CN110514841B

CN110514841B - Kit and protein chip for latent tuberculosis infection diagnosis

Info

Publication number: CN110514841B
Application number: CN201910655757.7A
Authority: CN
Inventors: 黄若磐; 杨倩婷; 张明霞; 陈骑; 张惠华; 易玉华
Original assignee: Third Peoples Hospital of Shenzhen; Raybiotech Inc
Current assignee: Reboo (Guangzhou) Biotechnology Co.,Ltd.; Third Peoples Hospital of Shenzhen
Priority date: 2019-07-19
Filing date: 2019-07-19
Publication date: 2020-05-12
Anticipated expiration: 2039-07-19
Also published as: CN110514841A

Abstract

The invention provides a kit and a protein chip for diagnosing latent tuberculosis infection, which can distinguish latent tuberculosis infectors from healthy people by detecting the expression levels of four proteins, namely interleukin-6, tumor necrosis factor receptor II, epidermal regulatory element and interferon inducible protein 10, and have higher sensitivity and specificity. The specific antibodies corresponding to the four proteins are fixed on a substrate to prepare a protein chip, the expression levels of the four proteins can be detected simultaneously, and the method has the advantages of low sample demand, simplicity and quickness. The application in molecular biological research and clinical detection of latent tuberculosis infection is beneficial to early diagnosis of latent tuberculosis infection and can more accurately discover the latent tuberculosis infection.

Description

Kit and protein chip for latent tuberculosis infection diagnosis

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a reagent kit and a protein chip for diagnosing latent tuberculosis infection.

Background

Tuberculosis is an infectious disease with the largest number of deaths caused by a single pathogen after AIDS, and one of the main reasons for the serious tuberculosis epidemic is the lack of an effective early diagnosis technology.

Not all people develop tuberculosis after infection, and a part of people can be classified as latent infection, namely tuberculosis latent infection. The tubercle bacillus exists continuously in the human body of the part of people, but the tubercle bacillus is controlled by the immunity of the organism and has no clinical manifestation. When the immunity of the organism is reduced, the tubercle bacillus latent in the organism can be burned again, and can be reproduced again in the organism, and finally, the tubercle bacillus can be changed into a tuberculosis patient. There is a 10% chance that a latently infected person will transform into a tuberculosis patient throughout life. About one fourth of the population in China is infected with tuberculosis bacteria, and if observation or intervention can be carried out on the part of population, the occurrence of tuberculosis can be greatly reduced. However, the current tuberculosis infection lacks a specific diagnosis method, and clinically, antibodies and cell immunity detection are available, and the cell immunity detection comprises PPD skin test and IGRA technology. The PPD skin test is simple, but has cross reaction with BCG, the false positive rate is higher, and the use efficiency is obviously limited particularly under the condition of wide inoculation of BCG (BCG) in China. The IGRA technology detects the expression of tuberculosis specific IFNg, although the specificity is obviously improved, the method is relatively more complex and time-consuming at present, the price is relatively more expensive, and the clinical popularization and use are seriously limited. Therefore, there is a need for a highly sensitive and specific method and product for diagnosing tuberculosis latently infected patients.

Disclosure of Invention

Based on this, the present invention aims to provide a kit for diagnosing a person with latent tuberculosis infection, which has high sensitivity and high specificity.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

the application of the reagent for detecting interleukin-6, tumor necrosis factor receptor II, epidermal regulatory factor and interferon inducible protein 10 in the preparation of the kit for diagnosing latent tuberculosis infection.

A reagent kit for diagnosing latent tuberculosis infection contains the reagent for quantitatively detecting interleukin-6, tumor necrosis factor receptor II, epidermal regulatory element and interferon inducible protein 10.

The invention also provides a protein chip for diagnosing latent tuberculosis infection, which has the following specific technical scheme:

a protein chip for diagnosing latent tuberculosis infection comprises: a substrate and a capture antibody immobilized on the substrate;

the capture antibody comprises: specific antibodies against interleukin-6, specific antibodies against tumor necrosis factor receptor II, specific antibodies against epidermal regulatory element, and specific antibodies against interferon inducible protein 10;

the substrate is coated with active epoxy groups.

