CN116298302A - Kit for detecting immunoglobulin G subclass, and preparation method and application method thereof - Google Patents

Kit for detecting immunoglobulin G subclass, and preparation method and application method thereof Download PDF

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CN116298302A
CN116298302A CN202211102818.5A CN202211102818A CN116298302A CN 116298302 A CN116298302 A CN 116298302A CN 202211102818 A CN202211102818 A CN 202211102818A CN 116298302 A CN116298302 A CN 116298302A
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human
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陈扬
徐陈槐
潘兴鑫
周延庆
罗浩
张现侠
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Hangzhou Saiji Biotechnology Co ltd
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Abstract

The invention provides a kit for detecting immunoglobulin G subclasses, which comprises at least four specific antibodies and a detection antibody, wherein the at least four specific antibodies comprise an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody and an anti-human IgG4 antibody, and the detection antibody comprises an anti-human IgG antibody. The kit can simultaneously complete detection of four immunoglobulin G subclasses of IgG1, igG2, igG3 and IgG4 in the same sample, can reduce reagent cost and labor cost, and solves the problem that the kit in the prior art can not accurately quantify IgG1, igG2, igG3 and IgG4 at the same time. The invention also provides a preparation method and a use method of the kit.

Description

Kit for detecting immunoglobulin G subclass, and preparation method and application method thereof
Technical Field
The invention relates to the technical field of biological medicine detection, in particular to a kit for detecting immunoglobulin G subclasses, a preparation method and a use method thereof.
Background
Immunoglobulin G (IgG) is the main contributing molecule to human immunity and represents about 75% of the total immunoglobulin in serum of healthy humans. Human IgG is divided into 4 different subtypes: igG1, igG2, igG3, igG4, at concentrations of IgG1 in serum: (60% -70%), igG2: (15% -20%), igG3: (5% -10%), igG4: (< 5%).
IgG1 is the most important subtype in IgG, has a strong ability to activate complement and bind phagocytes, and IgG1 deficiency or deficiency in combination with other IgG subclasses is associated with recurrent infections; igG2 dominates the immunity and response to polysaccharide antigens, and IgG2 deficiency may lead to deletion of IgG anti-saccharide antibodies, and selective IgG2 deficiency is manifested by increased incidence of viral and bacterial infection, indicative of functional deficiency of immune response, indicating that IgG2 has a role in protecting against these pathogens; igG3 often exhibits higher affinity than IgG1 in immune responses to protein and polypeptide antigens, and studies have shown that IgG3 levels are positively correlated with the severity of Graves' disease, autoimmune thyroiditis (AIT), thrombotic Thrombocytopenic Purpura (TTP); the studies have found that IgG 4-related diseases (IgG 4-RD) have significantly elevated IgG4 levels and have an infiltration of IgG 4-positive plasma cells, and that IgG 4-type autoantibodies or IgG 4-positive plasma cells are also observed in autoimmune diseases such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE).
Abnormalities in the concentration of IgG subclasses may be indicative of a disorder in normal autoimmune responses in humans. Various diseases cause an increase or decrease in the subclass of IgG, and defects or dysregulation of the subclass of IgG is an indicator of eliciting an immune response. The most common consequence of the absence of a certain subclass of IgG is insufficient humoral immunity, although this is not necessarily a clinical manifestation. Since a decrease in the level of one subclass of IgG may be accompanied by an increase in the level of one or more other subclasses of IgG, the total level of IgG remains normal. Therefore, even though the total amount of IgG is in the normal range, it is necessary to determine IgG subclass levels. The main clinical indicator of detection of IgG subclasses is that long-term or severe infections often occur and cannot be interpreted by general clinical and laboratory examination results. In general, the IgG subclass should be detected when clinically insufficient immune function is suspected. The dynamic detection of IgG subclasses has important clinical application value for diagnosis, pathogenesis and prognosis judgment of diseases.
Currently, the main methods for quantitatively detecting IgG subclasses in the market include an immunoturbidimetry method, a chemiluminescent method, an enzyme-linked immunosorbent assay method and a flow fluorescent method, wherein the immunoturbidimetry method is most frequently used, and the chemiluminescent method and the flow fluorescent method are less frequently used. The enzyme-linked immunosorbent assay method has complicated manual operation steps and poor result repeatability. The method has the advantages of simple and convenient operation of the immunotransmittance and turbidity, suitability for common automatic biochemical analyzers and common spectrophotometers, poor sensitivity and precision, large required antiserum amount and longer detection period. The immune scattering turbidimetry has the advantages of automatic operation and the defects of unsatisfactory sensitivity and detection range. Currently, the detection products of the subclass of IgG are most frequently detected by a single index, and thus a method capable of simultaneously detecting four indexes of IgG1, igG2, igG3 and IgG4 is required.
