity column with immunoglobulin G. The ionic strength provided by the mobile phase (A) under the action of these concentrations and pH conditions is more favorable for the retention of the immunoglobulin G and the removal of other components, thereby being favorable for improving the recovery rate of the final immunoglobulin G.

Further, after the purification of the one-dimensional chromatography system is completed, the kind of the mobile phase may be adjusted by the above-mentioned liquid phase pump (1D pump) to elute the affinity chromatography column so that the immunoglobulin G is eluted from the affinity chromatography column and then transported to the fraction collector by the mobile phase. For this purpose, the mobile phase (B) is supplied by a liquid phase pump (1D pump) of the one-dimensional chromatography system, said mobile phase (B) being an aqueous solution comprising acidic or buffering components, preferably buffering components to adjust the mobile phase pH. The buffering component may be a glycine buffering component or the like. The concentration of the buffering component in the mobile phase (B) may be 90 to 110 mM, for example, 100 mM, and the pH may be 4 or less, preferably 2.2 to 2.8. The change of the mobile phase (B) by the pH condition is mainly for increasing the precipitation of immunoglobulin G and the increase of particle size to facilitate elution.

Further, after the immunoglobulin G is enriched in the fraction collector by the mobile phase (B), a liquid phase pump (2D pump) of the two-dimensional chromatography system is started, and the mobile phase (C) is supplied to elute the immunoglobulin G enriched in the fraction collector to an antibody chromatography column in the two-dimensional chromatography system, thereby performing quantitative analysis. In the process, the multi-way valve group is used for realizing the series connection of the fraction collector and the antibody chromatographic group in the two-dimensional chromatographic system, and then the mobile phase (C) provides fluid so as to realize the separation and detection of the enriched components in the fraction collector on the antibody chromatographic column.

For the mobile phase (C), which may be an aqueous solution in some typical embodiments, and from the viewpoint of good peak profile (e.g., improved peak shape, overcoming problems of peak broadening and tailing) in the analysis of immunoglobulin G by an antibody column in a two-dimensional chromatography system, the mobile phase (C) may be an aqueous solution of a separation enhancement aid such as trifluoroacetic acid. In some specific embodiments, the concentration of the mobile phase (C) is generally 2% by mass or less, preferably 0.01 to 0.5% by mass.

In addition, from the viewpoint of facilitating the improvement of the separation efficiency, in the detection by the two-dimensional chromatography system, it is also preferable to use a polar solvent component (mobile phase (D)) in the mobile phase in addition to the above-mentioned mobile phase (C). For mobile phase (D) a polar solvent that does not cause protein denaturation may be used, and in some embodiments the polar solvent may be a nitrile based solvent, such as acetonitrile and the like.

Further, in some specific embodiments of the present invention, the volume fraction of the mobile phase (D) is 40 vol% or less, preferably 10 to 35 vol%, more preferably 15 to 30 vol% based on the volume of the total mobile phase supplied by the two-dimensional chromatography system.

Multi-way valve set and working path

The invention realizes the connection switching from the one-dimensional chromatographic system to the two-dimensional chromatographic system by using the multi-way valve group.

There are no particular restrictions on the number of multi-way valves of the multi-way valve block, and in some specific embodiments, the connection or switching of the working paths described above is achieved by means of one or two multi-way valves.

From the viewpoint of ease of operation, the present invention may use a six-way valve. The working path controlled by the six-way valve is described below with reference to fig. 1 to 3.

As shown in fig. 1, the six-way valve is in the connection state shown in fig. 1, after the two-dimensional chromatography device is subjected to sample injection, the liquid phase pump (1D pump) is firstly started, at this time, the component to be analyzed is sent to the affinity chromatography column 1D analytical column (1D analytical column) of the one-dimensional chromatography system through the supply mobile phase (a), the waste liquid is led to the outlet VWD through the six-way valve, under the action of the mobile phase (a), the immunoglobulin G component is retained on the 1D analytical column, and the main impurity component is discharged out of the system through the VWD. Furthermore, in some embodiments, the retention time of the immunoglobulin on the 1D analytical column can also be precisely determined by the connection state or working path as shown in FIG. 1, and can be monitored by connecting an ultraviolet detector to the end of the working path as shown in FIG. 1. Also, the precise determination of the retention time facilitates switching of the connection mode of the two-dimensional chromatography apparatus at the appropriate time to collect the protein components including (all) the immunoglobulin G components in the fraction collector (i.e. the fraction collection loop in the figure).

