WO2019169796A1 - Anti-torch-igg antibody profile chip and preparation method therefor, and torch detection kit - Google Patents

Abstract

The present invention relates to the technical field of biological detection, and provides an anti-torch-IgG antibody profile chip and a preparation method therefor, and a TORCH detection kit and a detection method. The anti-torch-IgG antibody profile chip can be used for simultaneously and quantitatively detecting the infection of related pathogenic microbes of TORCH. Moreover, by optimizing coating liquid and coating conditions of the chip and sealing stabilizing agents, the stability of the anti-torch-IgG antibody profile chip is better, and the service cycle is longer. The TORCH detection kit provided by the present invention has the advantages that the consumption of relevant antigens can be effectively reduced, the antigen cost can be reduced, and meanwhile, the stable period is longer, the use period is longer, the transportation cost is reduced, and the storage and transportation convenience is improved. The TORCH detection method can be used for simultaneously and quantitatively detecting the infection of various related pathogenic microbes of TORCH, the sensitivity is high, and the specificity is high.

Description

Anti-torch-IgG type antibody spectrum chip and preparation method thereof and kit for TORCH detection

This application claims to be filed on March 07, 2018, the Chinese Patent Office, Application No. 201101188210.6, the Chinese patent application entitled "An anti-torch-IgG type antibody spectrum chip and its preparation method and TORCH detection kit" Priority is hereby incorporated by reference in its entirety.

Technical field

The invention belongs to the technical field of biological detection, and particularly relates to an anti-torch-IgG type antibody spectrum chip, a preparation method thereof and a kit for detecting TORCH.

Background technique

TORCH is a pathogen causing perinatal malformation caused by congenital intrauterine infection and perinatal infection. It is an abbreviation of a group of pathogenic microorganisms, in which T (Toxoplasma) is Toxoplasma gondii and O (Others) is other pathogenic microorganisms. For example, Treponema pallidum, herpes zoster virus, parvovirus B19, Coxsackie virus, etc., R (Rubella.Virus) is a rubella virus, C (Cytomegalo.Virus) is a 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 newborns. It is called TORCH syndrome in perinatal medicine, and its infection is distributed worldwide. In our population, TORCH infection is widespread. Pregnant women have no obvious clinical symptoms after TORCH infection during pregnancy, but after fetal infection, it may cause developmental defects and functions in liver, kidney, heart, brain and other organs of the fetus or newborn. Obstacles may cause adverse consequences such as embryo/fetal abortion, premature delivery, intrauterine growth retardation, deformity, stillbirth and neonatal death, posing a great threat to prenatal and postnatal care and population quality. Therefore, in order to reduce the birth rate of disabled children and improve the quality of the birth population, serological screening of TORCH infection should be done to detect early pregnancy and treat it promptly. For newborns, TORCH testing should also be routinely conducted to understand the neonatal TORCH infection for early intervention and early treatment.

In the detection of TORCH, the infection of Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex I/II type four pathogenic microorganisms accounted for about 90%, and the remaining 10% were other pathogenic microorganisms, including parvovirus B19, Coxsackie virus, Infection with Treponema pallidum, Hepatitis B virus, Chlamydia, etc. At present, the detection methods for TPRCH mainly include ELISA, immunofluorescence assay (IFT), colloidal gold and gene chip. The most convenient and most commonly used early screening method in China is the use of ELISA diagnostic techniques. ELISA is a specific IgM and IgG antibody in human serum. Because IgM is an early infection indicator, it has a great impact on the fetus. Therefore, the detection of IgM has attracted much attention. The detection of specific IgM in the placenta is a reliable basis for the diagnosis of intrauterine infection. . ELISA reagents are widely used in general laboratories because of their stability, sensitivity, specificity, and low cost, but usually only a single indicator can be detected. IFT is generally also for the detection of individual indicators. The method of colloidal gold is generally only qualitative or semi-quantitative. Although the effect of gene chip technology is good, it is costly and generally expensive.

Summary of the invention

In view of this, the present invention provides a kit for anti-torch-IgG type antibody spectrum chip, a preparation method thereof and TORCH detection, in view of the defects in the prior art.

