CN108398554B - anti-TORCH-IgM antibody spectrum chip, preparation method thereof and TORCH detection kit - Google Patents
anti-TORCH-IgM antibody spectrum chip, preparation method thereof and TORCH detection kit Download PDFInfo
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- 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
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56905—Protozoa
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56927—Chlamydia
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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- 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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/19—Rubella virus
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
- G01N2333/45—Toxoplasma
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention belongs to the technical field of biological detection, and discloses an anti-TORCH-IgM antibody spectrum chip and a preparation method thereof, and a TORCH detection kit and a detection method. The anti-TORCH-IgM antibody spectrum chip not only can simultaneously quantitatively detect the infection of various related pathogenic microorganisms of TORCH, but also has better stability and longer service cycle by optimizing the coating liquid, the coating conditions and the sealing stabilizer of the chip. The kit for detecting the TORCH can effectively reduce the usage amount of the related antigen, reduce the antigen cost, simultaneously ensure that the stability period is longer, the service cycle is longer, reduce the transportation cost and improve the convenience of storage and transportation. The method for detecting TORCH can simultaneously and quantitatively detect the infection of various related pathogenic microorganisms of TORCH, and has high sensitivity and good specificity.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an anti-TORCH-IgM antibody spectrum chip, a preparation method thereof and a TORCH detection kit.
Background
TORCH is a pathogen causing congenital intrauterine infections and perinatal infections causing malformations in perinatal infants, and is an acronym for a group of pathogenic microorganisms, wherein t (toxoplasma) is toxoplasma gondii, o (other) is other pathogenic microorganisms such as treponema pallidum, herpes zoster virus, parvovirus B19, coxsackie virus, etc., R (rubella virus) is rubella virus, C (cytopegalo virus) is cytomegalovirus, and H (herpes virus) is herpes simplex type I/II. TORCH infection is one of the important factors that seriously endanger the health of newborn infants, and is called TORCH syndrome in the medicine of perilife, and the infection is distributed worldwide. In the population of China, the TORCH infection widely exists, a pregnant woman has no obvious clinical symptoms after the TORCH infection in the gestation period, but after the fetal infection, development defects and dysfunction of a plurality of organs such as the liver, the kidney, the heart, the brain and the like of a fetus or a newborn can be caused, adverse consequences such as embryo/fetal abortion, premature birth, intrauterine growth retardation, malformation, dead fetus, newborn death and the like can be caused, and great threats are formed on the prenatal and postnatal care and population quality. Therefore, in order to reduce the birth rate of sick and disabled children and improve the birth population quality, serological screening of TORCH infection should be done well in order to find the bad pregnancy early and to deal with it in time. The TORCH detection should be carried out routinely for neonates, and the TORCH infection condition of the neonates can be known so as to facilitate early intervention and early treatment.
The infection of four pathogenic microorganisms of Toxoplasma, rubella virus, cytomegalovirus and herpes simplex I/II in the detection of TORCH accounts for about 90%, and the rest 10% is other pathogenic microorganisms, including parvovirus B19, Coxsackie virus, treponema pallidum, hepatitis B virus, chlamydia and the like.
Disclosure of Invention
In view of the above, the present invention provides an anti-TORCH-IgM antibody spectrum chip, a method for preparing the same, and a kit for detecting TORCH, aiming at the defects in the prior art.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme.
An anti-TORCH-IgM antibody spectrum chip coated with a protein chip for detecting the antigen of the relevant pathogenic microorganism by TORCH.
The invention adopts protein chip technology to integrate various pathogens of the TORCH on the protein chip to prepare the TORCH-related antibody IgM chip with high flux, multiple detection indexes, stable performance, good repeatability and high accuracy.
Preferably, the pathogenic microorganism related to the TORCH detection is at least one of toxoplasma gondii (TOX), Cytomegalovirus (CMV), Rubella Virus (RV), Herpes Simplex Virus (HSV), human parvovirus B19, coxsackie virus, treponema pallidum, Hepatitis B Virus (HBV) and chlamydia.
