CN117092353A - Immunoglobulin G4 detection kit and preparation method thereof - Google Patents
Immunoglobulin G4 detection kit and preparation method thereof Download PDFInfo
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- CN117092353A CN117092353A CN202311174463.5A CN202311174463A CN117092353A CN 117092353 A CN117092353 A CN 117092353A CN 202311174463 A CN202311174463 A CN 202311174463A CN 117092353 A CN117092353 A CN 117092353A
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- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 65
- 238000001514 detection method Methods 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 65
- 239000004094 surface-active agent Substances 0.000 claims abstract description 33
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- 239000004005 microsphere Substances 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 12
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 11
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 11
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 10
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 9
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- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 claims description 5
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- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
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- 235000021240 caseins Nutrition 0.000 claims description 2
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- 239000005720 sucrose Substances 0.000 claims description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 2
- 229940033663 thimerosal Drugs 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- 241001529936 Murinae Species 0.000 claims 1
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- VGYFVNQYBUPXCQ-UHFFFAOYSA-N ethene;2-methyloxirane Chemical group C=C.CC1CO1 VGYFVNQYBUPXCQ-UHFFFAOYSA-N 0.000 description 1
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- 125000000647 trehalose group Chemical group 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
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- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000002888 zwitterionic surfactant Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses an immunoglobulin G4 detection kit, which comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises the following components: 0.01-0.06mol/L of first buffer solution; 10-40g/L of electrolyte; 30-80g/L of a thawing promoter; 1-10g/L of a first surfactant; 0.8-1.2g/L of a first preservative; the R2 reagent comprises the following components: anti-immunoglobulin G4 monoclonal antibody coats 0.1-2.0G/L of latex particles; 0.01-0.1mol/L of a second buffer solution; 5-30g/L of stabilizer; 0.8-1.2g/L of a second preservative; 0-4g/L of a second surfactant; and 0-4g/L of blocking agent. Based on the same inventive concept, the invention also discloses a preparation method of the immunoglobulin G4 detection kit. The preparation process is a one-step method, is simple, and has the advantages of prolonged mechanical unsealing stability to 40 days, good stability, cost saving and wide linearity.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to an immunoglobulin G4 detection kit and a preparation method thereof.
Background
IgG is the major component of immunoglobulin in serum, accounting for about 75% of the total immunoglobulin content in serum. IgG has the subtypes of IgG1, igG2, igG3 and IgG4 4, and compared with other immunoglobulin G subtypes, the concentration content of immunoglobulin G4 is lower, accounting for 4% -5% of the total immunoglobulin G.
While immunoglobulin G4 increases or decreases, as seen in various immune system diseases. Immunoglobulin G4-related diseases (G4-RD) are a new class of chronic, progressive inflammatory and fibrotic and sclerotic diseases with unknown etiology recently discovered, and are mainly characterized by significant increases in serum immunoglobulin G4 levels; the affected organ tissue undergoes swelling or nodular/sclerotic lesions due to massive lymphocyte and immunoglobulin G4 positive plasma cell infiltration accompanied by fibrosis. Serum elevated immunoglobulin G4 is clearly indicated in the expert consensus of diagnosis and treatment of immunoglobulin G4-related diseases (2021 edition) as an important indicator for diagnosis and disease assessment of immunoglobulin G4-RD. Therefore, the detection of serum immunoglobulin G4 concentration has a certain clinical value.
The main methodologies at present are chemiluminescence, immunonephelometry, latex-enhanced immunonephelometry, enzyme-linked immunosorbent assay and fluorescent immunochromatography. The sensitivity of the chemiluminescence method is high, but the stability of the reagent is slightly poor; the immune turbidimetry reagent is generally prepared by using antisera or polyclonal antibodies, so that more nonspecific proteins exist and cross reaction is easy to occur; the enzyme-linked immunosorbent assay is low in price, complex in operation process and long in time consumption; fluorescence immunochromatography is short in time but poor in reproducibility.