In order to achieve the object of the present invention, the present invention adopts the following technical solutions.

An anti-torch-IgG type antibody profile chip coated with a protein chip having an antigen of a pathogenic microorganism associated with TORCH detection.

The invention adopts protein chip technology, and integrates various pathogens of TORCH on a protein chip to prepare a TORCH-related antibody IgG chip with high throughput, multiple detection indexes, stable performance, good repeatability and high accuracy.

Wherein, preferably, the pathogenic microorganisms related to the TORCH detection are Toxoplasma gondii (TOX), cytomegalovirus (CMV), rubella virus (RV), herpes simplex virus (HSV), human parvovirus B19, Coxsackie virus, At least one of Treponema pallidum, Hepatitis B virus (HBV), and Chlamydia.

In some embodiments, the pathogenic microorganism associated with the TORCH detection is Toxoplasma gondii (TOX), cytomegalovirus (CMV), rubella virus (RV), herpes simplex virus (HSV), human parvovirus B19, Coxsackie virus There are 9 kinds of pathogenic microorganisms related to TORCH, Treponema pallidum, Hepatitis B virus (HBV) and Chlamydia. Herpes simplex virus (HSV) includes herpes simplex virus type I and herpes simplex virus type II.

The anti-torch-IgG type antibody profile chip of the present invention further comprises a quality control point and/or a reference point.

Wherein the quality control point comprises at least one positive property control point (PC), at least one negative nature control point (NC), at least one sample control point (SC), and/or at least one enzyme quality control point (EC) . The reference points comprise reference curve points (S1-S5) of different concentrations and/or at least one chip position reference point (Loc).

In some embodiments, the chip of the present invention comprises a positive property control point (PC), a negative nature control point (NC), a sample control point (SC), an enzyme quality control point (EC), 5 Reference curve points (S1-S5) and a chip position reference point (Loc).

In some embodiments of the invention, the positive property control point can be human IgG and the enzyme-linked immunolabel used is an enzyme label against human IgG. In some other embodiments, the positive control handle may also be coated with a BSA-DNP conjugate, and the enzyme-linked immunolabel used is a mixture of enzyme-labeled anti-human IgG and anti-DNP-BSA enzyme-labeled antibody. .

In some embodiments of the invention, the negative property control point can be a minor concentration of human IgG that is below the response signal value. Other unrelated proteins may be substituted in other embodiments.

In some embodiments of the invention, the sample control point can be a murine anti-human IgG. Other anti-human IgGs can also be used in other embodiments.

In some embodiments of the invention, the enzyme labeling control point can be a human IgG. Other anti-rabbit antibodies can also be used in other embodiments (the enzyme is labeled with rabbit anti-human IgG), such as a goat anti-rabbit IgG antibody.

In some embodiments of the invention, the reference curve points are five different concentrations of human IgG.

In the present invention, the position reference point of the chip itself is coated with a 2 μg/ml human IgG solution, which is mainly the positioning effect when the array is valued.

The invention also provides a preparation method of the anti-torch-IgG type antibody spectrum chip, wherein the antigen, the control point protein and/or the reference point protein of the related pathogenic microorganism are detected by diluting the TORCH with a coating buffer, and the spotting instrument is used to The dot array form is coated on a chip substrate.

The invention coats the antigens of 10 pathogenic microorganisms related to TORCH detection and the required quality control point proteins and reference point proteins on a chip substrate by a highly precise spotting instrument in a 4×5 dot array form.

Wherein, the coating buffer is a buffer containing PEG, water-soluble cyclodextrin, trehalose, preservative and glycerin.

In some embodiments, the buffer solution is CB buffer at pH 9.6, Tris buffer at pH 8.5, or PBS buffer at pH 7.4-7.6.

The coating buffer of the present invention contains PEG. In some embodiments, the PEG is PEG4000. The content of the PEG is preferably from 0.5% to 1%, more preferably 0.5%.