In some embodiments, the pathogenic microorganisms associated with the TORCH assay are toxoplasma gondii (TOX), Cytomegalovirus (CMV), Rubella Virus (RV), Herpes Simplex Virus (HSV), human parvovirus B19, coxsackie virus, treponema pallidum, Hepatitis B Virus (HBV), and chlamydia, for a total of 9. The Herpes Simplex Virus (HSV) includes herpes simplex virus-I and herpes simplex virus-II.
The anti-torch-IgM antibody spectrum chip of the present invention further comprises a quality control point and/or a reference point.
Wherein the quality control points comprise at least one positive quality control Point (PC), at least one negative quality control point (NC), at least one sample quality control point (SC) and/or at least one enzyme-labeled quality control point (EC), and the reference points comprise reference curve points (S1-S5) with different concentrations and/or at least one chip position reference point (L oc).
In some embodiments, the chip of the present invention comprises one positive control Point (PC), one negative control point (NC), one sample control point (SC), one enzyme-labeled control point (EC), 5 reference curve points (S1-S5), and one chip position reference point (L oc).
In some embodiments of the invention, the positive control point can be human IgM, and the enzyme-linked immunosorbent assay marker used is an enzyme label for anti-human IgM. In other embodiments, the positive control spots may be coated with a BSA-DNP conjugate, and the enzyme-linked immunosorbent assay is a mixture of enzyme-labeled anti-human IgM and enzyme-labeled anti-DNP-BSA antibody.
In some embodiments of the invention, the negative control point can be a trace concentration of human IgM that is below the reaction signal value. Other unrelated proteins may be substituted in other embodiments.
In some embodiments of the invention, the sample quality control point can be a murine anti-human IgM. In other embodiments, other anti-human IgM may be used.
In some embodiments of the invention, the enzyme-labeled quality control point may be human IgM. In other embodiments, other anti-rabbit antibodies (labeled with rabbit anti-human IgM), such as goat anti-rabbit IgM, may be used.
In some embodiments of the invention, the reference curve points are five different concentrations of human IgM. For example, 0.5. mu.g/ml, 1. mu.g/ml, 2.5. mu.g/ml, 5. mu.g/ml, 10. mu.g/ml human IgG.
In the invention, the position reference point of the chip is coated with 2 mu g/ml human IgM solution, which is mainly used for positioning when the array takes values.
The invention also provides a preparation method of the anti-TORCH-IgM antibody spectrum chip, which is characterized in that the antigen, quality control point protein and/or reference point protein of related pathogenic microorganism are detected by diluting TORCH with coating buffer solution, and the chip substrate is coated with the coating buffer solution in a dot array form by a spotting instrument.
The invention uses a high-precision sample applicator to coat the antigens of 10 pathogenic microorganisms related to the TORCH detection, the required quality control point protein and the reference point protein on a chip substrate in a dot array form of 4 × 5
Wherein the coating buffer solution is a buffer solution containing PEG, water-soluble cyclodextrin, trehalose, a preservative and glycerol.
In some embodiments, the buffer is CB buffer at pH9.6, Tris buffer at pH8.5, or PBS buffer at pH 7.4-7.6.
The coating buffer solution contains PEG. In some embodiments, the PEG is PEG 4000. The content of the PEG is preferably 0.5% to 1%, more preferably 0.5%.
The coating buffer solution contains water-soluble cyclodextrin, so that the coating is more stable and uniform, the coated points are more regular and more round, and the CV is smaller, preferably, the water-soluble cyclodextrin is 0.01-0.02% of Captisol, 0.02-0.04% of 2-hydroxy- β -cyclodextrin or carboxymethyl- β -cyclodextrin;
the coating buffer solution contains trehalose. Preferably, the content of the trehalose is 5-8%;
the coating buffer of the invention contains a preservative. In some embodiments, the preservative is Proclin 300. Preferably, the content of the preservative is 0.05%.
The coating buffer of the invention contains glycerol. Preferably, the content of glycerin is 15%.
Preferably, in the preparation method of the chip, the coating is 2-8 ℃, and the standing coating is 16-24 h.
The chip substrate of the invention includes but is not limited to enzyme labeling reaction plate, glass sheet, chemical film, porous silica gel. Other suitable carriers for protein attachment may also serve as the chip substrate.