Therefore, there is a need to develop a kit for detecting immunoglobulin G subclasses, and methods for preparing and using the same, to solve the above-mentioned problems in the prior art.
Disclosure of Invention
The invention aims to provide a kit for detecting immunoglobulin G subclasses, which solves the problem that the kit in the prior art cannot accurately quantify IgG1, igG2, igG3 and IgG4 at the same time.
To achieve the above object, the present invention provides a kit for detecting immunoglobulin G subclasses, comprising at least four specific antibodies including an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody and an anti-human IgG4 antibody, and a detection antibody including an anti-human IgG antibody.
The kit for detecting the immunoglobulin G subclass has the beneficial effects that: the kit solves the problem that the kit in the prior art can not accurately quantify IgG1, igG2, igG3 and IgG4 at one time, has good repeatability and batch difference, has a variation coefficient of the batch test of the kit of not more than 15 percent, has a variation coefficient of the batch test of not more than 15 percent, and has good application prospect.
Alternatively, the at least four specific antibodies are immobilized to a solid support.
Optionally, the solid phase carrier comprises any one of an ELISA plate micropore, a magnetic bead, an affinity membrane and a liquid phase chip, and the liquid phase chip comprises microspheres.
Optionally, the solid phase carrier is the microsphere, the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody are respectively fixed on the microsphere to obtain 4 different specific antibody microspheres, the 4 different specific antibody microspheres are mixed with a reaction buffer solution to obtain a specific antibody solution, and each milliliter of the specific antibody solution contains 5.0x10 5 ～8.0×10 5 And each microsphere. The beneficial effects are that: the greater or lesser number of microspheres per milliliter of specific antibody solution affects the potency of the specific antibody.
Optionally, the anti-human IgG antibody is conjugated with phycoerythrin to obtain a phycoerythrin conjugated antibody, and the phycoerythrin conjugated antibody is mixed with a reaction buffer to obtain a detection antibody solution.
Optionally, the mass ratio of the anti-human IgG antibody to the phycoerythrin is 1 (1-5). The beneficial effects are that: the subsequent detection result is clearer and discernable.
Optionally, the concentration of the detection antibody solution is (2-6) μg/ml. The beneficial effects are that: different concentrations of detection antibody solution affect the antibody titer.
Optionally, the signal detection method adopted by the kit is any one of a visible light color development method, a chemiluminescence method and a fluorescence method.
Optionally, the kit further comprises a washing buffer solution, a reaction buffer solution, a sample diluent, a calibrator and a quality control product.
It is still another object of the present invention to provide a method for preparing the kit, comprising the steps of:
s0: providing an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody, an anti-human IgG4 antibody, an anti-human IgG antibody, microspheres and phycoerythrin, wherein the microspheres are polystyrene fluorescent microspheres loaded with allophycocyanin at different concentrations;
s1: respectively fixing the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody on the microspheres to obtain 4 different specific antibody microspheres, and mixing the 4 different specific antibody microspheres with a reaction buffer solution to obtain a specific antibody solution;
s2: and coupling the anti-human IgG antibody with the phycoerythrin to obtain a phycoerythrin coupled antibody, and mixing the phycoerythrin coupled antibody with a reaction buffer solution to obtain a detection antibody solution.
The preparation method of the kit has the beneficial effects that: respectively fixing the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody on the microspheres to obtain 4 different specific antibody microspheres, and mixing the 4 different specific antibody microspheres with a reaction buffer solution to obtain a specific antibody solution; the anti-human IgG antibody is coupled with phycoerythrin to obtain phycoerythrin coupled antibody, and the phycoerythrin coupled antibody is mixed with a reaction buffer solution to obtain a detection antibody solution, so that the preparation process of the kit is simple and convenient, and the obtained kit can accurately and quantitatively detect IgG1, igG2, igG3 and IgG4 at one time.