The coating buffer of the invention contains water-soluble cyclodextrin, so that the coating is more stable, uniform, and the coating is more regular, more round, and the CV is smaller. Preferably, the water-soluble cyclodextrin is 0.01% to 0.02% Captisol, 0.02%-0.04% 2-hydroxy-β-cyclodextrin or carboxymethyl-β-cyclodextrin;

The coating buffer of the present invention contains trehalose. Preferably, the content of the trehalose is 5% to 8%;

The coating buffer of the present invention contains a preservative. In some embodiments, the preservative is Proclin 300. Preferably, the preservative is present in an amount of 0.05%.

The coating buffer of the present invention contains glycerin. Preferably, the glycerin is present in an amount of 15%.

Preferably, in the method for preparing the chip of the present invention, the coating is 2-8 ° C, and the coating is allowed to stand for 16-24 h.

The chip substrate of the present invention includes, but is not limited to, an enzyme-labeled reaction plate, a glass plate, a chemical film, and a porous silica gel. Other carriers suitable for protein attachment can also serve as a matrix for the chip.

Further, the method for preparing the chip of the present invention further comprises the step of blocking with a blocking stabilizer.

Preferably, the blocking stabilizer is 0.01% of pH 7.4 containing 1% to 5% BSA, 2% to 6% trehalose, 0.8% to 1% PVA 20000, 0.2% to 0.4% betaine, 0.05% NaN3. M PBS solution. In some embodiments, the blocking stabilizer is a 0.01 M PBS solution of pH 7.4 containing 1% BSA, 4% trehalose, 0.8% PVA 20000, 0.2% betaine, 0.05% NaN3.

The blocking is preferably blocked at room temperature for 1 h.

The invention also provides a kit for TORCH detection, comprising the anti-torch-IgG type antibody profile chip.

Further, the kit for detecting TORCH further comprises at least one of an enzyme label, an enzyme standard dilution stabilizer, a sample diluent, a washing solution, and a color developing solution. In some embodiments, the kit for TORCH detection further comprises an enzyme label, an enzyme standard dilution stabilizer, a sample diluent, a wash solution, and a color developing solution.

Preferably, the enzyme label is a horseradish peroxidase-labeled anti-human IgG antibody, such as a goat anti-human IgG antibody.

Preferably, the enzyme standard dilution stabilizer comprises 0.05 M citric acid, 2%-5% goat serum, 2%-4% PEG 10000, 0.2%-0.5% sodium p-hydroxybenzoate, 0.1%-0.2. % gum arabic, 0.05% Proclin 3000, pH 7.4, 1 M Tris solution. More preferably, the enzyme standard dilution stabilizer is pH 7 comprising 0.05 M citric acid, 5% goat serum, 2% PEG 10000, 0.2% sodium p-hydroxybenzoate, 0.1% gum arabic, 0.05% Proclin 3000. .4 of 1 M Tris solution.

Preferably, the sample diluent is a 0.02 M Tris solution of pH 7.4 comprising 0.15 M NaCl, 0.05% Tween 20, 0.01% casein.

Preferably, the wash liquor is a 0.02 M Tris solution of pH 7.4 comprising 0.15 M NaCl, 0.05% Tween 20.

Preferably, the color developing solution is a sedimentation type TMB.

The invention also provides a TORCH detecting method, wherein the sample to be tested is diluted with the sample diluent, added to the anti-torch-IgG type antibody spectrum chip, the enzyme label is added, the color developing solution is added to avoid light and color, and the fluorescence detection is performed. The device detects the result and calculates the anti-torch-IgG type antibody signal value in the sample to be tested.

Further, the detecting method includes the step of washing the washing liquid after the incubation before adding the enzyme label and adding the color developing liquid.

The detection method of the present invention detects the level of IgG-related antibody IgG in a subject sample by the immunological principle of the antigen-antibody reaction. The signal detecting system of the detecting method of the present invention is a chemiluminescent method, and the substrate can be read by a fluorescence detecting device or an instrument using a substrate such as a chemiluminescent substrate such as luminol.