Further, the preparation method of the chip also comprises a step of sealing by using a sealing stabilizer.
Preferably, the blocking stabilizer is 0.01M disodium hydrogen phosphate solution with pH7.4 containing 1% -4% mannitol, 2% -5% PVP40000, 1% -3% NH2-PEG, 0.05% NaN 3. In some embodiments, the blocking stabilizer is 1% mannitol, 2% PVP40000, 1% NH2PEG, 0.05% NaN3 in 0.01M disodium hydrogen phosphate, pH 7.4.
The blocking is preferably performed at room temperature for 1 h.
The invention also provides a kit for detecting TORCH, which comprises the anti-TORCH-IgM antibody spectrum chip.
Furthermore, the kit for detecting TORCH also comprises at least one of an enzyme label, an enzyme-labeled dilution stabilizer, a sample diluent, a washing solution and a developing solution. In some embodiments, the kit for the TORCH detection further includes an enzyme label, an enzyme-labeled dilution stabilizer, a sample diluent, a washing solution, and a developing solution.
Preferably, the enzyme label is an anti-human IgM antibody labeled with horseradish peroxidase, such as a goat anti-human IgM antibody.
Preferably, the enzyme-labeled dilution stabilizer is 100mM Tris solution with pH7.4 containing 0.05M citric acid, 3% -5% bovine serum gamma globulin, 2% -4% PEG10000, 0.05% -0.2% acacia gum, 1% -2% betaine and 0.05% Proclin 3000. More preferably, the enzyme-labeled dilution stabilizer is 100mM Tris solution with pH7.4 containing 0.05M citric acid, 3% bovine serum gamma globulin, 2% PEG10000, 0.05% acacia gum, 1% betaine and 0.05% Proclin 3000.
Preferably, the sample diluent is a 0.02M Tris solution at pH7.4 comprising 0.15M NaCl, 0.05% Tween20, 0.01% casein.
Preferably, the wash solution is a 0.02M Tris solution at pH7.4 comprising 0.15M NaCl, 0.05% Tween 20.
Preferably, the color-developing solution is a sedimentation type TMB.
The invention also provides a TORCH detection method, wherein a sample to be detected is diluted by the sample diluent and then added on the anti-TORCH-IgM antibody spectrum chip, an enzyme marker is added, a color development liquid is added for color development in a dark place, and a fluorescence detection device is used for detecting results and calculating the anti-TORCH-IgM antibody signal value in the sample to be detected.
Further, the detection method comprises a step of washing with a washing solution after incubation before adding the enzyme label and adding the color developing solution.
The detection method provided by the invention detects the level of the IgM (immunoglobulin M) related to the TORCH infection in a sample of a subject by the immunological principle of antigen-antibody reaction. The signal detection system of the detection method is in a chemiluminescence mode, and the result can be read by a fluorescence detection device or an instrument by using a chemiluminescence substrate such as luminol as a substrate.
According to the technical scheme, the invention provides an anti-TORCH-IgM antibody spectrum chip and a preparation method thereof, and a TORCH detection kit and a detection method. The anti-TORCH-IgM antibody spectrum chip of the present invention is coated with a protein chip for detecting antigens of relevant pathogenic microorganisms by TORCH. Further comprising a quality control point and/or a reference point. The anti-TORCH-IgM antibody spectrum chip is used for diluting TORCH with a coating buffer solution to detect antigens, quality control point proteins and/or reference point proteins of related pathogenic microorganisms, coated on a chip matrix in a dot array form through a spotting instrument, and further sealed by a sealing stabilizer. Compared with the traditional enzyme-linked immunosorbent assay, colloidal gold assay and the existing gene chip technology, the invention not only can simultaneously quantitatively detect the infection of various related pathogenic microorganisms of TORCH, but also can ensure that the anti-TORCH-IgM antibody spectrum chip has better stability (2-8 ℃ for 2 years in refrigeration, and can be stored for 8 months at room temperature) and longer service cycle by optimizing the coating liquid, the coating conditions and the sealing stabilizer of the chip. The kit for detecting TORCH comprises the anti-TORCH-IgM antibody spectrum chip and at least one of an enzyme marker, an enzyme-labeled dilution stabilizer, a sample diluent, a washing solution and a developing solution. The kit can effectively reduce the usage amount of the related antigens, reduce the antigen cost, optimize the kit, ensure that the kit has longer stable period, can be placed for two years at the temperature of 2-8 ℃ and can be placed for 8 months at room temperature, has better overall performance and longer service cycle, can further reduce the transportation cost, and improves the convenience of storage and transportation. The method for detecting TORCH can simultaneously and quantitatively detect the infection of various related pathogenic microorganisms of TORCH, has high sensitivity and good specificity, and provides a new high-efficiency and quick detection means for prenatal and postnatal care.
