CN115494240A - Method and reagent combination capable of simultaneously detecting different subtype immune globulin - Google Patents
Method and reagent combination capable of simultaneously detecting different subtype immune globulin Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention relates to the technical field of substance detection, in particular to a method and a reagent combination capable of simultaneously detecting different subtype immune globulins, wherein a solid phase carrier marked with antigen is added into a serum sample to be detected, and the mixture is uniformly mixed and incubated in a shaking way; adding a mixture of a fluorescein-labeled IgG secondary antibody and a fluorescein-labeled IgM secondary antibody into a serum sample, uniformly mixing, and carrying out vibration incubation in a dark place; the serum samples were examined by flow cytometry. Compared with the prior art, the method for simultaneously detecting the different subtype immunoglobulins simultaneously uses two fluoresceins as markers, wherein one fluorescein is used for marking an IgG secondary antibody, and the other fluorescein is used for marking an IgM secondary antibody, so that the IgG and the IgM can be simultaneously detected, the time can be saved, and the detection efficiency and the accuracy can be improved.
Description
[ technical field ] A
The invention relates to the technical field of substance detection, in particular to a method and a reagent combination for simultaneously detecting different subtype immune globulins.
[ background of the invention ]
Immunoglobulin (Ig), which refers to a globulin having the activity or chemical structure of an antibody (Ab), similar to antibody molecules, is mainly present in body fluids such as serum and can specifically bind to a corresponding antigen. Immunoglobulins can be classified into five classes by heavy chain type, namely immunoglobulin G (IgG), immunoglobulin a (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE).
IgG is the highest Ig content in the human body, widely distributed in vivo, and the main Ig in blood and tissue fluid. IgG, which is produced second to IgM only after antigen stimulation, is an antibody that plays a major role in humoral immune responses. IgG has a half-life of 21 days, is the antibody molecule with the highest concentration in serum, accounts for 70% of the immunoglobulin content, and is the only antibody molecule capable of providing maternal immunity to the newborn through the placenta. Most anti-infective antibodies and autoantibodies are of the IgG class.
IgM is the largest antibody actually found in the human circulatory system to date, and is also the first antibody to react first when contacted with an antigen. IgG is insufficient in the early stage of humoral immunity, and IgM plays a role in eliminating pathogens; when again exposed to foreign antigens, igM concentrations in the blood rapidly decline.
IgA is divided into serotype IgA and secretory IgA, and although serotype IgA has some functions of IgG and IgM, it does not show important immune functions in serum. IgD is contained in normal human serum in a very low level, and the biological function is not clear at present. IgE is the least abundant immunoglobulin in normal human serum, can cause type I hypersensitivity, and may be associated with anti-parasitic immunity of the body.
IgG class and IgM class antibodies are of great significance in clinical detection, and in the field of infectious diseases, igM is generally used as an index of primary infection and can be used for early diagnosis of infection, and IgG indicates previous infection. In autoimmune antibody detection, changes in IgG levels can indicate risks of diseases such as systemic lupus erythematosus, rheumatoid arthritis, muscular dystrophy, and the like, and some antibody subtypes with different projects are closely related to different diseases, for example, antiphospholipid antibodies are classified into three subtypes, namely IgA, igG, and IgM, according to immunoglobulin classes. IgG and IgM antibody positivity is related to clinical manifestation, and medium and high titer IgG antibody is closely related to occurrence of thrombus and pathological pregnancy; igM antiphospholipid antibodies are directly associated with venous thrombosis and autoimmune hemolytic anemia. IgG and IgM are antibody subtypes which are usually detected clinically, and simultaneous determination is often needed in some fields to assist in disease judgment.
Referring to fig. 1 to 3, methods for detecting antibodies in the prior art are classified into a capture method, an indirect method and a double antigen sandwich method according to different reaction modes. The capture method is to fix a certain subtype of secondary antibody on a solid phase carrier, capture the corresponding subtype of antibody in a sample, and then combine the antibody with a specific antigen, and is generally suitable for detecting low-content immunoglobulin, such as IgM.