According to the above technical solution, the present invention provides an anti-torch-IgG type antibody spectrum chip, a preparation method thereof and a TORCH detection kit and a detection method. The anti-torch-IgG type antibody profile chip of the present invention comprises a protein chip having an antigen of a pathogenic microorganism associated with TORCH detection. Further included control points and/or reference points. The anti-torch-IgG type antibody spectrum chip dilutes the antigen, the control point protein and/or the reference point protein of the pathogenic microorganism related to the TORCH with a coating buffer, and coats the chip substrate in a dot array by a spotting device. Further blocked with a blocking solution. Compared with the traditional enzyme-linked immunosorbent assay, colloidal gold assay and existing gene chip technology, the present invention can not only quantitatively detect the infection of various related pathogenic microorganisms of TORCH, but also optimize the coating solution and coating conditions of the chip, and close the membrane. The stabilizer makes the anti-torch-IgG type antibody spectrum chip more stable (2 years at 2-8 °C, 8 months at room temperature), and the use period is longer. The kit for detecting TORCH of the present invention comprises at least one of the anti-torch-IgG type antibody spectrum chip and the enzyme label, the enzyme standard dilution stabilizer, the sample diluent, the washing liquid, and the color developing liquid. The kit not only can effectively reduce the amount of related antigens, reduce the cost of antigen, but also optimize the kit to make it have a longer stabilization period. It can be placed at 2-8 °C for two years, and at room temperature for 8 months. Better performance and longer service life can further reduce transportation costs and improve storage and transportation convenience. The TORCH detection method of the invention can simultaneously quantitatively detect the infection of various related pathogenic microorganisms of TORCH, has high sensitivity and good specificity, and provides a new efficient and rapid detection means for prenatal and postnatal care.

Detailed ways

The invention discloses an anti-torch-IgG type antibody spectrum chip, a preparation method thereof and a kit for detecting TORCH. Those skilled in the art can learn from the contents of this document and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and product of the present invention have been described by the preferred embodiments, and it is obvious that those skilled in the art can change or appropriately modify and combine the methods described herein to implement and apply the present invention without departing from the spirit, scope and scope of the invention. Invention technology.

For a better understanding of the present invention, the embodiments of the present invention will be clearly and completely described in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, but not all embodiments. . All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.

Unless otherwise stated, the reagents involved in the examples of the present invention are all commercially available products, which are all commercially available. Wherein, the content of BSA is mass to volume ratio, in terms of g/ml, such as 5% BSA is 100 ml PBS contains 5 g BSA; Tween-20 content is volume ratio, such as 0.05% Tween-20 is 1 ml PBS containing 0.5 μl Tween-20; PEG4000 content mass to volume ratio, in g/ml, such as 0.5% PEG is 100ml buffer containing 0.5g PEG4000; trehalose is mass to volume ratio, in g/ml, such as 5% trehalose It is 5g trehalose in 100ml buffer; Captisol is mass to volume ratio, in g/ml, such as 0.02% Captisol is 100ml buffer containing 0.02g Captisol; Proclin300 is mass to volume ratio, in g/ml, such as 0.05 % Proclin300 contains 0.05 g of Proclin 300 in 100 ml buffer. Further, PVA, betaine, NaN3, sodium p-hydroxybenzoate, gum arabic, and casein are mass-to-volume ratios in terms of g/ml.

Example 1. Preparation of anti-torch-IgG type antibody profile chip kit

1, antigen coating:

1) The array design of the chip and the specific distribution of antigens and reference points are as follows (not limited to the following arrangement design):

2), the specific coating process:

First press the diluted antigen and related reference point protein:

The PC, NC, S1, S2, S3, S4, S5, and EC points in the array were coated at 2 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 0.5 μg/ml, 2 μg/ml, and 4 μg/ml, respectively. , 8 μg / ml, 2 μg / ml of human IgG, dilution buffer is pH 9.6 CB buffer (containing 0.5% PEG4000, 5% trehalose, 0.02% Captisol, 0.05% Proclin300, and 15%) Glycerin).

The SC spot was coated with 2 μg/ml of rabbit anti-human IgG antibody, and the dilution buffer was CB buffer at pH 9.6.

The Loc spot was coated with 2 μg/ml of human IgG, and the dilution buffer was CB buffer at pH 9.6.