Detailed Description
The invention discloses an anti-TORCH-IgM antibody spectrum chip, a preparation method thereof and a TORCH detection kit. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. Wherein, the content of BSA is mass volume ratio, and is calculated by g/ml, for example, 5 percent BSA is 5g BSA in 100ml PBS; the content of Tween-20 is volume ratio, such as 0.05% Tween-20 in 1ml PBS containing 0.5 μ l Tween-20; the content mass volume ratio of PEG4000 is calculated by g/ml, for example, 0.5g PEG4000 is contained in 100ml buffer solution of 0.5% PEG; the trehalose is in a mass-to-volume ratio, and is calculated by g/ml, for example, 5g of trehalose is contained in 100ml of buffer solution with 5% of trehalose; captisol is in a mass-to-volume ratio, and is calculated by g/ml, for example, 0.02g of Captisol is contained in 100ml of buffer solution; proclin300 is in terms of mass/volume ratio, e.g. 0.05% Proclin300 is 0.05g Proclin300 in 100ml buffer. In addition, PVA, betaine, NaN3, sodium p-hydroxybenzoate, gum arabic and casein are in mass-to-volume ratio in g/ml.
Example 1 preparation of anti-torch-IgM antibody Spectrum chip kit
1. Coating of antigen:
1) the array design of the chip and the specific distribution of the antigens and reference points are as follows (not limited to the following arrangement design):
2) and the specific coating process comprises the following steps:
the diluted antigens and associated reference point proteins were first pressed:
the PC, NC, S1, S2, S3, S4, S5, EC spots in the array were coated with 2. mu.g/ml, 0.01. mu.g/ml, 0.5. mu.g/ml, 1. mu.g/ml, 2.5. mu.g/ml, 5. mu.g/ml, 10. mu.g/ml, 2.5. mu.g/ml of human IgM, respectively, and the dilution buffer was CB buffer of pH9.6 (containing 0.5% PEG4000, 5% trehalose, 0.02% Captisol, 0.05% Proclin300, and 15% glycerol).
SC dots are coated with 2 ug/ml goat anti-IgM antibody and the dilution buffer is CB buffer with pH 9.6.
L the point on the gel was coated with 2. mu.g/ml human IgM and the dilution buffer was CB buffer pH 9.6.
Dilution of coating antigen: 50 mu g/ml of Tox purified antigen, 20 mu g/ml of HSV-I recombinant antigen, 20 mu g/ml of HSV-II recombinant antigen, 15 mu g/ml of RV recombinant antigen, 10 mu g/ml of CMV recombinant antigen, 8 mu g/ml of HBV antigen, 40 mu g/ml of TP recombinant antigen, 25 mu g/ml of B19 recombinant antigen, 30 mu g/ml of CV recombinant antigen and 25 mu g/ml of CT recombinant antigen.
The diluted antigens were filtered through 0.22 μm filters and coated onto arrays using a BioDot precision printer. After the complete array spotting, the chip was placed at 2-8 ℃ and coated for 16-24 h.
2. And (3) sealing:
the coated chip was taken out, washed 3 times with PBST wash solution of pH7.4, and 150. mu.l of blocking stabilizer (1% mannitol, 2% PVA40000, 1% NH2-PEG, 0.05% NaN3 preservative disodium hydrogen phosphate solution of pH7.4) was added to each well, blocked at room temperature for 1 hour, patted dry, allowed to stand at room temperature with a humidity of 15% or less, dried for 4 hours, sealed, and stored at 2-8 ℃.