The indirect method is to fix the antigen on a solid phase carrier, bind with specific antibody in the sample, and then bind with a certain subtype of secondary antibody. It can be seen from the principle that only one subtype can be detected in one reaction system of the capture method and the indirect method, and the simultaneous detection cannot be realized.
The principle of the double-antigen sandwich method is that an antigen 1 is fixed on a solid phase carrier, is combined with a specific antibody in a sample, and is then combined with an antigen 2, the method has stronger specificity, and the method can detect the existence of various subtypes and cannot distinguish the subtypes.
[ summary of the invention ]
In order to overcome the above problems, the present invention provides a method and a reagent combination capable of simultaneously detecting different subtypes of immunoglobulins, which can effectively solve the above problems.
The invention provides a technical scheme for solving the technical problems, which comprises the following steps: a method for simultaneously detecting different subtypes of immunoglobulin is provided, which comprises the following steps:
step S1, adding a solid phase carrier marked with antigen into a serum sample to be detected, uniformly mixing, and performing shake incubation;
step S2, adding a mixture of IgG secondary antibody marked by fluorescein I and IgM secondary antibody marked by fluorescein II into the serum sample in the step S1, uniformly mixing, and carrying out light-proof oscillation incubation;
s3, detecting the serum sample in the step S2 by using a flow cytometer;
and S4, reading signals of the fluorescein I and the fluorescein II by a flow cytometer in different fluorescence channels, wherein the signal reported by the fluorescein I represents the IgG concentration, and the signal reported by the fluorescein II represents the IgM concentration.
Preferably, in step S2, the secondary antibody is labeled with fluorescein by direct covalent labeling or indirectly by a biotin-avidin system.
Preferably, in step S2, the fluorescein-labeled IgG secondary antibody is bound to the microsphere-captured IgG-type antibody, and the fluorescein-labeled IgM secondary antibody is bound to the microsphere-captured IgM-type antibody.
Preferably, the solid phase carrier adopts capture microspheres with the particle size of 3-10 um.
A reagent combination capable of detecting different subtypes of immunoglobulin simultaneously comprises the following components:
the component one: an antigen coated microsphere;
and (2) component two: fluorescein-labeled IgG secondary antibody;
the component III is as follows: fluorescein-labeled IgM secondary antibody.
Preferably, the secondary antibody is labeled with the biotin using a direct covalent label or indirectly using a biotin-avidin system.
Preferably, the fluorescein-labeled IgG secondary antibody is bound to the microsphere-captured IgG-type antibody, and the fluorescein-labeled IgM secondary antibody is bound to the microsphere-captured IgM-type antibody.
Preferably, the microspheres are microspheres with a particle size of 3-10 um.
Preferably, phycoerythrin is used as the first fluorescein, and allophycocyanin is used as the second fluorescein.
Preferably, fluorescein isothiocyanate is adopted as the fluorescein I, and cyanine dye Cy5 is adopted as the fluorescein II.
Compared with the prior art, the method and the reagent combination for simultaneously detecting the different subtype immunoglobulins have the advantages that two fluorescein is used as the markers, wherein one fluorescein is used for marking an IgG secondary antibody, and the other fluorescein is used for marking an IgM secondary antibody, so that the IgG and the IgM can be simultaneously detected, the time can be saved, and the detection efficiency and the accuracy can be improved; the flow cytometer has the characteristic of a plurality of fluorescence detection channels with different wave bands, and a plurality of different fluorescein labels different secondary antibodies are used, so that a plurality of different subtype autoantibodies can be detected in the same reaction system.