Dilution of coated antigen: Tox purified antigen 50μg/ml, HSV-I recombinant antigen 20μg/ml, HSV-II recombinant antigen 20μg/ml, RV recombinant antigen 15μg/ml, CMV recombinant antigen 10μg/ml, HBV antigen 8μg/ml TP recombinant antigen 40 μg/ml, B19 recombinant antigen 25 μg/ml, CV recombinant antigen 30 μg/ml, CT recombinant antigen 25 μg/ml.

The diluted antigens were separately filtered through a 0.22 μm filter, and then coated with an array using a BioDot precision spotter. After all the arrays were spotted, the chip was placed at 2-8 ° C and coated for 16-24 h.

2. Closed:

The coated chips were removed, washed 3 times with PBST washing solution of pH 7.4, and then 150 μl of blocking stabilizer (1% BSA, 4% trehalose, 0.8% PVA20000, 0.2% betaine, and 0.05) were added to each well. %NaN3 preservative pH7.4 0.01M PBS), sealed at room temperature for 1 h, then patted dry, under 15% humidity, room temperature, dried for 4 h, sealed, and stored at 2-8 °C.

3. Preparation of enzyme standard reagent

Enzyme-labeled stabilizing buffer (0.1 M Tris at pH 7.4 containing 0.05 M citric acid, 5% goat serum, 2% PEG 10000, 0.2% sodium p-hydroxybenzoate, 0.1% gum arabic, and 0.05% Proclin 300) diluted horseradish peroxidase-labeled rabbit anti-human IgG antibody to 4000-fold.

4. Establishment of the reaction system of the kit

Remove the chip reagent and equilibrate to room temperature;

Loading: The negative and positive control sera and the sample dilution (0.02 M Tris, 0.15 M NaCl, 0.05% Tween 20, 0.01% casein, pH 7.4) were diluted 101 times of the sample to be tested, and 100 μl per well was added. Test the reaction in the well of the chip.

Incubation: reaction at room temperature for 30 min. 300 μl of washing solution (0.02 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4) was added and washed 3 times for 1 min each time.

Add enzyme-labeled secondary antibody (enzyme label): Add 50 μl of enzyme-labeled antibody to each well.

Incubation: reaction at room temperature for 30 min. Add 300 μl of washing solution and wash 3 times for 1 min each time.

Color development: 50 μl of TMB color developer was added to each well, and the reaction was carried out in the dark at room temperature for 30 min.

Measurement: Within 30 min, the signal value of the antibody corresponding to each well was read and calculated using a detector.

Example 2: Accuracy evaluation of anti-torch-IgG type antibody spectrum chip kit

1. 10 anti-toxoplasma IgG positive sera and 10 anti-toxoplasma IgG negative sera were screened by Diasorin ELISA kit, and tested with the anti-torch-IgG antibody microarray prepared in Example 1. The results are shown in Table 1 below. (+ means positive, - means negative).

Table 1 Anti-torch-IgG type antibody profile chip prepared in Example 1 was tested for anti-Toxoplasma IgG serum results

2. Ten anti-Rubella virus IgG-positive sera and 10 anti-Rubella virus IgG-negative sera were screened by Diasorin ELISA kit, and the anti-torch-IgG antibody microarray prepared in Example 1 was used. The results are shown in Table 2 below. + indicates positive, - indicates negative.

Table 2 Anti-torch-IgG type antibody profile chip prepared in Example 1 Test anti-Rubella virus IgG serum results

3. 10 anti-cytomegalovirus IgG-positive sera and 10 anti-cytomegalovirus IgG-negative sera were screened by Diasorin ELISA kit. The anti-torch-IgG antibody microarray prepared in Example 1 was tested. The results are shown in the following table. 3 (+ means positive, - means negative).

Table 3 Anti-torch-IgG type antibody profile chip prepared in Example 1 Test anti-cytomegalovirus IgG serum results

4. Ten anti-herpes simplex virus-I IgG positive serum and 10 herpes simplex virus-I IgG negative serum were screened by Diasorin ELISA kit, and the anti-torch-IgG antibody spectrum chip prepared in Example 1 was used for testing. The results are shown in Table 4 below (+ indicates positive, - indicates negative).