Another object of the present invention is to provide a method for using the kit, comprising the steps of:
s0: providing a specific antibody solution, a detection antibody solution, a diluted sample to be tested and a reaction buffer solution, wherein the specific antibody solution comprises at least four specific detection antibodies, the at least four specific detection antibodies comprise an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody and an anti-human IgG4 antibody, and the detection antibodies in the detection antibody solution comprise an anti-human IgG antibody;
s1: sequentially adding the reaction buffer solution, the diluted sample to be tested and the detection antibody solution to obtain a mixed solution;
s2: adding the specific antibody solution into the mixed solution, and then incubating at room temperature in a dark place to obtain a solution to be detected;
s3: detecting the fluorescence intensities of different microspheres in the solution to be detected to distinguish the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody, and detecting the fluorescence intensities of phycoerythrin in the solution to be detected to calculate the content of IgG1, igG2, igG3 and IgG4 in the sample to be detected.
The use method of the kit has the beneficial effects that: solves the problem that the kit in the prior art can not accurately quantify human IgG1, igG2, igG3 and IgG4 at the same time.
Drawings
FIG. 1 is a schematic diagram showing the relationship among specific antibodies, microspheres, detection antibodies and phycoerythrin in the examples of the present invention;
FIG. 2 is a graph showing the linearity of IgG1 in the linear region of the present invention;
FIG. 3 is a graph showing the linearity of IgG2 over the linear range of the present invention;
FIG. 4 is a graph showing the linearity of IgG3 in the linear region of the present invention;
FIG. 5 is a graph showing the linearity of IgG4 in the linear region of the present invention
FIG. 6 is a calibration graph of IgG1 of the examples of this invention;
FIG. 7 is a graph showing calibration of IgG2 in an embodiment of the invention;
FIG. 8 is a graph showing calibration of IgG3 in an embodiment of the invention;
FIG. 9 is a graph showing calibration of IgG4 in the examples of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless otherwise defined, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. As used herein, the word "comprising" and the like means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof without precluding other elements or items.
In an embodiment of the present invention, a kit for detecting immunoglobulin G subclasses is provided, the kit comprising at least four specific antibodies including an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody, and an anti-human IgG4 antibody, and a detection antibody including an anti-human IgG antibody.
Specifically, by comprising at least four specific antibodies and a detection antibody, wherein the at least four specific antibodies comprise an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody and an anti-human IgG4 antibody, and the detection antibody comprises an anti-human IgG antibody, the invention solves the problem that a kit in the prior art can not accurately quantify IgG1, igG2, igG3 and IgG4 at one time, and has good repeatability and batch difference, the variation coefficient of the batch test of the kit is not more than 15%, and the kit has good application prospect.
In some possible embodiments of the invention, the at least four specific antibodies are immobilized on a solid support.
In some possible embodiments of the invention, the solid support comprises any one of an elisa plate microwell, magnetic beads, affinity membrane, and a liquid phase chip comprising microspheres.
In some possible embodiments of the present invention, the solid support is the microsphere, the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody are immobilized on the microsphere to obtain 4 different specific antibody microspheres, and the 4 different specific antibody microspheres are mixed with a reaction buffer to obtain a specific antibody solution, wherein each milliliter of the specific antibody solution contains 5.0x10 5 ～8.0×10 5 And each microsphere. The greater or lesser number of microspheres per milliliter of specific antibody solution affects the potency of the specific antibody.
In some possible embodiments of the present invention, the anti-human IgG antibody is conjugated to phycoerythrin to obtain a phycoerythrin conjugated antibody, and the phycoerythrin conjugated antibody is mixed with a reaction buffer to obtain a detection antibody solution.
In some possible embodiments of the invention, the mass ratio of the anti-human IgG antibody to the phycoerythrin is 1 (1-5). The subsequent detection result is clearer and discernable.
In some possible embodiments of the invention, the concentration of the detection antibody solution is (2-6) μg/ml. Different concentrations of detection antibody solution affect the antibody titer.
In some possible embodiments of the present invention, the signal detection method adopted by the kit is any one of a visible light color development method, a chemiluminescence method and a fluorescence method.
In some possible embodiments of the invention, the kit further comprises a wash buffer, a reaction buffer, a sample diluent, a calibrator, and a quality control.
In some possible embodiments of the invention, the sample diluent is a 10 x sample diluent.
In some possible embodiments of the invention, the preparation steps of the 1 x sample dilution include: the 10 x sample dilution is diluted to the 1 x sample dilution in a 1:9 ratio using purified water, i.e., 1 part of the 10 x sample dilution plus 9 parts of purified water can be diluted to the 1 x sample dilution.