The invention also provides a preparation method of the protein chip for diagnosing the latent tuberculosis infection, and the specific technical scheme is as follows:

a preparation method of a protein chip for diagnosing latent tuberculosis infection comprises the following steps:

coating active epoxy groups on a substrate of the protein chip;

immobilizing a capture antibody on a substrate of the protein chip;

the capture antibody comprises: specific antibodies against interleukin-6, specific antibodies against tumor necrosis factor receptor II, specific antibodies against epidermal regulatory element, and specific antibodies against interferon inducible protein 10.

Based on the technical scheme, the invention has the following beneficial effects:

the inventor finds that the expression levels of four proteins, namely interleukin-6, tumor necrosis factor receptor II, epidermal regulatory element and interferon inducible protein 10, can be detected to distinguish tuberculosis latent infected people from healthy people, and the sensitivity and the specificity are high. The specific antibodies corresponding to the four proteins are fixed on a substrate to prepare a protein chip, the expression levels of the four proteins can be detected simultaneously, and the method has the advantages of low sample demand, simplicity and quickness. The application in molecular biological research and clinical detection of the latent tuberculosis infection is beneficial to early diagnosis of the latent tuberculosis infection and can more accurately discover the latent tuberculosis infection.

Drawings

FIG. 1 is a schematic diagram showing the distribution of spotting domains of a protein chip.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

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. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention relates to a protein chip for diagnosing latent tuberculosis infection, which comprises the following components: a substrate and a capture antibody immobilized on the substrate;

Optionally, the protein chip further comprises a positive control. Preferably, the positive control is calf IgG immobilized on the substrate.

Preferably, the positive control may be different concentrations of calf IgG. More preferably, the calf IgG is biotin-labeled calf IgG.

Preferably, the substrate is a substrate coated with active epoxy groups. The glass slide coated by the active epoxy group can adsorb the coated antibody on the surface of the chip more effectively, and the stability of the antibody is increased.

Preferably, the substrate comprises several non-interfering sample application areas.

More preferably, the several non-interfering sample application areas are separated by a removable frame. The specific frame structure is adopted before sample spotting, the chip lattice is optimized, so that samples can be detected in batches, the operation is simple and convenient, the sample consumption is small, and no cross contamination exists.

Further preferably, the substrate comprises 16-32 non-interfering sample application cells.

As shown in fig. 1, each spotting cell was spotted with a specific antibody against interleukin-6, a specific antibody against tumor necrosis factor receptor II, a specific antibody against epidermal regulatory element and a specific antibody against interferon-induced protein 10 and two positive control bovine IgG at different concentrations.

Specifically, each specific antibody was spotted 4 times in duplicate, as were positive control bovine IgG of two different concentrations.

In other embodiments, the number of repeated spotting can be reasonably set according to needs, for example, 3-10 repeated spotting are performed.

The invention relates to a preparation method of a protein chip for diagnosing latent tuberculosis infection, which comprises the following steps:

immobilizing the capture antibody on a substrate of the protein chip;

Preferably, the immobilization of the capture antibody on the substrate of the protein chip comprises the steps of:

spotting the solution of 100-1000pl capture antibody on the substrate, standing overnight at room temperature, and vacuum-drying for 1-4 hours;

the solution of the capture antibody contains 0.02-20ng of capture antibody and 0.01-10g/100ml of bovine albumin.

Among these, overnight standing at room temperature was beneficial for more efficient immobilization of the antibody on the slide.

Preferably, the vacuum drying time is 1.5-2.5 hours.

More preferably, the spotting conditions are: the temperature is 70-75F, and the humidity is 40-45%. Under the condition of the sample spotting condition, the spotting form can be improved, and the distribution of the antibody on the glass slide is more uniform.

Further, vacuum-pumping for drying, pumping for sealing with water vapor-resistant plastic pouch, and storing at 0-8 deg.C. Preferably, it is stored at (4. + -. 0.5) ℃ C. The chip preserved by the method enables the antibody to be more effectively fixed on the surface of the chip and effectively increases the preservation stability of the chip.