[ description of the drawings ]
FIG. 1 is a schematic diagram of a prior art capture method;
FIG. 2 is a schematic diagram of a prior art indirect method;
FIG. 3 is a schematic diagram of a double antigen sandwich method in the prior art;
FIG. 4 is a flowchart illustrating the steps of the method for simultaneously detecting different types of immunoglobulins according to the present invention;
FIG. 5 is a scatter diagram of the detection results of a first clinical specimen according to a first embodiment of the method for simultaneously detecting immunoglobulins of different subtypes;
FIG. 6 is a scatter plot of the detection results of a second clinical specimen in accordance with a first embodiment of the present invention;
FIG. 7 is a scattergram of 24 clinical samples of anti-phospholipid antibody syndrome patients tested according to an embodiment of the method for simultaneous detection of different subtypes of immunoglobulins of the present invention;
FIG. 8 is a scatter diagram showing the results of a first clinical specimen in a second embodiment of the method for simultaneously detecting immunoglobulins of different subtypes according to the present invention;
FIG. 9 is a scatter plot of the results of the second clinical sample of the second embodiment of the method for simultaneously detecting immunoglobulins of different subtypes according to the present invention;
FIG. 10 is a scatter plot of the results of the third clinical sample of the second embodiment of the method for simultaneously detecting immunoglobulins of different subtypes according to the present invention.
[ detailed description ] embodiments
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and implementation examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It should be noted that in embodiments of the present invention all directional indications (such as up, down, left, right, front, back \8230;) are limited to relative positions on a given view, not absolute positions.
In addition, the descriptions related to "first", "second", etc. in the present invention are only for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
Referring to fig. 4 to 10, the method for simultaneously detecting different subtypes of immunoglobulins according to the present invention comprises the following steps:
step S1, taking a serum sample to be detected, adding a solid phase carrier marked with an antigen, uniformly mixing, and oscillating and incubating. According to the actual content of the antibody to be detected, the serum sample can be pre-diluted in a certain proportion.
In the step S1, the solid phase carrier adopts capture microspheres with the grain diameter of 3-10um, and the capture microspheres can be magnetic to facilitate automatic detection and can also be non-magnetic. The surface of the capture microsphere can be activated by carboxyl, epoxy, tosyl, aldehyde and other groups as long as the capture microsphere can coat the antibody. The microsphere-coated antibody technology is conventionally known.
And S2, carrying out magnetic separation on the serum sample in the step S1, washing after the magnetic separation, adding a mixture of the IgG secondary antibody marked by the reporter molecule I and the IgM secondary antibody marked by the reporter molecule II, uniformly mixing, and carrying out vibration incubation in a dark place.
In the step S2, fluorescein is adopted as the reporter molecule. A reaction system of the traditional indirect method only has one reporter molecule, and the invention innovatively adopts two fluorescein as the reporter molecule and can report two detection signals of IgG and IgM at the same time. The emission spectra of the two fluorescein are not mutually interfered, and the fluorescein I is marked on an IgG secondary antibody and used for detecting IgG; and the secondary IgM antibody is marked by fluorescein II and is used for detecting IgM. The fluorescein can be used for labeling the secondary antibody by direct covalent labeling or indirect labeling by a biotin-avidin system.
In the step S2, the fluorescein-labeled IgG secondary antibody is bound to the IgG type antibody captured by the microsphere, and the fluorescein-labeled IgM secondary antibody is bound to the IgM type antibody captured by the microsphere.
And S3, carrying out magnetic separation on the serum sample in the step S2, washing after the magnetic separation, and detecting by using a flow cytometer.
And S4, reading signals of the fluorescein I and the fluorescein II by a flow cytometer in different fluorescence channels, wherein the signal reported by the fluorescein I represents the IgG concentration, and the signal reported by the fluorescein II represents the IgM concentration.
The method of the present invention for simultaneously detecting different subtypes of immunoglobulins is described below with reference to specific examples.
Example one
The reagent combination used in this example comprised the following components:
a component A1: capturing microspheres, namely coating cardiolipin antigen with polystyrene magnetic microspheres with the diameter of six microns, wherein the concentration of the cardiolipin antigen is 40 particles/[ mu ] L;
a component B1: PE-IgG (phycoerythrin-labeled IgG secondary antibody) and APC-IgM (allophycocyanin-labeled IgM secondary antibody), concentration: PE-IgG antibody 0.4ug/mL, concentration: the APC-IgM antibody is 0.4ug/mL, PE is the fluorescein I, and APC is the fluorescein II;
a component C1: PBST buffer, base solution of pH 7.0-7.4 PBS (phosphate buffer solution), containing 0.1% Tween-20.