In some possible embodiments of the present invention, the preparation steps of the diluted sample to be tested include: and continuously diluting a sample to be tested with the 1X sample diluent twice for 101 times, taking 10ul of serum, adding the diluted sample to be tested into 1ml of the 1X sample diluent, diluting and uniformly mixing, and then taking 10ul of diluted sample to be tested, adding the diluted sample to be tested into 1ml of the 1X sample diluent, diluting and uniformly mixing to obtain the diluted sample to be tested.
In an embodiment of the present invention, a method for preparing the kit is provided, including the following steps:
s0: providing an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody, an anti-human IgG4 antibody, an anti-human IgG antibody, microspheres and phycoerythrin, wherein the microspheres are polystyrene fluorescent microspheres loaded with allophycocyanin at different concentrations;
s1: respectively fixing the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody on the microspheres to obtain 4 different specific antibody microspheres, and mixing the 4 different specific antibody microspheres with a reaction buffer solution to obtain a specific antibody solution;
s2: and coupling the anti-human IgG antibody with the phycoerythrin to obtain a phycoerythrin coupled antibody, and mixing the phycoerythrin coupled antibody with a reaction buffer solution to obtain a detection antibody solution.
Specifically, the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody are respectively fixed on the microspheres to obtain 4 different specific antibody microspheres, and the 4 different specific antibody microspheres are mixed with a reaction buffer solution to obtain a specific antibody solution; the anti-human IgG antibody is coupled with phycoerythrin to obtain phycoerythrin coupled antibody, and the phycoerythrin coupled antibody is mixed with a reaction buffer solution to obtain a detection antibody solution, so that the preparation process of the kit is simple and convenient, and the obtained kit can accurately and quantitatively detect IgG1, igG2, igG3 and IgG4 at one time.
In some possible embodiments of the present invention, there is provided a method for using the kit, comprising the steps of:
s0: providing a specific antibody solution, a detection antibody solution, a diluted sample to be tested and a reaction buffer solution, wherein the specific antibody solution comprises at least four specific detection antibodies, the at least four specific detection antibodies comprise an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody and an anti-human IgG4 antibody, and the detection antibodies in the detection antibody solution comprise an anti-human IgG antibody;
s1: sequentially adding the reaction buffer solution, the diluted sample to be tested and the detection antibody solution to obtain a mixed solution;
s2: adding the specific antibody solution into the mixed solution, and then incubating at room temperature in a dark place to obtain a solution to be detected;
s3: detecting the fluorescence intensities of different microspheres in the solution to be detected to distinguish the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody, and detecting the fluorescence intensities of phycoerythrin in the solution to be detected to calculate the content of IgG1, igG2, igG3 and IgG4 in the sample to be detected. Solves the problem that the kit in the prior art can not accurately quantify human IgG1, igG2, igG3 and IgG4 at the same time.
In some embodiments of the invention, the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody, the anti-human IgG4 antibody, and the anti-human IgG antibody are all murine monoclonal antibodies.
In some embodiments of the invention, the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody, the anti-human IgG4 antibody, and the anti-human IgG antibody are each selected from any one of an ovine, a rabbit, or a camel antibody.
FIG. 1 is a schematic diagram showing the relationship among specific antibodies, microspheres, detection antibodies and phycoerythrin in the examples of the present invention.
In some embodiments of the present invention, referring to fig. 1, the specific antibody 2 is immobilized on the microsphere 1, the detection antibody 4 is coupled to the phycoerythrin 5 to obtain a phycoerythrin coupled antibody, and the specific antibody 2 immobilized on the microsphere 1 is specifically bound to the sample 3 to be tested first, and then is specifically bound to the phycoerythrin coupled antibody.
In some embodiments of the present invention, the apparatus for detecting the fluorescence intensity of the different microspheres in the solution to be detected and detecting the fluorescence intensity of the phycoerythrin in the solution to be detected is a flow cytometry, which comprises the following steps: and irradiating the solution to be detected by two beams of excitation light with different wavelengths emitted by the flow cytometry to obtain the fluorescence intensity of the microsphere and the fluorescence intensity of the phycoerythrin, determining the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody according to the fluorescence intensities of the microsphere, and determining the content of IgG1, igG2, igG3 and IgG4 in the sample to be detected according to the fluorescence intensities of the phycoerythrin.