Table 4 Anti-torch-IgG type antibody profile chip prepared in Example 1 Tested for anti-herpes simplex virus-I IgG serum results

5. Ten anti-herpesvirus-II IgG-positive sera and 10 anti-herpes simplex virus-II IgG-negative sera were screened by Diasorin ELISA kit, and the anti-torch-IgG antibody microarray prepared in Example 1 was tested. The results are shown in Table 5 below (+ indicates positive, - indicates negative).

Table 5 Anti-torch-IgG type antibody profile chip prepared in Example 1 Tested for anti-herpes simplex virus-II IgG serum results

6. 10 cases of anti-parvovirus B1 IgG positive serum and 10 anti-parvovirus B1 IgG negative sera were screened by using the EUROIMMUN ELISA kit. The anti-torch-IgG type antibody microarray prepared in Example 1 was tested. The results were as follows: Table 6 (+ indicates positive, - indicates negative).

Table 6 Anti-torch-IgG type antibody profile chip prepared in Example 1 Test anti-parvovirus B1 IgG serum results

7. Ten anti-coxsackie virus IgG-positive sera and 10 anti-coxsackie virus IgG-negative sera were screened by the EUROIMMUN ELISA kit, and the anti-torch-IgG type antibody spectrum chip prepared in Example 1 was used for testing. The results are shown in Table 7 below (+ indicates positive, - indicates negative).

Table 7 Anti-torch-IgG type antibody profile chips prepared in Example 1 Test anti-Coxsackie virus IgG serum results

8. 10 anti-treponema pallidum (TP) IgG positive sera and 10 anti-treponema pallidum (TP) IgG negative sera were screened by EUROIMMUN ELISA kit, and the anti-torch-IgG antibody microarray prepared in Example 1 was used. The results are shown in Table 8 below (+ indicates positive, - indicates negative).

Table 8 Anti-torch-IgG type antibody profile chip prepared in Example 1 Test anti-treponema IgG serum results

9. 10 anti-hepatitis B virus (HBV) core antibody IgG positive serum and 10 anti-hepatitis B virus core antibody IgG negative serum were screened by Diasorin ELISA kit, and the anti-torch-IgG type antibody spectrum chip prepared in Example 1 was used. The test was carried out and the results are shown in Table 9 below (+ indicates positive, - indicates negative).

Table 9 Anti-torch-IgG type antibody profile chip prepared in Example 1 Test anti-HBV core antibody IgG serum results

10. Ten anti-Chlamydia trachomatis (CT) IgG-positive sera and 10 anti-Chlamydia trachomatis (CT) IgG-negative sera were screened by IBL ELISA kit, and the anti-torch-IgG antibody microarray prepared in Example 1 was used. The results are shown in Table 10 below (+ indicates positive, - indicates negative).

Table 10 Anti-torch-IgG type antibody profile chips prepared in Example 1 Test anti-Chlamydia trachomatis IgG serum results

In summary, the anti-torch-IgG type antibody spectrum chip prepared by the invention tests the accuracy of each anti-pathogenic IgG to meet the requirements and has high accuracy.

Example 3, specific evaluation of anti-torch-IgG type antibody spectrum chip kit

The anti-torch-IgG type antibody profile chip kit of the present invention is compared with a conventional ELISA kit. Taking the Toxoplasma gondii project as an example, 20 anti-toxoplasma IgG antibody clinical negative sera were selected, and the anti-torch-IgG type antibody spectrum chip prepared in Example 1 was simultaneously tested with Diasorin ELISA kit. The test data are shown in Table 11 below (" +" indicates positive, and "±" indicates weak yang "-" indicates negative).

Table 11 Anti-torch-IgG type antibody spectrum chip specific comparison results prepared in Example 1

The results showed that the anti-torch-IgG type antibody microarray prepared in Example 1 tested one case of false positive in 20 negative sera, while the Diasorin ELISA kit on the market tested 3 cases of negative sera in 3 cases of false positivity. It is shown that the anti-torch-IgG type antibody spectrum chip of the present invention is more specific than the Diasorin ELISA kit.