A method for simultaneously detecting different subtypes of immunoglobulins, comprising the steps of:
step X1, diluting 5 microliters of a serum sample to be detected by 20 times with the component C1, adding 50ul of the component A1 into 20 microliters of the diluted serum sample, uniformly mixing, and performing shake incubation for 30min at 25 ℃;
step X2, washing the captured microspheres for 2 times by using the component C1 after magnetic separation, adding 100ul of the component B1, uniformly mixing, and carrying out shake incubation for 30min in a dark place at 25 ℃;
step X3, washing the captured microspheres for 1 time by using the component C1 after magnetic separation, and detecting by using a flow cytometer;
and step X4, reading signals of the PE and the APC in different fluorescence channels by a flow cytometer, wherein the signal reported by the PE represents the IgG concentration, and the signal reported by the APC represents the IgM concentration.
Please refer to fig. 5 and 6. In FIG. 5, both PE and APC channels fluoresced higher, as indicated by high cardiolipin IgG concentration, high cardiolipin IgM concentration; in FIG. 6, both PE and APC channel fluorescence were moderate, as indicated by moderate cardiolipin IgG concentration and moderate cardiolipin IgM concentration.
Referring to FIG. 7, 24 clinical specimens of anti-phospholipid antibody syndrome patients were tested using the reagents and test methods of example I. PE channel fluorescence signals, which characterize the concentration of cardiolipin IgG antibodies; the APC channel fluorescence signal represents the cardiolipin IgM antibody concentration, and since there is no recognized reference and traceability system, concentration conversion is not performed, and a scatter plot is plotted for 24 samples with the PE channel (cardiolipin IgG) fluorescence value as the horizontal axis and the APC channel (cardiolipin IgM) fluorescence value as the vertical axis, as shown in fig. 6. In 3 case samples in the area 1 in the figure, the cardiolipin IgG signal value is high, but the IgM fluorescence signal is low, and diseases such as thrombus and pathological pregnancy can be possibly caused; in 5 case samples in the area 2, the IgG degree of cardiolipin is relatively low, but the IgM fluorescence signal is high, and diseases such as venous thrombosis, autoimmune hemolytic anemia and the like can exist.
Example two
The reagent combination used in this example comprised the following components:
a component A2: the microsphere is captured, polystyrene magnetic microsphere with diameter of 6 microns is adopted to coat rubella virus antigen, and the concentration is 40 particles/mu L;
and (3) a component B2: FITC-IgG (fluorescein isothiocyanate labeled IgG secondary antibody) and Cy5-IgM (cyanine dye Cy5 labeled IgM secondary antibody) mixture, concentration: FITC-IgG antibody 0.5ug/mL, concentration: 0.5ug/mL of Cy5-IgM antibody, wherein FITC is the fluorescein I, and Cy5 is the fluorescein II;
and (3) a component C2: PBST buffer, base solution of pH 7.0-7.4 PBS (phosphate buffer solution), containing 0.1% Tween-20.
A method for simultaneously detecting different subtypes of immunoglobulins, comprising the steps of:
step Y1, diluting 5 microliters of the serum sample to be detected by 50 times with the component C2, adding 50ul of the component A2 into 20 microliters of the diluted serum sample, uniformly mixing, and performing shake incubation for 30min at 25 ℃;
step Y2, washing the captured microspheres for 2 times by using the component C2 after magnetic separation, adding 100ul of the component B2, uniformly mixing, and carrying out shake incubation for 30min in a dark place at 25 ℃;
step Y3, washing the captured microspheres for 1 time by using the component C2 after magnetic separation, and detecting by using a flow cytometer;
and step Y4, reading signals of FITC and Cy5 by a flow cytometer in different fluorescence channels, wherein the signal reported by FITC represents IgG concentration, and the signal reported by Cy5 represents IgM concentration.