In other embodiments of the present invention, the detection limit of IgG1 in the diluted sample to be measured detected by the flow cytometer is not higher than 650 μg/mL, the detection limit of IgG2 in the diluted sample to be measured is not higher than 250 μg/mL, the detection limit of IgG3 in the diluted sample to be measured is not higher than 15 μg/mL, and the detection limit of IgG4 in the diluted sample to be measured is not higher than 18 μg/mL.
In some possible embodiments of the present invention, the linear interval of IgG1 in the diluted sample to be measured detected by the flow cytometer is not narrower than [800,40000] μg/mL, the linear interval of IgG2 in the diluted sample to be measured is not narrower than [300,10000] μg/mL, the linear interval of IgG3 in the diluted sample to be measured is not narrower than [20,2600] μg/mL, and the linear interval of IgG4 in the diluted sample to be measured is not narrower than [20,5500] μg/mL.
In some possible embodiments of the present invention, the absolute value of the linear correlation coefficient R in the linear interval is not less than 0.990.
The manufacturer, model or make of the instrument used in the examples is shown in Table 1.
Table 1 instrument manufacturer and model or make thereof
Manufacturing factories
Flow cytometer Shenzhen Mindray Bio-Medical Electronics Co.,Ltd.
Centrifugal machine Hunan Xiangxin Instrument Co., Ltd.
Constant temperature oscillation incubator TUOHE ELECTROMECHANICAL TECHNOLOGY (SHANGHAI) Co.,Ltd.
The manufacturer, model or make of the reagents used in the examples are shown in Table 2.
Table 2 reagent manufacturers and their model or make
Composition of the components Manufacturing factories
Fluorescent microsphere BioLegend,Inc.
Phycoerythrin Sigma
Boric acid Sinopharm Group Chemical Reagent Co., Ltd.
Sodium borate Sinopharm Group Chemical Reagent Co., Ltd.
Proclin300 Sigma
Tween-20 SHANGHAI ALADDIN BIOCHEMICAL TECHNOLOGY Co.,Ltd.
Example 1 preparation of the kit
The kit comprises a specific antibody solution, a detection antibody solution, a sample diluent, a reaction buffer solution, a washing buffer solution, a calibrator and a quality control product. The sample dilution was 10 x sample dilution and the wash buffer was 10 x wash buffer.
Preparation of specific antibody solutions: by activating carboxyl groups on the surfaces of different microspheres, forming covalent bonds between the carboxyl groups and amino groups of an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody and an anti-human IgG4 antibody to obtain 4 different microsphere specific antibodies, diluting the 4 different microsphere specific antibodies by 500 times with a reaction buffer solution to obtain a specific antibody solution, wherein each milliliter of the specific antibody solution contains 6.0X10 5 And each microsphere. Among them, anti-human IgG1 antibodies, anti-human IgG2 antibodies, anti-human IgG3 antibodies, and anti-human IgG4 antibodies were purchased from BD corporation.
Preparation of detection antibody solution: coupling an anti-human IgG antibody with phycoerythrin to obtain a phycoerythrin coupled antibody, controlling the coupling of each 1 mg of the anti-human IgG antibody with 5 mg of phycoerythrin, optimizing the phycoerythrin coupled antibody, diluting the optimized phycoerythrin coupled antibody by 50 times by using a reaction buffer solution to obtain a detection antibody solution, and controlling the concentration of the detection antibody solution to be 4 mug/ml. Among them, anti-human IgG antibodies were purchased from madia biotechnology limited.
Preparation of sample dilutions: is prepared from boric acid-borax and Proclin300 buffer solution through mixing.
Preparation of reaction buffer: is prepared from boric acid-borax, proclin300 and Tween-20 buffer solution through mixing.
Preparation of the washing buffer: is prepared from phosphate and Tween-20 buffer solution through mixing.
The calibrator comprises human IgG1 protein, human IgG2 protein, human IgG3 protein and human IgG4 protein with different concentrations, and is used for establishing calibration curve during detection.
The quality control product comprises high-value quality control products containing human IgG1 protein, human IgG2 protein, human IgG3 protein and human IgG4 protein and median quality control products containing human IgG1 protein, human IgG2 protein, human IgG3 protein and human IgG4 protein, and is used for judging the reliability of detection results.
Human IgG1 protein, human IgG2 protein, human IgG3 protein, and human IgG4 protein in the calibrator and quality control were purchased from NOVUS.