Preferably, the protein chip substrate comprises a plurality of non-interfering sample application regions separated by a well-detachable frame.

Preferably, the preparation method further comprises: a positive control capture antibody, which is calf IgG, is immobilized on the substrate of the protein chip.

Preferably, the present invention employs double positive controls of different concentrations. Meanwhile, different microarrays are standardized by using signals with different intensities of the two microarrays, so that the sensitivity and the repeatability of the chip can be obviously improved.

More preferably, the method for normalizing a microarray using the double positive control is:

calculating the positive control signal value POS of each microarray as (POSl +4 XPOS 2)/2; wherein POS1 is the signal value for the positive control at the first concentration and POS2 is the signal value for the positive control at the second concentration;

all data were then normalized with the positive control signal value POS: corrected value is original value (sample average signal value POS)_ave) Positive control signal value POS.

The invention relates to a protein chip kit for diagnosing latent tuberculosis infection, which comprises the following components:

the protein chip as described above or the protein chip prepared by the preparation method as described above, and a detection antibody;

the detection antibody comprises: specific antibodies against interleukin-6, specific antibodies against tumor necrosis factor receptor II, specific antibodies against epidermal regulatory element, and specific antibodies against interferon inducible protein 10;

wherein the detection antibody and the capture antibody for the same protein recognize different epitopes of the protein.

Preferably, the detection antibody is labeled with biotin, HRP, or colloidal gold.

Preferably, the kit further comprises: protein standards and/or fluorescein Cy3 labeled streptavidin. More preferably, the protein standard is a mixture of interleukin-6, tumor necrosis factor receptor II, epidermal regulatory element, and interferon inducible protein 10.

In the specific embodiment of the invention, the full-automatic sample dropping instrument is a product produced by Germany Scienion company; the substrate of the protein chip adopts a standard tissue slide, and the slide is a product of the United states Corning company. In other embodiments, other possible deposition instruments and substrates may be used.

Example 1 binding of proteins associated with latent infection

Sex Hormone Binding Globulin (SHBG), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor receptor II (TNF RII), epidermal regulatory element (Epiregulin), interferon inducible protein 10(IP-10), serine protease inhibitor (SERPINA3) and tumor necrosis factor a (TNFa) protein standard are respectively taken, and after gradient dilution, fluorescence values are measured by a fluorescence method in the prior art to obtain fluorescence signal values and corresponding concentrations of a standard curve, wherein the fluorescence signal values and the corresponding concentrations are shown in Table 1.

TABLE 1 Standard Curve fluorescence Signal values and corresponding concentrations of protein standards

From the data in the table above, the concentration and the fluorescence signal intensity are respectively subjected to logarithmic transformation, a standard curve is drawn, and a linear regression equation is calculated, wherein x is Log (standard concentration) and Y is Log (fluorescence signal intensity), and the linear regression equation is specifically shown in table 2.

TABLE 2 protein standards Standard Curve

	Regression equation	R²
			SHBG	y＝41.91x-8946.5	0.97
IL2	y＝1.4909x-1135.8	0.95
			IL-6	y＝0.9522x+2.1817	0.97
IL8	y＝96.374x-2357	0.99
			TNF RII	y＝0.883x+1.8571	0.98
Epiregulin	y＝0.7995x-0.5405	0.99
			IP-10	y＝0.7323x+3.257	0.93
SERPINA3	y＝1.2738x-5829.2	0.95
			TNFa	y＝3.3867x-135.1	0.98

Peripheral blood serum of clinically confirmed healthy patients and latent tuberculosis infection patients is collected respectively, wherein 63 healthy patients (HC) and 21 latent tuberculosis patients (LTBI) are obtained, and the content of SHBG, IL-2, IL-6, IL-8, TNF RII, Epiregulin, IP-10, SERPINA3 and TNFa in the serum is respectively determined by a fluorescence method in the prior art.

And the resulting concentrations were calculated according to the above linear regression equation, as shown in table 3.