Chinese patent publication No. CN 106771251A discloses a method for modifying mouse anti-IgG 4 monoclonal antibody by glycosylation in a kit, which can give consideration to high sensitivity and specificity. Chinese patent publication No. CN 114859063a discloses that by optimizing the coupling process of latex microsphere and antibody, i.e. adding a carrier molecule between microsphere and reduced antibody, the antibody extends from the surface of microsphere, thereby reducing the steric hindrance of antigen and antibody binding to realize directional coupling of antibody F (c) end and latex microsphere, greatly improving the sensitivity of the reagent. However, the latex reagents have complex process, are difficult to reproduce after repeated centrifugal resuspension ultrasonic processes, and have high cost. In addition, the reagent unsealing stability can be greatly influenced through complex chemical reaction, so that the reagent is short in machine time and the reagent is wasted.
Therefore, there is still a need to develop a G4 detection kit with simple process, good stability and linear range, so as to meet the use requirements of the current clinical detection.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides an immunoglobulin G4 detection kit and a preparation method thereof. The invention uses latex immunoturbidimetry as a detection principle, and aims to provide an immunoglobulin G4 detection kit and a preparation method thereof, which overcome the defects of the prior art and determine the content of immunoglobulin G4 in a human body. The preparation process is a one-step method, is simple, and has the advantages of prolonged mechanical unsealing stability to 40 days, good stability, cost saving and wide linearity.
In order to achieve the above object, the present invention is realized as follows:
an immunoglobulin G4 detection kit comprising an R1 reagent and an R2 reagent, the R1 reagent comprising the following components: 0.01-0.06mol/L of first buffer solution; 10-40g/L of electrolyte; 30-80g/L of a thawing promoter; 1-10g/L of a first surfactant; 0.8-1.2g/L of a first preservative; the R2 reagent comprises the following components: anti-immunoglobulin G4 monoclonal antibody coats 0.1-2.0G/L of latex particles; 0.01-0.1mol/L of a second buffer solution; 5-30g/L of stabilizer; 0.8-1.2g/L of a second preservative; 0-4g/L of a second surfactant; and 0-4g/L of blocking agent. Preferably, the R1 reagent comprises the following components: 0.02mol/L of first buffer solution; 20g/L of electrolyte; 60g/L of a thawing promoter; a first surfactant 2g/L; 1.0g/L of a first preservative; the R2 reagent comprises the following components: anti-immunoglobulin G4 monoclonal antibody coats 1.6G/L of latex particles; 0.02mol/L of a second buffer; 20g/L of stabilizer; 1.0g/L of a second preservative; 4g/L of a second surfactant; 4g/L of blocking agent.
The immunoglobulin G4 detection kit is characterized in that the first buffer solution and the second buffer solution are respectively one or more of TRIS buffer solution, boric acid-borax buffer solution, HEPES buffer solution, MES buffer solution and PB buffer solution. Preferably, the first buffer is a HEPES buffer, and the second buffer is a HEPES buffer.
The immunoglobulin G4 detection kit is characterized in that the fusion promoting agent is one or more of PEG6000, PEG8000, PEG12000 and PEG20000. Preferably, the fusion promoter is PEG20000.
The immunoglobulin G4 detection kit is characterized in that the first surfactant and the second surfactant are one or more of T-20, triton-100, S9 and glycerol respectively. Preferably, the first surfactant is glycerin, and the second surfactant is any one of T-20, triton-100 and S9. More preferably, the first surfactant is glycerin and the second surfactant is S9.
The immunoglobulin G4 detection kit is characterized in that the first preservative and the second preservative are one or more of Proclin300, sodium azide and thimerosal respectively. Preferably, the first preservative is sodium azide and the second preservative is Proclin300.
The immunoglobulin G4 detection kit is characterized in that the blocking agent is one or more of BSA, casein, ethanolamine and CE 510. Preferably, the blocking agent is CE510, and the CE510 is purchased from JSR corporation. After the coupling of the antibody and the latex in the R2 reagent is finished, CE510 is adopted as a blocking agent, so that the nonspecific adsorption of the protein can be inhibited, the background signal can be reduced, and the activity of the antibody can be kept, thereby achieving the effect of enhancing the reactivity.
