CN111537710B - Marker combination, antibody chip and kit for detecting hand-foot-and-mouth disease - Google Patents

Abstract

The invention provides a marker combination, an antibody chip and a kit for detecting hand-foot-and-mouth disease, the marker combination for detecting hand-foot-and-mouth disease comprises at least one of the following markers: OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1, pentraxin 3. The marker combination can reliably reflect the hand-foot-and-mouth disease state of an organism, and has higher sensitivity, specificity and accuracy for detecting the hand-foot-and-mouth disease. The marker combination is used for detecting the hand-foot-and-mouth disease, only dozens to hundreds of microliters of peripheral blood is needed, samples can be easily collected through venous blood collection or simpler peripheral blood collection, and the pain and the fund cost of the children patients are greatly reduced.

Description

Marker combination, antibody chip and kit for detecting hand-foot-and-mouth disease

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a marker combination, an antibody chip and a kit for detecting hand-foot-and-mouth disease.

Background

Hand-foot-and-mouth disease (HFMD) is a common child infectious disease caused by enteroviruses, also known as eruptive vesicular stomatitis, which mostly occurs in infants and children 3 years old and below, and can cause herpes or skin rash in the hands, feet, oral cavity and other parts, and a few children can cause complications such as myocarditis, pulmonary edema, aseptic meningitis and the like. If the disease of an individual critically ill child progresses rapidly, the child is easy to die. The disease has the main clinical symptoms of herpes of hand, foot and oral mucosa or ulcer after ulceration. The hand-foot-and-mouth disease is an infectious disease caused by enteroviruses, has strong infectivity, complex transmission route and quick transmission speed, and can cause pandemics in a short time. There are more than 20 enteroviruses causing hand-foot-and-mouth disease, among which coxsackievirus type a16 (Cox a 16) and enterovirus type 71 (EV 71) are the most common.

For the virus type of HFMD, the currently common diagnostic methods are serological neutralization test, pathogenic cell inoculation, molecular biological reverse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR (rRT-PCR), etc. These methods play a corresponding important role in different fields, but the corresponding problems are also revealed: these methods are complicated, have large requirements on corresponding reagents and clinical samples, have complicated sample components such as pharyngeal test cells and anal test cells, low viral load, high detection cost, easy generation of cross reaction, and need of professional operation, otherwise, the accuracy of the final result is difficult to ensure.

Disclosure of Invention

The invention aims to provide a marker combination, a detection chip and a kit for detecting hand-foot-and-mouth disease, so as to realize high-sensitivity and high-specificity detection for the hand-foot-and-mouth disease.

According to one aspect of the present invention, there is provided a marker combination for detecting hand-foot-and-mouth disease, comprising at least one of the following markers: OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1, pentraxin 3.

Preferably, the following nine markers are included simultaneously: OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1, pentraxin 3.

According to another aspect of the present invention, there is provided an antibody chip for detecting hand-foot-and-mouth disease: the kit comprises a solid phase carrier and a capture antibody coated on the surface of the solid phase carrier, wherein the capture antibody is a specific antibody aiming at the marker combination for detecting the hand-foot-and-mouth disease.

Preferably, the solid support is a glass slide.

Preferably, the solid phase carrier is a glass slide coated with amino groups.

According to another aspect of the present invention, there is provided a kit for detecting hand-foot-and-mouth disease: comprises a capture antibody, wherein the capture antibody is a specific antibody aiming at the marker combination for detecting the hand-foot-and-mouth disease.

Preferably, a solid phase carrier is included, and the capture antibody is coated on the surface of the solid phase carrier.

Preferably, the following components are also included: an antibody mixture containing a biotin-labeled detection antibody as a specific antibody against the marker combination for detecting hand-foot-and-mouth disease; streptavidin: the streptavidin is labeled with a fluorescent dye, which is Cy3 or a fluorescent dye with a similar absorption wavelength.

Preferably, further comprising a marker standard comprising a marker combination for detecting hand-foot-and-mouth disease according to claim 1 or 2.

The marker combination for detecting the hand-foot-and-mouth disease provided by the invention can reliably reflect the hand-foot-and-mouth disease state of an organism, and has higher sensitivity, specificity and accuracy for detecting the hand-foot-and-mouth disease. The marker combination is used for detecting the hand-foot-and-mouth disease, only dozens to hundreds of microliters of peripheral blood is needed, samples can be easily collected through venous blood collection or simpler peripheral blood collection, and the pain and the fund cost of the children patients are greatly reduced. The high-throughput antibody chip and the kit prepared by the marker combination are used for judging the hand-foot-and-mouth disease of children, so that healthy children, children with the hand-foot-and-mouth disease and children with the hand-foot-and-mouth disease with the severe hand-foot-and-mouth disease can be rapidly distinguished, reliable data support is provided for prevention and treatment of the hand-foot-and-mouth disease, early discovery and early diagnosis of the hand-foot-and-mouth disease are promoted, and the clinical treatment effect of patients with the hand-foot-and-mouth disease is favorably improved.

Drawings

FIG. 1 is a flowchart of screening for markers for hand-foot-and-mouth disease in example 1;

FIG. 2 is a ROC plot for nine marker factors, respectively, wherein (a) is a ROC plot for OPN, (b) is a ROC plot for APRIL, (c) is a ROC plot for TRAIL R1, (d) is a ROC plot for MIP-3b, (e) is a ROC plot for TWEAK, (f) is a ROC plot for BLC, (g) is a ROC plot for ICOS, (h) is a ROC plot for ICAM-1, and (i) is a ROC plot for Pentraxin 3;

FIG. 3 is a ROC plot of nine marker factors obtained based on NNET;

FIG. 4 is a ROC plot of nine marker factors obtained based on SVM;

fig. 5 is a graph of ROC curves for nine marker factors obtained based on LDA.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.