In fig. 2, the six-way valve is switched to form the connection mode of fig. 2, at this time, the 1D analytical column is connected to the fraction collector through the six-way valve, and further the fraction collector is connected to the VWD through the six-way valve, at this time, the liquid phase pump (1D pump) stops supplying the mobile phase (a) to the one-dimensional chromatography system, and instead, the mobile phase (B) is supplied, at this time, the protein component including the immunoglobulin G remaining in the 1D analytical column is eluted into the fraction collector by the mobile phase (B), that is, the immunoglobulin G completes the enrichment or the secondary purification on the fraction collector, and some impurities are further discharged from the VWD by the mobile phase (B) through the six-way valve block.

In fig. 3, the six-way valve is switched to form the connection of fig. 3, in which case a liquid phase pump (2D pump) is connected in series with the fraction collector and the reverse phase chromatography column (2D analytical column). Further, the liquid phase pump (2D pump) is started, which supplies mobile phase (C) (and optionally mobile phase (D) in the proportions described hereinbefore). The mobile phase coming from the liquid phase pump (2D) is now mainly a polar organic solvent and an aqueous solution containing the separation enhancement aid mentioned above, which can deliver immunoglobulin G enriched in the fraction separator to the 2D analytical column for analytical detection.

Detector and optional other auxiliary units

In the present invention, the immunoglobulin G eluted from the antibody column of the two-dimensional chromatography system is detected by an ultraviolet light detector capable of detecting light absorption of 380 nm or less, and it is preferable that ultraviolet light having a wavelength of about 280nm is used for final analysis of the immunoglobulin G eluted from the two-dimensional chromatography system, and further, quantitative analysis is performed by the absorption peak.

In some preferred embodiments of the present invention, the detector of the present invention may be a ultraviolet Diode Array Detector (DAD).

In addition, other auxiliary units in the two-dimensional chromatography apparatus of the present invention are not particularly limited. In some particular embodiments, the auxiliary units optionally may include:

automatic sample injector: to realize automatic sample introduction detection of samples

The column oven may be used to store each column and adjust the temperature of each column, and typically may be used to maintain the operating temperature of the affinity column at 42 ℃ or lower, preferably 20 to 40 ℃ during operation, and may be used to maintain the operating temperature of the reverse phase column in two-dimensional chromatography at 45 ℃ or higher, preferably 60 to 82 ℃ during operation.

And the automatic control device can switch the positions of the multi-way valves in the multi-way valve group at proper time by a method of programming and the like so as to realize the switching of each working path.

The liquid phase pump flow control device is used for adjusting the liquid phase pump to supply different mobile phases and the flow rate of the mobile phases, and the total flow rate of the mobile phases in each stage of the invention can be controlled within 0.5-2 ml/min, preferably 0.6-1.5 ml/min.

Quantitative and linear limits

Under optimized measuring conditions, the limit of quantitation (S/N = 10) of the immunoglobulin G in raw milk, pasteurized milk and milk powder is 50.0 mg/kg. In the comparison verification work, a standard sample with the lowest concentration point of 50.0 mg/kg is prepared and added into the sterilized milk, and the standard sample is processed and tested on a computer, so that the signal-to-noise ratio is greater than 10 when the sample addition concentration is 50.0 mg/kg. Thus, the standard method has a limit of 50.0 mg/kg for the quantitation of IgG in raw, pasteurized and powdered milk.