The immunoglobulin G4 detection kit is characterized in that the stabilizer is one or more of trehalose, sucrose and EDTA-2 Na. Preferably, the stabilizer is trehalose. The second surfactant S9 is added to the R2 reagent to enhance the repeatability and stability of the latex reagent by improving the latex dispersibility. In addition, the second surfactant S9 is compounded with the anhydrous trehalose, so that the bottle opening stability can be up to 40 days, and reagent waste caused by poor machine stability of the reagent after bottle opening is avoided.
Alternatively, the pH of reagent R1 is 7.0 and the pH of reagent R2 is 7.2.
Preferably, the immunoglobulin G4 detection kit comprises the following components in percentage by weight:
reagent R1:
the concentration of the components in the reagent R1 is the final concentration of the components in the reagent R1;
reagent R2:
the component concentration in the above reagent R2 is the final concentration of the component in the reagent R2.
Based on the same inventive concept, the invention also provides a preparation method of the immunoglobulin G4 detection kit, which comprises the following steps:
(1) Preparation of reagent R1
According to the component content of the reagent R1, sequentially and respectively dissolving a first buffer solution, an electrolyte, a thawing promoting agent, a first surfactant and a first preservative in pure water, uniformly stirring, adjusting the pH to 6.0-7.4, and stirring at room temperature overnight to obtain the reagent R1;
(2) Preparation of reagent R2
Placing latex microsphere with particle diameter of 180-220nm in second buffer solution, shaking at 10-40deg.C, 100-300r/min, shaking for 5-10min; adding the EDC and NHS solution, placing in a shaker at 10-40deg.C, 100-300r/min, and activating latex for 10-20min; under the condition of stirring, adding a second buffer solution and a monoclonal antibody, placing in a shaking table at 10-40 ℃ for 100-300R/min, taking out from the shaking table after coupling for 3-4h, placing on a stirrer, adding a sealing agent, incubating in the shaking table at 10-40 ℃ for 20min, taking out from the shaking table, placing on the stirrer, respectively adding a second surfactant and a stabilizer, adding one substance after incubating in the shaking table at 10-40 ℃ for 20min, adding the other substance, finally adding a preservative, adjusting the pH to 6.0-7.4, and stabilizing in the shaking table at 10-40 ℃ for one night to obtain the reagent R2.
The preparation method of the immunoglobulin G4 detection kit comprises the step of preparing a monoclonal antibody, wherein the monoclonal antibody is a mouse anti-human immunoglobulin G4 antibody.
The preparation method of the immunoglobulin G4 detection kit comprises the steps of: (1-3). Preferably, the mass ratio between the latex microsphere and the monoclonal antibody is 80:1.5. preferably, the latex microsphere has a particle size of 200nm.
Compared with the prior art, the invention has the following effects and advantages:
1. after the coupling of the antibody and the latex in the reagent R2 is finished, the CE510 is adopted as a blocking agent in the preferred scheme, and the CE510 is a synthetic water-soluble high molecular compound provided by JSR and can block blank sites on the latex microsphere, inhibit the non-specific adsorption of the protein and reduce background signals. Meanwhile, refolding of the antibody can be promoted, correct stereoscopic conformation of the antibody can be maintained, and activity of the antibody can be maintained, so that the effect of enhancing the reactivity can be achieved. The effect is obviously better than that of the common blocking agent BSA. Meanwhile, the second surfactant S9 is added into the R2, so that the repeatability and the stability of the latex reagent are enhanced by improving the dispersibility of the latex. The second surfactant S9 is a propylene oxide-ethylene oxide-vinyl diamine copolymer, belongs to typical zwitterionic surfactants, has an HLB value of more than 24 and has excellent water solubility. The good interfacial activity is not easily affected by inorganic electrolyte; the good emulsifying capacity can form a hydration film on the surface of the protein, thereby playing the roles of protecting the protein and increasing the stability of the protein, and the good dispersing capacity ensures that the latex is dispersed more uniformly. In addition, the second surfactant S9 is compounded with the anhydrous trehalose, so that the bottle opening stability can be up to 40 days, the requirements of clinical laboratory doctors are met far, and the reagent waste caused by poor on-machine stability of the reagent after bottle opening is avoided.