TABLE 3

The average values of SHBG, IL-2, IL-6, IL-8, TNF RII, Epiregulin, IP-10, SERPINA3, TNFa expression levels were calculated for healthy subjects and latent tuberculosis patients, respectively, and the p-value was calculated, as shown in Table 4.

TABLE 4 average values of the expression levels of respective proteins in healthy subjects and latent tuberculosis patients

It can be seen that the SHBG, IL2, IL-6, TNF RII, Epiregulin, IP-10, SERPINA3, TNFa expression levels of healthy and latent tuberculosis patients are biologically different.

The results of each set of antibody pairing assays for detecting SHBG, IL2, IL-6, IL8, TNF RII, Epiregulin, IP-10, SERPINA3 and TNFa cytokines were as follows:

TABLE 5

As shown in Table 5, the SHBG-coated antibody cross-reacted with TNF RII and the IP-10 antibody; TNFa has cross reaction with SERPINA3 and Epiregulin, IL8 antibody has low correlation with binding latent infection, and antibody pairs cannot be successfully paired, so that the TNFa and the SERPINA3 and Epiregulin are not suitable for combined application detection. In conclusion, four groups of antibody pairs, namely interleukin-6 (IL-6), tumor necrosis factor receptor II (TNF RII), epidermal regulatory element (Epiregulin) and interferon inducible protein 10(IP-10), are finally selected to form a chip for detection.

EXAMPLE 2 preparation of protein chip kit for diagnosis of latent tuberculosis infection

In order to detect the presence or absence of 4 proteins of the present invention in a sample, a slide glass immobilized with specific antibodies against the following 4 proteins was prepared: interleukin-6 (IL-6), tumor necrosis factor receptor II (TNF RII), epidermal regulatory element (Epiregulin), and interferon inducible protein 10 (IP-10).

1. Sources of antibodies

The use, source and concentration of capture and detection antibodies corresponding to specific proteins for interleukin-6 (IL-6), tumor necrosis factor receptor II (TNF RII), epidermal regulin (Epiregulin) and Interferon inducible protein 10(IP-10) are detailed in Table 6. Wherein the capture antibody and the detection antibody recognize different epitopes of the protein, respectively.

The specific method for labeling the detection antibody with biotin comprises the following steps: the detection antibody to be labeled is dialyzed three times for at least 6 hours each in a large amount of 1 XPBS buffer (1 ml of antibody in 1 liter of PBS). After the antibody concentration was measured, 80. mu.g of a biotin DMSO solution per mg of the antibody was added, mixed, and reacted at room temperature for 4 hours. The biotin-labeled antibody was dialyzed against PBS solution to remove free biotin and to calibrate the concentration of the biotin-labeled detection antibody.

TABLE 6 name of specific antibody, information on use, origin and concentration of antibody

2. Preparation and preservation of protein chips

(1) Determination of spotting concentration of capture antibody: the capture antibody was diluted at 2 × concentration in different buffers (sterile water, PBS buffer, PBS containing bovine albumin at different concentrations, PBS buffer containing glycerol at different concentrations, etc.), spotted on the chip surface using a non-contact spotting instrument, and then compared for its activity and stability using a sandwich ELISA method. Experiments show that the capture antibody diluted by PBS buffer containing 0.01-10g/100ml of bovine albumin has the best linearity rate and stability.

(2) And the condition control of sample application: when the capture antibody is directly contacted on the surface of the slide glass at room temperature, hollow spots are easily generated at the capture antibody points, the activity difference of different antibody points is large, and a tailing phenomenon is often found in a finally generated image. The present invention controls the spotting temperature to 70-75F and the antibody spot spotted at 40-45% humidity to have the best shape, and finds that the spotted slide is left to stand overnight at room temperature, dried by vacuum pumping for 2 hours the next day, and then the whole chip is packaged with an airtight pouch, and the slide capture antibody prepared by this method has the best activity and can be stable at 4 ℃ for at least six months.