3. Preparation of enzyme-labeled reagent
Horse radish peroxidase-labeled rabbit anti-human IgM antibody was diluted 6000-fold with an enzyme-labeled dilution stabilizer (100mM Tris containing 50mM citric acid, 3% bovine serum gamma globulin, 2% PEG10000, 0.05% gum arabic, 1% betaine, and 0.05% Proclin 300).
4. Establishment of reaction System of kit
Taking out the chip reagent, and balancing to room temperature;
sample adding: negative and positive control sera and a 101-fold dilution of the test sample with a sample diluent (0.02M Tris, 0.15M NaCl, 0.05% Tween20, 0.01% casein, pH7.4) were added to the test chip wells at 100. mu.l per well for reaction.
And (3) incubation: the reaction was carried out at room temperature for 30 min. Add 300. mu.l of washing solution (0.02M Tris, 0.15M NaCl, 0.05% Tween20, pH7.4) and wash 3 times for 1min each.
Enzyme-labeled secondary antibody (enzyme label): 50 μ l of enzyme-labeled antibody was added to each well.
And (3) incubation: the reaction was carried out at room temperature for 30 min. Add 300. mu.l of washing solution and wash 3 times for 1min each time.
Color development: 50 mul of TMB color developing agent is added into each hole, and the reaction is carried out for 30min at room temperature in a dark place.
And (3) determination: within 30min, the signal value of the corresponding antibody of each reaction well is read and calculated by a detector.
Example 2 evaluation of accuracy of anti-torch-IgM antibody chip kit
1. anti-Toxoplasma IgM positive sera and 10 anti-Toxoplasma IgM negative sera were determined by 10 screening samples using the Diasorin ISA kit E L, and the results of the test using the anti-torch-IgM type antibody panel chip prepared in example 1 are shown in Table 1 below (+ indicates positive-indicates negative).
TABLE 1 anti-torch IgM serum test results of anti-Toxoplasma IgM spectrum chip prepared in example 1
2. The results of the determination of anti-rubella virus IgM positive sera and 10 anti-rubella virus IgM negative sera using 10 selected by the Diasorin ISA kit E L are shown in Table 2 below (+ positive and-negative) using the anti-torch-IgM type antibody panel chip prepared in example 1.
TABLE 2 results of testing anti-rubella virus IgM serum by anti-torch-IgM antibody profiling chip prepared in example 1
3. The results of the tests carried out on the anti-torch IgM type antibody panel chip prepared in example 1 using 10 anti-cytomegalovirus IgM positive sera and 10 anti-cytomegalovirus IgM negative sera screened with the Diasorin ISA kit E L are shown in Table 3 below (+ positive and-negative).
TABLE 3 anti-torch IgM type antibody profiling chip prepared in example 1 test results against cytomegalovirus IgM serum
4. Anti-herpes simplex virus-I IgM positive sera and 10 anti-herpes simplex virus-I IgM negative sera were determined by 10 screening samples using the Diasorin ISA kit E L, and tested by the anti-torch-IgM antibody panel chip prepared in example 1, and the results are shown in the following Table 4(+ positive and-negative).
TABLE 4 anti-torch-IgM type antibody profiling chip prepared in example 1 test results against herpes simplex virus-I IgM serum
5. The results of the tests carried out on the anti-torch-IgM type antibody panel chip prepared in example 1 using 10 anti-herpes simplex virus-II IgM positive sera and 10 anti-herpes simplex virus-II IgM negative sera screened with the Diasorin ISA kit E L are shown in Table 5(+ indicates positive-indicates negative) below.
TABLE 5 anti-torch-IgM type antibody profiling chip prepared in example 1 test results against herpes simplex virus-II IgM serum
6. Infection with an anti-parvovirus B1IgM positive serum and 10 anti-parvovirus B1IgM negative sera were determined in 10 cases screened with the EUROIMMUN corporation E L ISA kit, and the test was performed using the anti-torch-IgM type antibody spectrum chip prepared in example 1, and the results are shown in the following Table 6(+ indicates positive, -indicates negative).