Referring to FIGS. 8-10, cy5 fluorescence is detected on the flow cytometer using the APC channel and is therefore labeled APC. In fig. 8, the FITC channel fluorescence was higher and the APC channel fluorescence was lower, which is indicated by high rubella virus IgG concentration and low rubella virus IgM concentration; in fig. 9, both the FITC channel and the APC channel fluoresced high, as indicated by high rubella virus IgG concentration and high rubella virus IgM concentration; in FIG. 10, FITC channel fluoresces moderately and APC channel fluoresces highly, as indicated by moderate rubella virus IgG concentration and high rubella virus IgM concentration.
Compared with the prior art, the method and the reagent combination for simultaneously detecting the different subtype immunoglobulins have the advantages that two fluorescein is used as the markers, wherein one fluorescein is used for marking an IgG secondary antibody, and the other fluorescein is used for marking an IgM secondary antibody, so that the IgG and the IgM can be simultaneously detected, the time can be saved, and the detection efficiency and the accuracy can be improved; the flow cytometer has the characteristic of a plurality of fluorescence detection channels with different wave bands, and a plurality of different fluorescein labels different secondary antibodies are used, so that a plurality of different subtype autoantibodies can be detected in the same reaction system.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and any modifications, equivalents and improvements made within the spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for simultaneously detecting different subtypes of immunoglobulins, comprising the steps of:
step S1, adding a solid phase carrier marked with antigen into a serum sample to be detected, uniformly mixing, and performing shake incubation;
step S2, adding a mixture of IgG secondary antibody marked by fluorescein I and IgM secondary antibody marked by fluorescein II into the serum sample in the step S1, uniformly mixing, and carrying out light-proof oscillation incubation;
s3, detecting the serum sample in the step S2 by using a flow cytometer;
and S4, reading signals of the fluorescein I and the fluorescein II by a flow cytometer in different fluorescence channels, wherein the signal reported by the fluorescein I represents the IgG concentration, and the signal reported by the fluorescein II represents the IgM concentration.
2. The method according to claim 1, wherein in step S2, the secondary antibody is labeled with fluorescein either directly or indirectly via biotin-avidin system.
3. The method according to claim 1, wherein in step S2, the fluorescein-labeled IgG secondary antibody is bound to the IgG type antibody captured by the microspheres, and the fluorescein-labeled IgM secondary antibody is bound to the IgM type antibody captured by the microspheres.
4. The method of claim 1, wherein the solid phase carrier comprises capture microspheres with a particle size of 3-10 um.
5. Combination of reagents for simultaneous detection of immunoglobulins of different subtypes for use in a method according to any one of claims 1 to 4, comprising the following components:
the component one: an antigen coated microsphere;
and (2) component two: fluorescein-labeled IgG secondary antibody;
the component III is as follows: and (3) fluorescein-labeled IgM secondary antibody.
6. The reagent combination for simultaneously detecting immunoglobulins of different subtypes according to claim 5, wherein the secondary antibody is labeled with fluorescein either directly by covalent labeling or indirectly by a biotin-avidin system.
7. The reagent combination for simultaneously detecting immunoglobulins of different subtypes according to claim 5, wherein the fluorescein-labeled IgG secondary antibody is conjugated to the IgG type antibody captured by the microspheres, and the fluorescein-labeled IgM secondary antibody is conjugated to the IgM type antibody captured by the microspheres.
8. The reagent combination for simultaneously detecting immunoglobulins of different subtypes according to claim 5, wherein said microspheres are microspheres with a particle size of 3-10 um.
9. The reagent kit for simultaneously detecting immunoglobulins of different subtypes according to claim 5, wherein one of the fluoresceins is phycoerythrin and the other one of the fluoresceins is allophycocyanin.
10. The reagent combination for simultaneously detecting immunoglobulins of different subtypes according to claim 5, wherein fluorescein isothiocyanate is used as the first fluorescein, and cyanine dye Cy5 is used as the second fluorescein.
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| CN117074678A (en) * | 2023-08-16 | 2023-11-17 | 中拓生物有限公司 | Kit for detecting tPSA and fPSA by using flow cytometry |
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