(3) And sample application: 100-1000pl PBS buffer (containing 0.02-2ng of specific antibody) was spotted on the slide using a full-automatic spotting instrument using the spotting conditions described above. Biotin-labeled bovine IgG served as a positive control. Each antibody had 4 replicates per chip and two positive controls at different concentrations had 4 replicates per chip. The spotted slides were allowed to stand overnight at room temperature and then air dried in a desiccator for 2 hours. The dried slides were mounted with a matched 16-well frame to divide a slide into 16 non-interfering cells. After the frame is closed with an adhesive film, the entire chip is sealed with an airtight pouch and stored at 2 ℃ to 8 ℃ for future use.

3. Sources of protein standards tested:

the names and sources used for the recombinant proteins listed in Table 7 are detailed in Table 2:

TABLE 7 name, Source of recombinant proteins in protein standards

Recombinant protein name	Origin of origin	Goods number
			Interleukin-6 (IL-6)	Raybiotech	230-00011
Tumor necrosis factor receptor II (TNF RII)	Raybiotech	228-20264
			Epidermal regulin (Epiregulin)	Raybiotech	230-10130
Interferon inducible protein 10(IP-10)	Raybiotech	230-00211

Diluting the above recombinant proteins with phosphate buffer containing 0.1% calf albumin, mixing at a certain amount, packaging, lyophilizing, and storing at-80 deg.C.

Example 3 expression level of 4 proteins in serum quantitatively determined by protein chip

1. Collection of peripheral blood supernatant

1.1 heparin anticoagulated venous blood 250ml, centrifugating at 2000 rpm for 5 minutes, and collecting supernatant for use.

2. Complete drying of slide chips

Taking out the slide chip from the box, balancing at room temperature for 20-30min, opening the packaging bag, uncovering the sealing strip, and then placing the chip in a vacuum drier or drying at room temperature for 1-2 hours.

3. Gradient dilution of protein standards

3.1 Add 500. mu.L of sample dilution (pH 7.2, containing 5% by mass of dextrose anhydride, 1% by mass of glycine, 5mM Tris buffer) to the vial of the standard mix and redissolve the standard. Before opening the tubule, it is rapidly centrifuged and gently pipetted up and down to dissolve the powder, marking the tubule as Std 1.

3.2 label 6 clean centrifuge tubes as Std2, Std3 to Std7, respectively, and add 200. mu.L of sample diluent to each vial.

3.3 draw 100. mu.L of Std1 to Std2 and mix gently, then draw 100. mu.L from Std2 to Std3, and so dilute to Std 7.

3.4 draw 100. mu.L of sample dilution into another new centrifuge tube, labeled CNTRL, as a negative control.

Note: since the initial concentration of each protein was different, the concentration of each protein series was different after gradient dilution from Std1 to Std 7. In this example, the concentrations of the gradient recombinant protein dilutions are shown in table 8.

Table 8 concentrations of recombinant protein standards used to prepare standard curves after dilution with gradients are shown below

Name of protein	Control	Std1	Std2	Std3	Std4	Std5	Std6	Std7	Unit of
										IL-6	0	2000	667	222	74	25	8	3	Pg/mL
TNF RII	0	40000	13333	4444	1481	494	165	55	Pg/mL
										Epiregulin	0	400000	133333	44444	14815	4938	1646	549	Pg/mL
IP-10	0	10000	3333	1111	370	123	41	14	Pg/mL

4. Chip operation process

4.1 Add 100. mu.L of sample dilution to each well, incubate for 30min on a shaker at room temperature, and block the quantitative protein chip.

4.2 draw out the sample dilution from each well, add 100. mu.L of standard and sample to the wells, and incubate overnight on a shaker at 4 ℃. The sample is serum naturally precipitated after venous blood collection, and is diluted by a diluent 1:1 before use.

4.3 cleaning

And (3) drawing out the standard substance or sample in each hole, washing with 1 × washing solution I for 5 times, shaking with a shaking table at room temperature for 5min each time, drawing out 150 μ L of 1 × washing solution I in each hole, and diluting with deionized water to obtain 20 × washing solution I.

And (3) pumping out the 1 Xwashing liquid I in each hole, adding the 1 Xwashing liquid II to wash for 2 times, shaking the 1 Xwashing liquid II in a shaking table at room temperature for 5min every time, pumping out the washing liquid II in 150 mu L of each hole, and diluting the 20 Xwashing liquid II with deionized water.