TABLE 6 anti-torch-IgM type antibody Spectroscopy chip prepared in example 1 results of testing anti-parvovirus B1IgM serum
7. Anti-coxsackievirus IgM positive sera and 10 anti-coxsackievirus IgM negative sera were determined by 10 cases screened with the EUROIMMUN company E L ISA kit, and tested with the anti-torch-IgM type antibody spectrum chip prepared in example 1, and the results are shown in the following Table 7(+ indicates positive, -indicates negative).
TABLE 7 anti-torch-IgM type antibody Spectroscopy chip prepared in example 1 results of testing anti-Coxsackie Virus IgM serum
8. anti-Treponema Pallidum (TP) IgM positive sera and 10 anti-Treponema Pallidum (TP) IgM negative sera were determined by 10 cases screened with E L ISA kit of EUROIMMUN, and the results of the test using the anti-torch-IgM type antibody spectrum chip prepared in example 1 are shown in Table 8 below (+ represents positive, -represents negative) — Table 8 the anti-torch-IgM type antibody spectrum chip prepared in example 1 shows the results of the test on anti-Treponema pallidum IgM sera
9. The results of the tests using the anti-torch-IgM type antibody spectrum chip prepared in example 1 were shown in Table 9 below (+ indicating positive-indicating negative) in which anti-Hepatitis B Virus (HBV) core antibody IgM serum was determined in 10 cases screened with the Diasorin ISA kit E L, Inc. and the results of the tests using the anti-torch-IgM type antibody spectrum chip prepared in example 1 are shown in Table 9 below (results of testing anti-hepatitis B virus core antibody IgM serum using the anti-torch-IgM type antibody spectrum chip prepared in example 1 are shown in Table 9 below
10. anti-Chlamydia Trachomatis (CT) IgM positive sera and 10 anti-Chlamydia Trachomatis (CT) IgM negative sera were determined by 10 cases screened with the IB L corporation E L ISA kit, and the test was performed by the anti-torch-IgM type antibody spectrum chip prepared in example 1, and the results are shown in the following Table 10(+ represents positive, -represents negative).
TABLE 10 results of testing anti-Chlamydia trachomatis IgM serum by anti-torch-IgM type antibody Spectroscopy chip prepared in example 1
In conclusion, the anti-torch-IgM antibody spectrum chip prepared by the invention meets the requirement on the accuracy of testing various anti-pathogen IgM antibodies, and is high in accuracy.
Example 3 anti-torch-IgM type antibody Spectroscopy chip kit specificity evaluation
Taking the Toxoplasma project as an example, 20 clinical negative sera of the anti-Toxoplasma IgM antibody are selected, the anti-Toxoplasma IgM antibody spectrum chip prepared in example 1 and the E L ISA kit of Diasorin company are used for testing at the same time, and the test data are as the following table 11 (the "+" represents positive, and the "+ -" represents weak positive "-" represents negative).
TABLE 11 comparison results of specificity of anti-torch-IgM type antibody spectrum chip prepared in example 1
The result shows that 20 negative sera tested by the anti-torch-IgM antibody spectrum chip prepared in example 1 have no false positive, while 2 false positive tested by the Diasorin company E L ISA kit on the market tested by the 20 negative sera, which shows that the anti-torch-IgM antibody spectrum chip of the present invention has better specificity than the Diasorin company E L ISA kit.
Example 4 evaluation of stability of anti-torch-IgM antibody chip kit
The anti-torch-IgM type antibody spectrum chip kits prepared in example 1 were placed at 2 to 8 ℃ for 0 month, 6 months, 12 months, 18 months and 24 months, respectively, and after 0 month, 2 months, 4 months, 6 months and 8 months at 18 to 28 ℃ for 0 month, 2 months, 4 months and 8 months, signal value data were measured with anti-corresponding antigen IgM quality control serum, respectively, and the results are shown in tables 12 and 13.