Preparation of the kit: 2.5 ml of specific antibody solution is filled into the first packaging bottle, 2.5 ml of detection antibody solution is filled into the second packaging bottle, 21 ml of 10 x sample diluent is filled into the third packaging bottle, 2.5 ml of reaction buffer is filled into the fourth packaging bottle, 15 ml of 10 x washing buffer is filled into the fifth packaging bottle, calibrator is filled into the sixth packaging bottle, quality control is filled into the seventh packaging bottle, and the first packaging bottle, the second packaging bottle, the third packaging bottle, the fourth packaging bottle, the fifth packaging bottle, the sixth packaging bottle and the seventh packaging bottle are filled into the packaging box to obtain the kit. Wherein the kit is 100 parts per kit.
The kit can be optionally added with no calibrator and no quality control.
Intra-batch and inter-batch experiments of the kit
10 kits were obtained from lot I and labeled 1-10 on each kit, and the results of the in-lot test are shown in Table 3 in μg/ml.
TABLE 3 data for in-batch testing
Figure BDA0003841351490000121
As can be seen from Table 3, the coefficient of variation in the in-batch test of the kit was not more than 15%.
10 of the kits obtained in batch I, batch II and batch III were each extracted and labeled 1 to 10 on the kit, respectively, and the results of the batch-to-batch test are shown in Table 4 in μg/ml.
TABLE 4 data from batch to batch
Figure BDA0003841351490000122
Figure BDA0003841351490000131
As can be seen from Table 4, the coefficient of variation in the batch test of the kit was not more than 15%.
Example 2 methods of Using the kit
Preparation of 1 x sample dilutions: the 10X sample diluent is diluted into 1X sample diluent according to the ratio of 1:9 by using purified water, namely 1 part of 10X sample diluent and 9 parts of purified water can be diluted into 1X sample diluent for standby.
Preparation of the sample to be tested after dilution: and (3) continuously diluting the sample to be tested with 1 Xsample diluent twice for 101 times, adding 10ul of serum into 1ml of 1 Xsample diluent for dilution and uniform mixing, and then adding 10ul of diluted serum sample into 1ml of 1 Xsample diluent for dilution and uniform mixing to obtain diluted sample to be tested for later use.
The application method of the kit comprises the following specific steps:
(1) Add 25. Mu.l reaction buffer to each flow tube;
(2) Respectively adding 25 mu l of diluted sample to be tested into the flow tube;
(3) Add 25. Mu.l of detection antibody solution to each flow tube;
(4) Adding 25 mu l of specific antibody solution which is fully and uniformly mixed into a flow tube respectively, fully and uniformly mixing, and oscillating for more than 30 seconds by an oscillator;
(5) Incubating for 1h at room temperature in a constant temperature shaking incubator in a dark place;
(6) Placing the flow tube in a centrifuge, centrifuging for 5min under 300g, discarding supernatant, and retaining microspheres;
(7) Adding 300 μl of 1 Xwashing buffer into the flow tube, re-suspending the microspheres by vortex, mixing thoroughly, and oscillating with an oscillator for more than 30 seconds;
(8) Detection using a merry BriCyte E6 flow cytometer;
(9) Using FCAPArray TM The detection result was analyzed by Software Version 3.0.
The final test results obtained by the above procedure are shown in tables 5 and 6.
TABLE 5 fluorescence intensity of class-corresponding microspheres of anti-human IgG antibodies
Class of anti-human IgG antibodies Fluorescence intensity of microspheres
Anti-human IgG1 antibodies 300000
Anti-human IgG2 antibodies 100000
Anti-human IgG3 antibodies 70000
Anti-human IgG4 antibodies 10000
As is clear from Table 5, when the fluorescence intensity of the microspheres was 300000, the anti-human IgG1 antibody was assigned, when the fluorescence intensity of the microspheres was 100000, the anti-human IgG2 antibody was assigned, when the fluorescence intensity of the microspheres was 70000, the anti-human IgG3 antibody was assigned, and when the fluorescence intensity of the microspheres was 10000, the anti-human IgG4 antibody was assigned.
TABLE 6 content of IgG1, igG2, igG3 and IgG4 in samples to be tested after dilution
Fluorescence intensity of phycoerythrin Antibody content
IgG1 1000-46000 800-40000
IgG2 1000-40000 300-10000
IgG3 1000-70000 20-2600
IgG4 800-55000 20-5500
Note that: the unit of antibody content in Table 6 is μg/mL.