Preparing 7 working curves with different concentrations: the immunoglobulin G standard intermediate solution (500 mg/L) was diluted with the eluent to prepare standard series working solutions at concentrations of 0.0 mg/L, 40.0 mg/L, 80.0 mg/L, 120.0 mg/L, 160.0 mg/L, 200.0 mg/L and 300.0 mg/L. And (3) carrying out sample injection measurement under the test condition determined by the invention to obtain the peak area of the immunoglobulin G. The concentration X (mg/L) of the immunoglobulin G is used as a horizontal coordinate, the peak area Y of the immunoglobulin G is used as a vertical coordinate, a standard curve is drawn, good linear relation is formed in the range of 0.0-300.0 mg/L, and the correlation coefficient is larger than 0.998.

(on-line detection)

Furthermore, the invention also provides an online quantitative detection method for automatically detecting the content of the immunoglobulin G in the dairy product, which comprises the above quantitative detection method for the immunoglobulin G.

The online detection method of the invention can be carried out in a full-automatic manner, a semi-automatic manner, etc. to detect whether the content of immunoglobulin G contained in the dairy product production meets the preset target or product requirements during a suitable time interval.

Further, according to the on-line quantitative detection result, if the detection result is different from a preset target or product requirement, the correction can be carried out through the adjustment of the production line.

Examples

The invention will be further illustrated by the following specific examples:

1. reagents and materials

Sodium hydroxide (NaOH);

hydrochloric acid (HCl): 37 percent;

trifluoroacetic acid (CF) ₃ COOH, TFA): the chromatogram is pure;

acetonitrile (CH) ₃ CN): carrying out chromatographic purification;

potassium dihydrogen phosphate (KH) ₂ PO ₄ ): the superior grade is pure;

glycine (NH) ₂ CH ₂ COOH): the superior grade is pure;

sodium chloride (NaCl);

immunoglobulin IgG control: the purity is more than or equal to 95.0 percent, and the beauty treatment is P/N MSS0378.

One-dimensional analytical column:

agilent Bio-Monolith Protein G liquid chromatography column, internal diameter: 5.2mm; length: 5 mm;

fraction collection ring: 1 mL;

two-dimensional analytical column:

GL MonoSelectRP-mAb, internal diameter: 2.1 mm; length: 20 mm; particle size: 1. mu m; pore diameter: 60 nm (length)

Mobile phase A: contains 50mM KH ₂ PO ₄ pH =7;

mobile phase B: contains 100 mM glycine buffer, pH = 2.50;

and (3) mobile phase C:0.1% aqueous TFA;

a mobile phase D: and (3) acetonitrile.

2. Test conditions

First-dimensional gradient conditions:

Time	A	B	flow rate
				0 min	100.00%	0.00%	1.000 mL/min
1.00 min	100.00%	0.00%	1.000 mL/min
				1.50 min	0.00%	100.00%	1.000 mL/min
7.00 min	0.00%	100.00%	1.000 mL/min
				8.00 min	100.00%	0.00%	1.000 mL/min
14.00 min	100.00%	0.00%	1.000 mL/min

Second-dimensional gradient conditions:

Time	C	D	flow rate
				0 min	75.00%	25.00%	0.800 mL/min
3.21 min	75.00%	25.00%	0.800 mL/min
				10.20 min	0.00%	100.00%	0.800 mL/min
11.20 min	75.00%	25.00%	0.800 mL/min
				14.00 min	75.00%	25.00%	0.800 mL/min

Valve position:

0.00 min: position shown in figure 1;

2.3 min: position shown in figure 2;

3.2 min: the position is shown in figure 3.

And (3) detecting the wavelength:

uv detector (uv diode array detector): 280nm;

column temperature:

a first dimension: 35 ℃; a second dimension: at 80 ℃.

Sampling amount each time:

50.00 µL；

sample processing method：

Weighing 5 g of milk powder, adding 30 mL of warm water at 40 ℃, and carrying out vortex oscillation for 5 min; weighing about 30 g of liquid milk, accurately adjusting pH to 4.5 with 50% acetic acid solution, diluting to 50 mL, shaking, filtering with filter paper, filtering the filtrate with 0.22 μm filter membrane, and detecting on a sample injection bottle.

Standard calibration mode:

1. sodium chloride solution (0.15 mol/L): 2.19 g of sodium chloride was weighed, dissolved in water and made to a volume of 250 mL.