TABLE 12 EXAMPLE 1 preparation of anti-torch-IgM type antibody Spectroscopy chip kit 2-8 ℃ stability evaluation results
TABLE 13 EXAMPLE 1 preparation of anti-torch-IgM type antibody Spectroscopy chip kit the results of the evaluation of the stability at 18-28 deg.C
The result shows that the anti-torch-IgM antibody spectrum chip kit of the present invention has a stabilization period of two years at 2-8 ℃ and one year at 18-28 ℃.
Example 5 Effect of blocking stabilizer and enzyme-labeled diluent on stability in anti-torch-IgM type antibody Spectroscopy chip kit
The anti-torch-IgM antibody spectrum chip kit prepared in example 1 of the present invention and the anti-torch-IgM antibody spectrum chip comparison kit (other method steps of the kit are the same as example 1) composed of a common blocking solution (PBS (pH: 7.40.01M) containing 5% BSA) and an enzyme-labeled diluent (PBS (pH: 7.40.01M) containing 0.05% tween-20 and 1% BSA) were respectively placed at 2-8 ℃ for 6 months, 12 months, 18 months and 24 months, and placed at 18-28 ℃ for 2 months, 4 months, 6 months and 8 months, and then signal value data were respectively tested using anti-corresponding antigen IgM quality control serum, and the results are shown in tables 14 and 15.
TABLE 142-8 ℃ stability evaluation results of blocking stabilizer and enzyme-labeled dilution stabilizer
TABLE 1518-28 deg.C stability evaluation results of blocking stabilizer and enzyme-labeled diluting stabilizer
The result shows that the anti-torch-IgM antibody spectrum chip detection kit prepared by using the common confining liquid and the enzyme-labeled diluent is obviously reduced in the signal intensity by using the corresponding quality control serum after being placed at 2-8 ℃ for 12 months or at 18-28 ℃ for 2 months, while the anti-torch-IgM antibody spectrum chip kit provided by the invention is also shown to have good performance by using the corresponding quality control serum after being placed at 2-8 ℃ for 12 months or at 18-28 ℃ for 2 months. The detection kit for the anti-torch-IgM antibody spectrum chip of the invention is proved to have better sealing stabilizer and enzyme-labeled diluent stabilizer than common sealing liquid and enzyme-labeled diluent, and can greatly improve the stability of the product.
Example 6 comparison of antigen costs used in anti-torch-IgM antibody Panel kit and colloidal gold kit
Comparing the antigen cost with the colloidal gold rapid test paper disclosed in the chinese patent with application No. 201110220541:
calculating the antigen dosage used by the fluorescence immunochromatography kit:
toxoplasma antigen coating is taken as an example: in the patent CN201110220541, the Toxoplasma gondii antigen is coated at the concentration of 1.5mg/ml, and the width of each test strip is 0.4cm, so that the average dosage of the antigen per person is about 0.6 mu g.
The antigen dosage used by the kit of the invention is calculated as follows:
the invention uses a Biodot precise spotting instrument to coat the antigen, and the coating volume of each point is only 10n L, so the amount of the antigen used for coating each person is 50 mug/ml × 10n L× 10-6-0.005 mug of toxoplasma purified antigen.
Therefore, the using amount of the toxoplasma gondii antigen in the invention is obviously lower than 2 orders of magnitude of the colloidal gold rapid detection test paper. The other antigens were used in a similar manner. In conclusion, the anti-torch-IgM antibody spectrum chip kit provided by the invention can effectively reduce the usage amount of related antigens, and reduce the usage cost of the related antigens while ensuring the sensitivity and specificity of the kit.
Claims (9)
1. An anti-TORCH-IgM antibody spectrum chip coated with a protein chip for detecting antigens of relevant pathogenic microorganisms by TORCH; the preparation method of the chip comprises the steps of diluting the TORCH with a coating buffer solution to detect the antigen, the quality control point protein and/or the reference point protein of related pathogenic microorganisms, coating the antigen, the quality control point protein and/or the reference point protein on a chip substrate in a dot array form by a spotting instrument, and sealing the chip substrate with a sealing stabilizer; the coating buffer is a CB buffer containing 0.5 percent of PEG4000, 0.02 percent of Captisol, 5 percent of trehalose, 0.05 percent of Proclin300 and 15 percent of glycerol with pH of 9.6; the sealing stabilizer contains 1% of mannitol, 2% of PVP40000 and 1% of NH2PEG, 0.05% NaN3 in 0.01M disodium hydrogen phosphate, pH 7.4.