2. According to the immunoglobulin G4 detection kit provided by the invention, the monoclonal antibody is adopted for coupling latex, so that the specific binding of antigen and antibody is realized, the interference of analogues (IgG 1, igG2 and IgG 3) in a sample is prevented, and the cross reaction is avoided.
3. According to the immunoglobulin G4 detection kit provided by the invention, the latex reagent adopts the microspheres with the particle size of 180-220nm, and the screened latex microspheres can simultaneously give consideration to sensitivity and linearity, so that compared with the prior art adopting 2 latex microspheres with different particle sizes, the cost is saved, and the process is simpler.
4. According to the preparation method of the immunoglobulin G4 detection kit, provided by the invention, the preparation process of the R2 latex reagent adopts a one-step process, complicated steps such as centrifugal resuspension and ultrasound are not needed, and the method is simple to operate, easy to reproduce and low in production cost.
Drawings
FIG. 1 is a calibration graph of the kit of example 1.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, 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 defined otherwise, all 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 terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used in this specification includes any and all combinations of one or more of the associated listed items.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1:
1. the preparation method of the immunoglobulin G4 detection kit provided by the embodiment comprises the following steps:
(1) Preparation of R1:
100mL HEPES buffer was weighed and placed on a stirrer, and 2.0g NaCl, PEG20000 6g, 0.2g glycerol, 0.1g NaN were added in order 3 . Adding another substance after completely dissolving, adjusting pH to 7.0 after completely dissolving, and incubating in a shaking table at 37deg.C for one night to obtain reagent R1.
After the completion of the preparation, the concentrations of the components of the reagent R1 were respectively: HEPES buffer 0.02mol/L; naCl 20g/L; PEG20000 60g/L; glycerol 2g/L; naN (NaN) 3 1g/L；
(2) Preparation of reagent R2:
latex microspheres with a particle size of 200nm 0.008g in 2mL HEPES buffer at pH7.0 at 0.02M were placed in a shaking table at 37℃for 2min at 200 r/min. Adding the mixture into the EDC and NHS solution, placing in a shaking table at 37 ℃ for 200r/min, shaking for 15min to activate the latex; the concentration of EDC and NHS was 0.05M and the addition volume ratio was 1:1. Then, 3mL of HEPES buffer (0.02M pH 7.2) and an antibody solution were added with stirring, and the total amount of the mouse anti-human immunoglobulin G4 antibody added was 30mg/L. Placing in a shaking table at 37 ℃ for 200r/min, and coupling for 3h. Taking out from the shaker, placing on a stirrer, adding 0.02g CE510, incubating at 37deg.C for 20min, taking out from the shaker, and placing on the stirrer. Adding 0.02g S9 and 0.1g trehalose respectively, adding one substance after the addition of the other substance is completed, incubating for 20min at 37 ℃ in a shaking table, adding 0.005g Proclin300 finally, adjusting the pH to 7.2 after the substances are completely dissolved, and incubating for one night in the shaking table at 37 ℃ to obtain the reagent R2.
After the preparation is completed, the concentration of each component of the reagent R2 is as follows: anti-immunoglobulin G4 monoclonal antibody coating latex particles 1.6G/L, HEPES buffer 20mM/L, CE510 4G/L, S9 4G/L, trehalose 20G/L, proclin 300.0G/L.
Example 2:
referring to the preparation method of example 1, the difference from example 1 is that only blocking agent CE510 was replaced with BSA having a mass fraction of 5%.
Example 3:
referring to the preparation method of example 1, the following immunoglobulin G4 detection kit of 3 experimental groups was prepared according to the conditions of table 1 below, wherein experimental group 1 is a technical scheme of example 1. Experimental group 2 and experimental group 3 differ only in: the selection of the second surfactant is different, see in particular table 1 below.