As is clear from Table 6, when the fluorescence intensity of phycoerythrin is 1000-46000, the corresponding IgG1 content in the diluted sample to be measured is 800-40000. Mu.g/mL, when the fluorescence intensity of phycoerythrin is 1000-40000, the corresponding IgG2 content in the diluted sample to be measured is 300-10000. Mu.g/mL, when the fluorescence intensity of phycoerythrin is 1000-70000, the corresponding IgG3 content in the diluted sample to be measured is 20-2600. Mu.g/mL, and when the fluorescence intensity of phycoerythrin is 800-55000, the corresponding IgG4 content in the diluted sample to be measured is 20-5500. Mu.g/mL.
Reference range studies were performed on 153 healthy adult serum samples to obtain normal reference value ranges for the four immunoglobulin G subclasses in healthy humans, specific values are detailed in table 7, and units in table 7 are μg/mL.
TABLE 7 Normal reference value Range
Normal reference value range
IgG1 4100-10100
IgG2 1700-7900
IgG3 110-850
IgG4 36-2000
For reference, each laboratory can establish its own reference range due to differences in geography, race, gender, age, etc.
The detection limit of IgG1 in the diluted sample to be detected by the flow cytometry is not higher than 650 mug/mL, the detection limit of IgG2 in the diluted sample to be detected is not higher than 250 mug/mL, the detection limit of IgG3 in the diluted sample to be detected is not higher than 15 mug/mL, and the detection limit of IgG4 in the diluted sample to be detected is not higher than 18 mug/mL.
FIG. 2 is a graph showing the linearity of IgG1 in the linear region of the present invention; FIG. 3 is a graph showing the linearity of IgG2 over the linear range of the present invention; FIG. 4 is a graph showing the linearity of IgG3 in the linear region of the present invention; FIG. 5 is a graph showing the linearity of IgG4 in the linear region of the present invention.
The linear interval of IgG1 in the diluted sample to be measured detected by a flow cytometer is not narrower than [800,40000] mug/mL, the linear interval of IgG2 in the diluted sample to be measured is not narrower than [300,10000] mug/mL, the linear interval of IgG3 in the diluted sample to be measured is not narrower than [20,2600] mug/mL, the linear interval of IgG4 in the diluted sample to be measured is not narrower than [20,5500] mug/mL, and the absolute value of the linear correlation coefficient R in the linear interval is not smaller than 0.990. Referring to fig. 2, the linear correlation coefficient R in the linear section of igg1 is 0.9985; referring to fig. 3, the linear correlation coefficient R in the linear section of igg2 is 0.9952; referring to fig. 4, the linear correlation coefficient R in the linear section of igg3 is 0.9988; referring to fig. 5, the linear correlation coefficient R in the linear section of igg4 is 0.9997.
Example 3 calibration curves for four immunoglobulin G subclasses IgG1, igG2, igG3, and IgG4 were established using the kits provided herein
The calibrator comprises human IgG1 protein, human IgG2 protein, human IgG3 protein and human IgG4 protein with different concentrations, and is used for establishing calibration curve during detection.
FIG. 6 is a calibration graph of IgG1 of the examples of this invention; FIG. 7 is a graph showing calibration of IgG2 in an embodiment of the invention; FIG. 8 is a graph showing calibration of IgG3 in an embodiment of the invention; FIG. 9 is a graph showing calibration of IgG4 in the examples of the present invention.
The step of establishing a calibration curve includes:
(1) 8 flow tubes were taken, 25. Mu.l of reaction buffer was added to each flow tube, and tube 1 was a blank tube.
(2) To the other 7 flow tubes, 25 μl of calibrator A solution, calibrator B solution, calibrator C solution, calibrator D solution, calibrator E solution, calibrator F solution, and calibrator G solution were added sequentially.
(3) Steps (3) to (8) in example 2;
(4) Using FCAP Array TM The detection results were analyzed by Software Version 3.0 and Graphpad Prism 9 and a calibration curve was drawn.
Calibration curve for IgG1, see fig. 6; calibration curve for IgG2, see fig. 7; calibration curve for IgG3, see fig. 8; calibration curve for IgG4, see fig. 9.