2. Standard solution preparation

2.1 IgG standard stock solution:

weighing 5 mg of bovine IgG standard substance into a 10 mL volumetric flask, and recording the mass of the bovine IgG standard substance as W _S Adding sodium chloride solution to dissolve, fixing the volume to 10 mL, and recording the mass of the solution as W _T Vortex mixingAnd (6) homogenizing. IgG stock concentration calibration methods are as follows.

2.2 Preparing IgG standard working solution:

accurately transferring a certain amount of standard stock solution, diluting with sodium chloride solution to constant volume with dilution times of 0.1, 0.2, 0.4, 0.6, 0.8 and 1, and preparing at present.

After the IgG standard solution is prepared, the concentration of the IgG standard solution needs to be corrected before use, and the specific operation is as follows:

the absorbance of the test solution was measured at 280nm and 320 nm using a 1 cm quartz cuvette and a sodium chloride solution as a blank reference.

The percentage content of IgG CIgG% in the test solution is calculated according to the formula B.1:

………………………（B.1）

in the formula:

C _IgG % IgG percentage,%;

a280-ultraviolet absorbance value of test solution at 280nm;

a320-ultraviolet absorbance value of test solution at 320 nm;

1.4-0.1% extinction coefficient of bovine IgG solution in 1 cm cuvette;

1.7 Rayleigh scattering correction factor;

W _T -mass of solution in grams (g);

W _S -bovine IgG standard mass in grams (g).

3. Analysis results

FIG. 4 shows the DAD spectrum of the separation assay using affinity chromatography column alone when detecting bovine colostrum;

FIG. 5 shows a DAD profile of the same bovine colostrum of FIG. 4 when tested using a C4 column instead of the fraction collection loop of the present invention;

FIG. 6 shows a DAD profile when IgG standard solution (200.0 mg/L) was tested using the detection method of the present invention;

FIG. 7 shows that the IgG standard solution (160.0 mg/L) was analyzed directly by using an affinity column in a one-dimensional chromatography system, and the number of peaks was large.

4. Application example

The two-dimensional chromatography apparatus of the present invention was used to perform quantitative immunoglobulin G measurement on the following samples under the same conditions as above, and the results are shown in table 1 below.

Table 1:

industrial applicability

The detection method of the present invention can be industrially used for the quantitative detection of immunoglobulin G.

Claims

1. A method for quantitatively detecting immunoglobulin G, comprising:

a pretreatment step of removing at least casein from a test object to obtain a sample containing immunoglobulin G;

and, in the step of detecting:

the igg in the igg-containing sample is enriched in the fraction collector after being purified in the igg-specific affinity chromatography column, and then detected by the detector after passing through the two-dimensional chromatography system.

2. The method of claim 1, further comprising determining a retention time of immunoglobulin G on the immunoglobulin G-specific affinity chromatography column using the detector.

3. The method according to claim 1 or 2, wherein the detection object is selected from liquid milk or powdered milk; in the pretreatment step, the detection object is further subjected to degreasing and/or delactomy.

4. The method of claim 1 or 2, wherein the immunoglobulin G-specific affinity chromatography column is selected from Protein G affinity chromatography columns.

5. The method of claim 1 or 2, wherein the antibody chromatography column in the two-dimensional chromatography system is selected from a monoclonal antibody chromatography column.

6. The method of claim 1 or 2, wherein the multi-way valve set is a six-way valve.

7. A method according to claim 1 or 2, wherein the detector comprises one or more ultraviolet light detectors.

8. The method of claim 1 or 2, wherein the two-dimensional chromatography device further comprises one or more pumps to provide mobile phase to the one-dimensional chromatography system and the two-dimensional chromatography system.

9. A method for the online quantitative detection of igg, comprising the method according to any one of claims 1 to 8.

10. A method for producing dairy products, wherein the method comprises the steps of using the method for online quantitative determination according to claim 9 to online determine whether the content of immunoglobulin G in the dairy products meets a preset standard, and adjusting or not adjusting the method for producing dairy products according to the result of the method for online quantitative determination.