Example 4: Stability evaluation of anti-torch-IgG type antibody spectrum chip kit

The anti-torch-IgG type antibody spectrum chip kit prepared in Example 1 was placed at 2-8 ° C for 0 months, 6 months, 12 months, 18 months, and 24 months, and placed at 18- The signal value data were respectively tested with anti-corresponding antigen IgG quality control serum after 28 months, 2 months, 4 months, 6 months, and 8 months. The results are shown in Tables 12 and 13.

Table 12 Example 1 Preparation of anti-torch-IgG type antibody spectrum chip kit 2-8 ° C stability evaluation results

Table 13 Example 1 Preparation of anti-torch-IgG type antibody spectrum chip kit 18-28 ° C stability evaluation results

The results show that the anti-torch-IgG type antibody spectrum chip kit of the present invention has a stable period of 2 years at 2-8 ° C and a stable period of 18 years at 18-28 ° C.

Example 5: Effect of blocking solution and enzyme standard dilution on anti-torch-IgG type antibody spectrum chip kit on stability

The anti-torch-IgG type antibody spectrum chip kit prepared in Example 1 of the present invention is used with a general blocking solution (pH=7.4 0.01M PBS containing 5% BSA) and an enzyme standard dilution solution (pH=7.4 0.01M PBS containing 0.05). Anti-torch-IgG type antibody spectrum chip comparison kit consisting of % Tween-20 and 1% BSA) (other steps of the kit are the same as in Example 1), placed at 2-8 ° C for 6 months, 12 months respectively. After 18 months, 24 months, placed at 18-28 ° C, 2 months, 4 months, 6 months, 8 months later, the signal value data were tested with anti-corresponding antigen IgG quality control serum. The results are shown in Tables 14 and 15.

Table 14 Stability evaluation results of 2-8 °C blocking stabilizer and enzyme standard dilution stabilizer

Table 15 Stability Evaluation Results of 18-28°C Blocking Stabilizer and Enzyme Standard Dilution Stabilizer

The results showed that anti-torch-IgG type antibody microarray detection kit was prepared at 2-8 ° C for 12 months or 18-28 ° C using a general blocking solution and an enzyme standard dilution (0.01 M pH 7.4 PBS, 10% BSA). After 2 months, the signal intensity of the test was significantly decreased using the corresponding quality control serum, and the anti-torch-IgG type antibody chip kit of the present invention was placed at 2-8 ° C for 12 months or 18-28 ° C for 2 months. The corresponding quality control serum test also showed good performance. It is indicated that the blocking stabilizer and the enzyme standard dilution stabilizer in the anti-torch-IgG type antibody spectrum chip detection kit of the present invention are better than the general blocking solution and the enzyme standard dilution liquid, and can greatly improve the stability of the product.

Example 6. Comparison of Antigen Costs Between Anti-torch-IgG Type Antibody Profile Chip Kit and Fluorescent Immunochromatography Kit

Compare the antigen cost with the fluorescent immunochromatography kit disclosed in Chinese Patent Application No. 201510487338.9:

Calculation of the amount of antigen used in the fluorescent immunochromatographic kit:

Toxoplasma antigen coating as an example: CN201510487338.9 patent mentions the concentration of Toxoplasma antigen coating 0.5-0.8mg/ml, coated at 1μl/cm, each test strip is 0.4cm wide, so the average person is The amount of antigen used is about 0.2-0.32 μg.

The amount of antigen used in the kit of the present invention is calculated:

The invention uses BioDot precision spotting instrument for antigen coating, and the volume of coating at each point is only 10nL, so the amount of antigen used for coating each part is: Toxoplasma purifying antigen dosage: 50μg/ml×10nL ×10-6 = 0.005 μg.

It can be seen that the amount of Toxoplasma antigen used in the present invention is significantly lower than that of the fluorescent immunochromatography kit by two orders of magnitude. Other antigen usage levels are similar. In summary, the anti-torch-IgG antibody microarray kit of the present invention can effectively reduce the amount of related antigens, and reduce the sensitivity and specificity of the kit while reducing the associated antigen use cost.