TABLE 1
| A second surfactant | |
| Experiment group 1 | S9 (i.e. example 1) |
| Experiment group 2 | T-20(4g/L) |
| Experiment group 3 | Triton-100 (4 g/L) |
Example 4:
referring to the preparation method of example 1, the following 3 experimental groups of immunoglobulin G4 detection kits were prepared according to the conditions of table 2 below, wherein experimental group 4 is the technical scheme of example 1. Experimental group 2 and experimental group 3 differ only in: the choice of secondary surfactant and stabilizer is different, see in particular table 1 below.
TABLE 2
| Different combinations of secondary surfactant and stabilizer | |
| Experiment group 4 | S9+ trehalose (i.e., example 1) |
| Experiment group 5 | S9 (trehalose without stabilizer) |
| Experiment group 6 | Trehalose (without second surfactant S9) |
Example 5: application method of immunoglobulin G4 detection kit
The biochemical analyzer is used for detection, and the following detection is performed by using the Hitachi 7180 biochemical analyzer as an example, and the specific method is as follows:
measuring the wavelength to 570nm, respectively taking calibrator solutions (2 uL) with different concentrations, adding an immunoglobulin G4R 1 reagent (180 uL), uniformly mixing, incubating at 37 ℃ for 5 minutes, adding an immunoglobulin G4R 2 reagent (60 uL), uniformly mixing, incubating at 37 ℃ for 30 seconds, reading absorbance value A1 of each tube, incubating for 300 seconds, reading absorbance value A2 of each tube, calculating absorbance difference delta A=A2-A1, repeatedly measuring for 2 times per tube, taking the average value of absorbance difference delta A measured for 2 times per calibration tube as the vertical coordinate, corresponding calibrator concentration as the horizontal coordinate, and drawing a calibration curve of concentration-absorbance difference.
And taking a serum sample to be measured, measuring the absorbance difference value of the sample by the same method, and substituting the absorbance difference value into a calibration curve to calculate the content of the immunoglobulin G4 in the sample to be measured. The quality control product and the sample can be applied with the automatic dilution function of the instrument (the automatic dilution can be performed without automatic dilution or by 100 times).
The kit provided by the invention is not only suitable for Rili 7180, but also suitable for semi-automatic and full-automatic biochemical analyzers of other brands and models, and specific parameters can be adjusted according to the instruments.
Example 6: quality evaluation of immunoglobulin G4 detection kit
In this example, the immunoglobulin G4 detection kit prepared in the above-described examples was tested, and the quality of the immunoglobulin G4 detection kit of the present invention was evaluated mainly from the aspects of standard curve formulation, linear range, reactivity, reproducibility, stability, and the like.
1. Standard curve making
(1) Experimental method
The concentrations of the calibrator are respectively as follows: 0g/L,0.75g/L,1.5g/L,3.0g/L,4.5g/L,6g/L. The standard curve of the immunoglobulin G4 calibrator of the present invention was determined as in example 5. The experimental subject is the immunoglobulin G4 detection kit prepared in example 1.
(2) Experimental results and analysis
The calibration curve of the kit of example 1 of the present invention is shown in FIG. 1.
2. Linear range
(1) Experimental method
The high concentration calibrator (6 g/L) near the upper limit of the linear range and distilled water (0 g/L) near the lower limit of the linear range are used, namely, the calibrator Xmax is 6g/L, the calibrator Xmin is 0g/L, and at least 3 dilution concentrations (x) are prepared according to different volume ratios of the calibrator Xmax and the calibrator Xmin of 5:1, 1:1 and 1:5. And testing the kit respectively, testing each diluted concentration for 2 times, and respectively calculating the average value (y) of the detection results. And (3) taking the dilution concentration (x) as an independent variable and taking the average value (y) of the detection result as a dependent variable to solve a linear regression equation. The experimental subject is the immunoglobulin G4 detection kit prepared in example 1.