Example 4 quality control Using the kit provided by the invention
The quality control product comprises high-value quality control products containing human IgG1 protein, human IgG2 protein, human IgG3 protein and human IgG4 protein and median quality control products containing human IgG1 protein, human IgG2 protein, human IgG3 protein and human IgG4 protein, and is used for judging the reliability of detection results. The quality control product 1 is a high-value quality control product containing human IgG1 protein, human IgG2 protein, human IgG3 protein and human IgG4 protein; quality control 2 is a median quality control containing human IgG1 protein, human IgG2 protein, human IgG3 protein and human IgG4 protein, and specific parameters are shown in table 8.
Table 8 parameters of IgG1 quality control, igG2 quality control, igG3 quality control and IgG4 quality control
Figure BDA0003841351490000171
Figure BDA0003841351490000181
As can be seen from Table 8, the contents of the 4 antibodies were all within the target value range, indicating that the quality control of the kit was passed.
While embodiments of the present invention have been described in detail hereinabove, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. It is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention described herein is capable of other embodiments and of being practiced or of being carried out in various ways.

Claims (11)

1. A kit for detecting immunoglobulin G subclasses, comprising at least four specific antibodies including an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody, and an anti-human IgG4 antibody, and a detection antibody including an anti-human IgG antibody.
2. The kit of claim 1, wherein the at least four specific antibodies are immobilized on a solid support.
3. The kit of claim 2, wherein the solid support comprises any one of an elisa plate microwell, magnetic beads, affinity membrane, and a liquid phase chip comprising microspheres.
4. The kit according to claim 3, wherein the solid support is the microsphere, the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody are immobilized on the microsphere to obtain 4 different specific antibody microspheres, and the 4 different specific antibody microspheres are mixed with a reaction buffer to obtain a specific antibody solution, wherein each milliliter of the specific antibody solution contains 5.0X10 s 5 ～8.0×10 5 And each microsphere.
5. The kit of claim 1, wherein the anti-human IgG antibody is conjugated to phycoerythrin to provide a phycoerythrin conjugated antibody, and wherein the phycoerythrin conjugated antibody is mixed with a reaction buffer to provide a detection antibody solution.
6. The kit according to claim 5, wherein the mass ratio of the anti-human IgG antibody to the phycoerythrin is 1 (1-5).
7. The kit according to claim 5, wherein the concentration of the detection antibody solution is (2-6) μg/ml.
8. The kit according to claim 1, wherein the signal detection method is any one of a visible light color development method, a chemiluminescent method and a fluorescent light method.
9. The kit of claim 1, further comprising a wash buffer, a reaction buffer, a sample diluent, a calibrator, and a quality control.
10. A method of preparing a kit according to any one of claims 1 to 9, comprising the steps of:
s0: providing an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody, an anti-human IgG4 antibody, an anti-human IgG antibody, microspheres and phycoerythrin, wherein the microspheres are polystyrene fluorescent microspheres loaded with allophycocyanin at different concentrations;
s1: respectively fixing the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody on the microspheres to obtain 4 different specific antibody microspheres, and mixing the 4 different specific antibody microspheres with a reaction buffer solution to obtain a specific antibody solution;
s2: and coupling the anti-human IgG antibody with the phycoerythrin to obtain a phycoerythrin coupled antibody, and mixing the phycoerythrin coupled antibody with a reaction buffer solution to obtain a detection antibody solution.
11. A method of using the kit of any one of claims 1 to 9, comprising the steps of:
s0: providing a specific antibody solution, a detection antibody solution, a diluted sample to be tested and a reaction buffer solution, wherein the specific antibody solution comprises at least four specific detection antibodies, the at least four specific detection antibodies comprise an anti-human IgG1 antibody, an anti-human IgG2 antibody, an anti-human IgG3 antibody and an anti-human IgG4 antibody, and the detection antibodies in the detection antibody solution comprise an anti-human IgG antibody;
s1: sequentially adding the reaction buffer solution, the diluted sample to be tested and the detection antibody solution to obtain a mixed solution;
s2: adding the specific antibody solution into the mixed solution, and then incubating at room temperature in a dark place to obtain a solution to be detected;
s3: detecting the fluorescence intensities of different microspheres in the solution to be detected to distinguish the anti-human IgG1 antibody, the anti-human IgG2 antibody, the anti-human IgG3 antibody and the anti-human IgG4 antibody, and detecting the fluorescence intensities of phycoerythrin in the solution to be detected to calculate the content of IgG1, igG2, igG3 and IgG4 in the sample to be detected.
CN202211102818.5A 2022-05-13 2022-09-09 Kit for detecting immunoglobulin G subclass, and preparation method and application method thereof Pending CN116298302A (en)

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