Claims (13)

1. An anti-torch-IgG type antibody profile chip coated with a protein chip having an antigen of a pathogenic microorganism associated with TORCH detection.
2. The chip according to claim 1, wherein the pathogenic microorganism associated with the TORCH detection is Toxoplasma gondii (TOX), cytomegalovirus (CMV), rubella virus (RV), herpes simplex virus (HSV), human parvovirus B19, ke Saqi virus, at least one of Treponema pallidum, Hepatitis B virus (HBV), and Chlamydia.
3. The chip according to claim 1 or 2, wherein the protein chip further comprises a quality control point and/or a reference point; the quality control point comprises at least one positive property control point (PC) and at least one negative nature control point (NC) At least one sample control point (SC) and/or at least one enzyme control point (EC); the reference point comprising different concentration reference curve points (S1-S5) and/or at least one chip position reference point (Loc).
4. The chip according to claim 3, wherein said positive property control is coated with human IgG or coated with a BSA-DNP conjugate; said negative nature control point is coated with a trace concentration of human IgG or other lower than the reaction signal value a protein unrelated to TORCH for detecting pathogenic microorganisms; said sample control point is coated with anti-human IgG; said enzyme-labeled control point is coated with human IgG or anti-rabbit antibody; said reference curve points are coated with different concentrations Human IgG; the chip position reference point is coated with a known concentration of human IgG solution.
5. The method for preparing a chip according to any one of claims 1 to 4, wherein the antigen, the control point protein and/or the reference point protein of the related pathogenic microorganism are detected by diluting TORCH with a coating buffer, and are arranged in a dot array by a spotting device. The coating is on the chip substrate.
6. The preparation method according to claim 5, wherein the coating buffer is a buffer containing PEG, water-soluble cyclodextrin, trehalose, preservative and glycerin; the coating is 2-8 ° C, and the package is set. It is 16-24h; the chip substrate is an enzyme standard reaction plate, a glass plate, a chemical film, and a porous silica gel.
7. The preparation method according to claim 6, wherein the buffer solution is a CB buffer of pH 9.6, a Tris buffer of pH 8.5 or a PBS buffer of pH 7.4-7.6; the PEG is PEG4000; the PEG The content is 0.5% to 1%; the water-soluble cyclodextrin is 0.01% to 0.02% Captisol, 0.02% to 0.04% 2-hydroxy-β-cyclodextrin or carboxymethyl-β-cyclodextrin; The content of the trehalose is 5% to 8%; the preservative is Proclin 300; the content of the preservative is 0.05%; and the content of the glycerin is 15%.
8. The preparation method according to any one of claims 5 to 7, further comprising the step of blocking with a blocking stabilizer.
9. The preparation method according to claim 8, wherein the blocking stabilizer is 1% to 5% BSA, 2% to 6% trehalose, 0.8% to 1% PVA 20000, 0.2% to 0.4% betaine, 0.05%. NaN3 was pH7.4 in 0.01 M PBS.
10. A kit for TORCH detection, comprising the anti-torch-IgG type antibody profile chip according to any one of claims 1 to 4.
11. The kit according to claim 10, further comprising at least one of an enzyme label, an enzyme standard dilution stabilizer, a sample diluent, a washing solution, and a color developing solution.
12. The kit according to claim 11, wherein the enzyme marker is horseradish peroxidase-labeled anti-human IgG antibody (goat anti-human); the enzyme-labeled dilution stabilizer is 0.05 M citric acid, 2%- 5% goat serum, 2%-4% PEG10000, 0.2%-0.5% sodium p-hydroxybenzoate, 0.1%-0.2% gum arabic, 0.05% Proclin 3000, pH 7.4, 1 M Tris solution; sample dilution The solution was a 0.02 M Tris solution of pH 7.4 containing 0.15 M NaCl, 0.05% Tween 20, 0.01% casein; the washing solution was a 0.02 M Tris solution of pH 7.4 containing 0.15 M NaCl, 0.05% Tween 20; The liquid is a settling type TMB.
13. A TORCH detection method, wherein the sample to be tested is diluted with a sample diluent, and then added to the anti-torch-IgG type antibody spectrum chip according to any one of claims 1 to 4, and the enzyme label is added, and the color developing solution is added to protect the light from color. The fluorescence detecting device detects the result, and calculates an anti-torch-IgG type antibody signal value in the sample to be tested.