4.4 incubation of detection antibody mixtures

And centrifuging the detection antibody mixed solution small tube, adding 1.4ml of sample diluent, uniformly mixing, and quickly centrifuging again. Add 80. mu.L of detection antibody to each well and incubate for 2 hours on a shaker at room temperature.

4.5 cleaning

And (3) extracting the detection antibody in each hole, washing with 1 Xwashing liquid I for 5 times, shaking in a shaking table at room temperature for 5min every time, extracting clean washing liquid from 150 mu L of 1 Xwashing liquid I in each hole, then adding 1 Xwashing liquid II for washing for 2 times, shaking in a shaking table at room temperature for 5min every time, extracting clean washing liquid from 150 mu L of 1 Xwashing liquid II in each hole, and washing for each time.

4.6 incubation of Cy 3-streptavidin

The Cy 3-streptavidin vial was centrifuged, then 1.4ml of sample diluent was added, mixed well and then rapidly centrifuged again. Add 80. mu.L of Cy 3-streptavidin to each well, wrap the slide with aluminum foil and incubate in the dark for 1 hour on a shaker at room temperature.

4.7 cleaning

Cy 3-streptavidin was removed from each well and the 1 XWash I was washed 5 times, 5min at room temperature with shaking, 150. mu.L of 1 XWash I per well, and the wash was removed for each wash.

4.8 fluorescence detection

I) The slide frame was removed, taking care not to touch the antibody-printed side of the slide by hand.

2) The slide was placed in a slide wash tube, and about 30ml of 1 XWash I was added to cover the slide entirely, shaking for 15min on a room temperature shaker, discarding 1 XWash I, adding about 30ml of 1 XWash II, and shaking for 5min on a room temperature shaker.

3) The residual wash solution of the slide was removed. The slides were placed in slide wash/dry tubes without lid and centrifuged at 1000rpm for 3 min.

4) The signal is scanned using a laser scanner, such as an Innopsys scanner, using either Cy3 or a green channel (excitation frequency of 532 nm).

4.9 data extraction of the chip and data analysis with analysis software.

1) The fluorescence values of the biochip were read with Mapix software. The microarray parameters of the chip were 3 (rows) × 8 (columns), and the spot diameters were 120 μm.

2) The value selected after reading is the Median reading (F532 media-LocalBack) with the local background removed. The specific quantitative chip calculation software QAH-CUST-SW was used to generate a standard curve for each recombinant protein.

The data were normalized using the same two positive control spots on each chip as a reference frame before calculating the concentration of different proteins in different sample sera. The signal values of the two positive controls differ by a factor of approximately 4.

The normalization process steps are as follows:

all data were then normalized with the positive control signal value POS: correction value (original value x sample average signal value POS)_ave) Positive control signal value POS. Mean signal value of sample POS_aveIs the average signal value of all samples in the microarray.

Example 4 Experimental data processing for quantitative determination of 4 proteins in the supernatant of blood in example 3

1. The concentration of 4 proteins in each sample was calculated from the standard curve

Taking the results of one clinical serum sample experiment as an example, the fluorescence readings of the eight standard samples are shown in table 9.

TABLE 9 standard photopeak readings for three-fold gradient dilution (F532 media-Local Background)

Taking Epiregulin as an example, the relationship between the protein concentration and the fluorescence signal of Epiregulin is known from the information in tables 8 and 9 and is shown in the following table.

TABLE 10 relationship between protein concentration and fluorescence signal of Epiregulin

The standard curve is plotted in Table 10, and r is calculated²0.8589. The regression equation is that y is 0.3176x +12020

The sample No. 1 (1-1) was tested, the signal intensity was 4255, and the Epiregulin content was calculated to be 13371.388 pg/ml.

2. Determination of detection result

And (3) detecting the concentration of the protein in each sample, processing data according to the method, obtaining the detection concentration of 4 proteins in each sample, establishing a machine learning model Support Vector Machine (SVM) through an R language algorithm package kernalab, and judging the type of the sample.