(2) Experimental results and analysis
TABLE 3 Linear Range results
As shown in Table 3, the kit of the invention has better linearity and linearity range: 0-6g/L. The regression equation is: y= 1.0415x-0.0696, r 2 ＝0.9995。
3. Contrast of reactivity
(1) Experimental method
The immunoglobulin G4 detection kits prepared in example 1 and example 2 were tested for 6 concentrations of calibrator at 0G/L,0.75G/L,1.5G/L,3.0G/L,4.5G/L, and 6G/L, respectively, and each calibrator was tested for 2 times, and the absorbance difference results were averaged for comparison.
(2) Experimental results and analysis
The test results are shown in Table 4.
TABLE 4 calibrator reactivity test results for immunoglobulin G4 detection kits
As shown in the experimental results of Table 4, the reactivity of each point of the calibrator measured in example 1 is obviously higher than that of example 2, which indicates that the blocking agent CE510 used in the R2 reagent has better effect than that of BSA with the mass fraction of 5%, and is beneficial to improving the reactivity of the reagent.
4. Detection of repeatability
(1) Experimental method
The test was repeated 10 times for each concentration using immunoglobulin G4 samples with a measurement value of 0.4G/L, 0.8G/L, and 1.78G/L from the Pronocon company as low, medium and high value samples, and the measurement means and standard deviations (S) were calculated, respectively, to calculate the coefficients of variation, and the test kits of example 1 and example 3 were subjected to repeated investigation.
Since the reproducibility of the kit is mainly related to the R2 reagent, the experiment mainly examined the reproducibility of the three sets of kits prepared in example 3.
(2) Experimental results and analysis
TABLE 5 repeatability test results of immunoglobulin G4 detection kits
As shown in the results of Table 5, the reproducibility of the kit for the low, medium and high value samples was not as good as that for the second surfactant S9 after T-20 and triton-100 were used as the second surfactants in the experimental group 2 and the experimental group 3, respectively, and the reproducibility of the low value samples was significantly improved after the second surfactant S9 was added in example 1.
5. Anti-interference experiment
(1) Experimental method
(1) Immunoglobulin G4 samples with the concentration of 0.78G/L are taken, the same volumes of interfering substances with different concentrations are respectively added, the final concentration of the added triglyceride interfering substances is 5000mg/L, the final concentration of bilirubin is 300 mu mol/L and the final concentration of hemoglobin is 5000mg/L, the kit prepared in example 1 is used for repeatedly measuring each prepared sample for 3 times, the concentration values of the three times are measured to obtain an average value, and the relative deviation between the added interfering substances and the measured value of the immunoglobulin G4 after the addition of the physiological saline is observed compared with the samples with the same volumes of physiological saline.
(2) The samples IgG1, igG2, and IgG3 were repeatedly assayed 3 times using the kit prepared in example 1, respectively, and averaged.
(2) Experimental results and analysis
TABLE 6 anti-interference experimental results
Note that: the relative deviation is not more than +/-10 percent and basically reaches the standard.
TABLE 7 Cross test results
As can be seen from the experimental results in Table 6, when a certain concentration of interfering substances such as triglyceride, bilirubin and hemoglobin is present in the test sample, it has no influence on the measurement results of the kit of the present invention, indicating that the present invention has a strong anti-interference ability.
From the results in table 7, it is clear that the reagent has no cross-reaction with the IgG1, igG2, and IgG3 samples, all of which are tested near 0.
6. Stability of
(1) Failure stability:
the immunoglobulin G4 detection kit is placed in a water bath at 37 ℃, and calibration is carried out on the calibrator before storage (namely when the kit is not destroyed), after storage for 3 days, 5 days and 7 days respectively, and the concentration value of the split-charging sample is tested and analyzed.
(2) Results and analysis of stability-to-failure experiments
TABLE 8 results of stability-to-failure experiments
From the results shown in Table 8, the kit of example 1 still maintained more than 98% of reactivity after storage at 37℃for 7 days, indicating that the immunoglobulin G4 detection kit has good stability at normal temperature and increased the practicability of the kit for clinical diagnosis. The stability of the kit of example 2 was not as excellent as that of example 1, and the results also indicate that the use of blocking agent CE510 significantly improved the stability of the kit.