2. The chip of claim 1, wherein the pathogenic microorganism related to the TORCH detection is at least one of toxoplasma gondii (TOX), Cytomegalovirus (CMV), Rubella Virus (RV), Herpes Simplex Virus (HSV), human parvovirus B19, coxsackie virus, treponema pallidum, Hepatitis B Virus (HBV), and chlamydia.
3. The chip of claim 1 or 2, wherein the protein chip further comprises quality control points and/or reference points, the quality control points comprise at least one positive quality control Point (PC), at least one negative quality control point (NC), at least one sample quality control point (SC) and/or at least one enzyme-labeled quality control point (EC), and the reference points comprise reference curve points (S1-S5) with different concentrations and/or at least one chip position reference point (L oc).
4. The chip of claim 3, wherein the positive control spots are coated with human IgM or coated with BSA-DNP conjugates; the negative quality control point is coated with trace concentration of human IgM or other proteins irrelevant to TORCH detection pathogenic microorganisms, wherein the trace concentration of human IgM is lower than a reaction signal value; the sample quality control point is coated antihuman IgM; the enzyme-labeled quality control points are coated with human IgM or anti-rabbit antibodies; the reference curve points are coated with human IgM at different concentrations; the chip position reference points are coated with human IgM solutions of known concentrations.
5. The method for preparing a chip according to any one of claims 1 to 4, wherein antigens, control point proteins and/or reference point proteins of pathogenic microorganisms of interest are detected by diluting the TORCH with a coating buffer, coated on a chip substrate in the form of a dot array by a spotting instrument, and blocked with a blocking stabilizer; the coating buffer is a CB buffer containing 0.5 percent of PEG4000, 0.02 percent of Captisol, 5 percent of trehalose, 0.05 percent of Proclin300 and 15 percent of glycerol with pH of 9.6; the sealing stabilizer contains 1% of mannitol, 2% of PVP40000 and 1% of NH2PEG, 0.05% NaN3 in 0.01M disodium hydrogen phosphate, pH 7.4.
6. The preparation method according to claim 5, wherein the coating is carried out at 2-8 ℃ for 16-24 h; the chip substrate is an enzyme-labeled reaction plate, a glass sheet, a chemical membrane and porous silica gel.
7. A kit for TORCH detection, comprising the anti-TORCH-IgM type antibody panel chip according to any one of claims 1 to 4.
8. The kit of claim 7, further comprising at least one of an enzyme label, an enzyme-labeled dilution stabilizer, a sample diluent, a washing solution, and a developing solution.
9. The kit of claim 8, wherein the enzyme label is an anti-human IgM antibody labeled with horseradish peroxidase; the enzyme-labeled diluent stabilizer is 100mM Tris solution with pH7.4 containing 0.05M citric acid, 3% -5% bovine serum gamma globulin, 2% -4% PEG10000, 0.05% -0.2% Arabic gum, 1% -2% betaine and 0.05% Proclin 3000; the sample diluent was a 0.02M Tris solution at ph7.4 containing 0.15M NaCl, 0.05% Tween20, 0.01% casein; the washing solution was 0.02M Tris solution, pH7.4, containing 0.15M NaCl, 0.05% Tween 20; the color development liquid is a sedimentation type TMB.
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| PCT/CN2018/092768 WO2019169792A1 (en) | 2018-03-07 | 2018-06-26 | Anti-torch-igm antibody repertoire chip, preparation method therefor and torch detection kit |
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| CN112748242B (en) * | 2020-12-21 | 2023-07-21 | 珠海碳云智能科技有限公司 | Secondary antibody buffer sealing solution for polypeptide chip technology platform detection, kit comprising secondary antibody buffer sealing solution and application of secondary antibody buffer sealing solution |
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| CN1412557A (en) * | 2001-10-11 | 2003-04-23 | 南京集慧科技开发有限责任公司 | ToRCH protein chip and its preparation method |
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