And judging the type of the sample. The judgment standard is as follows: when R is greater than 0.23, the tuberculosis infection is judged. Otherwise, it is a healthy control.

Example 5 resolution of the sensitivity and specificity of latent infection with tuberculosis

In order to test the efficacy of the kit in distinguishing latent tuberculosis infection patients, different clinical samples are detected by adopting the kit prepared in the embodiment 2 of the invention, and the sensitivity and specificity of the kit in distinguishing latent tuberculosis infection are judged.

Clinically confirmed healthy control HC (63 cases) and latent tuberculosis infected patients LTBI (21 cases) were collected separately, peripheral blood serum was collected separately, and expression levels of 4 proteins were measured using the kits, respectively, and the measurement method and data processing method were as described in example 3 to example 4. And substituting the result into the SVM model, calculating the result and the judgment result of each sample, and diagnosing LTBI when the result value is more than 0.23. The result shows that 57 of 63 HC detected by the kit are judged to be HC, and the true positive rate is 90%, which indicates that the protein chip detection method discovered by the invention has good sensitivity; in 21 cases of the detected LTBI, 20 cases are judged as LTBI, and the false positive rate is 5.24%, which shows that the protein chip detection method disclosed by the invention has good specificity.

TABLE 11 results of different types of clinical specimens

In conclusion, the invention discloses a protein chip kit for diagnosing latent tuberculosis infection. The kit uses a standard tissue slide as a surface carrier, and can complete the reaction of multiple sandwich ELISA on the surface of the slide. The developed protein chip diagnostic kit for latent tuberculosis infection can detect 4 proteins on one protein chip simultaneously. The sensitivity and specificity of the kit for detecting 4 proteins can reach the level of single-factor ELISA. And 4 proteins are combined to develop a protein chip diagnostic kit which has high sensitivity and good specificity and can be used for diagnosing latent tuberculosis infectors. The kit is suitable for detecting the expression of a plurality of specific proteins from human peripheral blood, thereby being used for early diagnosis of tuberculosis latent infection and screening of healthy people.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the technical features should be considered as the scope of the present description.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The application of the reagent for detecting interleukin-6, tumor necrosis factor receptor II, epidermal regulatory element and interferon inducible protein 10 in the preparation of the kit for diagnosing latent tuberculosis infection.

2. The use according to claim 1, wherein the kit comprises:

can quantitatively detect interleukin-6, tumor necrosis factor receptor II, epidermal regulatory element and interferon inducible protein 10.

3. The use according to claim 2, wherein the kit comprises:

specific antibodies against interleukin-6, specific antibodies against tumor necrosis factor receptor II, specific antibodies against epidermal regulatory element, and specific antibodies against interferon inducible protein 10.

4. The use according to claim 3, wherein the kit comprises:

capture antibodies against interleukin-6, tumor necrosis factor receptor II, epidermal regulatory elements, interferon inducible protein 10;

a detection antibody against interleukin-6, a detection antibody against tumor necrosis factor receptor II, a detection antibody against epidermal regulatory element, and a detection antibody against interferon inducible protein 10;

5. The use according to any one of claims 1 to 4, wherein the kit further comprises:

protein standards and/or fluorescein Cy3 labeled streptavidin;

the protein standard substance is a mixture of interleukin-6, tumor necrosis factor receptor II, epidermal regulatory element and interferon inducible protein 10.

6. The use of claim 1, wherein the kit is a protein chip kit comprising:

a substrate and a capture antibody immobilized on the substrate;

7. Use according to claim 6, wherein the substrate is a substrate coated with active epoxy groups.

8. The use of claim 6, wherein the protein chip further comprises a positive control; the positive control is calf IgG immobilized on the substrate.

9. The use according to any one of claims 6 to 8, wherein the preparation method of the protein chip comprises the following steps:

coating active epoxy groups on a substrate of the protein chip;

immobilizing a capture antibody on a substrate of the protein chip;

10. The use of claim 9, wherein the immobilization of the capture antibody on the substrate of the protein chip comprises the steps of:

the solution of the capture antibody contains 0.02-20ng of the capture antibody and 0.01-10g/100ml of bovine albumin.