(3) Stability of unsealing
Kits of experiment group 4, experiment group 5 and experiment group 6 were placed in biochemical instruments, and calibrators were calibrated and tested for quality control and analyzed on day 0, day 5, day 10, day 15, day 20, day 25, day 30, day 35, and day 40.
(4) Unsealing stability test results and analysis
TABLE 9 results of the opening stability test
From the opening stability data of table 9: experimental group 4 > experimental group 6 > experimental group 5, namely the immunoglobulin G4 detection kit of example 1 has the best unsealing stability, which indicates that the unsealing stability can be up to 40 days after S9 and trehalose are added into the R2 reagent at the same time, and the waste of the reagent is avoided.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. An immunoglobulin G4 detection kit is characterized by comprising an R1 reagent and an R2 reagent,
the R1 reagent comprises the following components: 0.01-0.06mol/L of first buffer solution; 10-40g/L of electrolyte; 30-80g/L of a thawing promoter; 1-10g/L of a first surfactant; 0.8-1.2g/L of a first preservative;
the R2 reagent comprises the following components: anti-immunoglobulin G4 monoclonal antibody coats 0.1-2.0G/L of latex particles; 0.01-0.1mol/L of a second buffer solution; 5-30g/L of stabilizer; 0.8-1.2g/L of a second preservative; 0-4g/L of a second surfactant; and 0-4g/L of blocking agent.
2. The immunoglobulin G4 detection kit of claim 1, wherein the first buffer and the second buffer are each one or more of TRIS buffer, boric acid-borax buffer, HEPES buffer, MES buffer, and PB buffer.
3. The immunoglobulin G4 detection kit of claim 1 or 2, wherein the fusogenic agent is one or more of PEG6000, PEG8000, PEG12000, PEG20000.
4. The immunoglobulin G4 detection kit of claim 1, wherein the first surfactant and the second surfactant are each one or more of T-20, triton-100, S9, and glycerol.
5. The immunoglobulin G4 detection kit of claim 1, wherein the first preservative and the second preservative are one or more of Proclin300, sodium azide, and thimerosal, respectively.
6. The immunoglobulin G4 detection kit of claim 1, wherein the stabilizer is one or more of trehalose, sucrose, EDTA-2 Na.
7. The immunoglobulin G4 detection kit of claim 1, wherein the blocking agent is one or more of BSA, casein, ethanolamine, CE 510.
8. The method for preparing an immunoglobulin G4 detection kit according to any one of claims 1 to 7, comprising the steps of:
(1) Preparation of reagent R1
According to the component content of the reagent R1, sequentially and respectively dissolving a first buffer solution, an electrolyte, a thawing promoting agent, a first surfactant and a first preservative in pure water, uniformly stirring, adjusting the pH to 6.0-7.4, and stirring at room temperature overnight to obtain the reagent R1;
(2) Preparation of reagent R2
Placing latex microsphere with particle diameter of 180-220nm in second buffer solution, shaking at 10-40deg.C, 100-300r/min, shaking for 5-10min; adding the EDC and NHS solution, placing in a shaker at 10-40deg.C, 100-300r/min, and activating latex for 10-20min; under the condition of stirring, adding a second buffer solution and a monoclonal antibody, placing the mixture on a shaking table at 10-40 ℃ for 100-300R/min, taking out the mixture from the shaking table after coupling for 3-4h, placing the mixture on a stirrer, adding a sealing agent, incubating the mixture on the shaking table at 10-40 ℃ for 20min, taking out the mixture from the shaking table, placing the mixture on the stirrer, respectively adding a second surfactant and a stabilizer, adding one substance after incubating the mixture on the shaking table at 10-40 ℃ for 20min, adding the other substance, finally adding a second preservative, adjusting the pH value to 6.0-7.4, and stabilizing the shaking table at 10-40 ℃ for one night to obtain the reagent R2.
9. The method of claim 8, wherein the monoclonal antibody is a murine anti-human immunoglobulin G4 antibody.
10. The method for preparing an immunoglobulin G4 detection kit according to claim 8, wherein the mass ratio between the latex microsphere and the monoclonal antibody is (60-100): (1-3).
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