Example 1 screening of markers for hand-foot-and-mouth disease

Fig. 1 shows a general flow involved in the screening process of the present embodiment, and the specific process is as follows.

S1, sample collection

Firstly, selecting healthy children, mild hand-foot-mouth disease and severe children as research objects, selecting 30 volunteers of the healthy children, the mild hand-foot-mouth disease and the severe children through comprehensive investigation of individual indexes (such as age, sex and the like) and clinical indexes (such as hand-foot-mouth disease staging), and collecting serum of the volunteers.

S2, quantitative detection of biological factors

The antibody adsorbed on the surface of the substrate, the biological factor to be detected and a detection antibody marked by biotin form an antibody-protein-antibody sandwich (sandwich ELISA technology), streptavidin-marked fluorescein is added, and scanning imaging is performed under laser excitation, so that 440 biological factors in a serum sample are accurately and quantitatively detected, wherein the biological factors comprise cytokines, angiogenesis factors, inflammatory factors, growth factors, chemotactic factors, apoptosis factors, receptor cytokines, hormones and the like.

S3, data arrangement and analysis

The data obtained from the protein chip is first normalized by an internal reference, which is a set of protein mixtures of known concentrations. And obtaining a standard substance curve of the target factor by adopting the internal reference standard substance, and determining the protein concentration according to the standard substance curve. The data are compared among different experimental groups and p values are calculated, indexes with p values less than 0.05 and obvious differences are selected for cluster analysis, so that biomarker groups which can distinguish different experimental groups are searched, and a differential expression protein spectrum is obtained. And carrying out receiver operating characteristic curve (ROC) analysis and model establishment on the obtained biomarker group to find valuable biomarkers for distinguishing the hand-foot-mouth disease states.

The combination of markers obtained from the screening was compared with the results of clinical tests, literature reported markers, including differences in biomarker expression levels and pathological tests to further correct the biomarker combinations.

And (4) screening results: nine markers obtained by screening are valuable for evaluating the state of the hand-foot-and-mouth disease, namely Osteopontin (OPN), proliferation-inducing ligand (APRIL), tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL R1), macrophage inflammatory protein 3 beta (MIP-3B), tumor necrosis factor-like apoptosis weak inducing factor (TWEAK), human B-lymphocyte chemotactic factor (BLC), inducible costimulatory molecule (ICOS), intercellular adhesion molecule-1 (ICAM-1) and inflammatory factor Pentraxin 3 (Pentraxin 3).

Example 2 modeling of marker combinations

Modeling mode

5-fold cross validation (5-fold cross validation) was used and the classification model was established 5 times (5-time repeat): and randomly and averagely dividing the sample N into a training group k-1 and a testing group 1 in each round, repeating for 5 times to obtain 5 models, and taking the average of the classification accuracy of the final testing group of the 5 models as the performance index of the classified sample. The advantage of this screening method is that almost all samples in each round are used for training the model, and therefore the distribution of the closest original samples, so the results obtained by evaluation are reliable; no random factors influence the experimental data in the experimental process, and the experimental process is ensured to be reproducible. The model is derived from the training set and verified using the test set. And (3) respectively evaluating the sensitivity, specificity and accuracy of the selected factors by using 3 groups of models including neural network analysis (NNET), linear Discriminant Analysis (LDA) and Support Vector Machine (SVM) for the biomarkers obtained by screening.

Establishing a sample group: selecting a plurality of healthy children, children with moderate hand-foot-and-mouth disease and children with severe hand-foot-and-mouth disease volunteers, and respectively establishing a training group and a testing group. Collecting the serum of a volunteer; the training group comprises 31 healthy children, 30 children with moderate hand-foot-and-mouth disease and 19 children with severe hand-foot-and-mouth disease; the test group included 28 healthy children, 30 infants with moderate hand-foot-and-mouth disease and 20 infants with severe hand-foot-and-mouth disease.

(II) modeling results

The method is characterized in that nine marker factors, namely OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1 and Pentraxin 3, are respectively used as single markers for statistical modeling, the operating characteristic curve (ROC curve) of a subject of a training set corresponding to the nine marker factors is shown in figure 2, the AUC values corresponding to the nine marker factors are all larger than 0.8, and the fact that the model established by respectively using the nine marker factors as the single markers has high accuracy in judgment of the hand-foot-mouth disease is shown. Referring to fig. 3-5, the AUC values corresponding to the marker combination composed of the above 9 marker factors are all greater than the AUC values corresponding to the 9 marker factors as single markers respectively and close to 1, which indicates that the model established by the marker combination composed of the above 9 marker factors has a high accuracy for the hand-foot-and-mouth disease determination.

Judging the detection result: and (3) detecting the concentration of the markers in each sample, processing data according to the method, obtaining the detection concentration of 9 markers in each sample, wrapping caret through an R language algorithm, respectively establishing a machine learning model Neural Network (NNET), a Support Vector Machine (SVM) and a Linear Discriminant Analysis (LDA), and judging the type of the sample. Judging the type of the sample, wherein the judgment standard is as follows: (1) NNET model: when p is greater than 0.82, the hand-foot-and-mouth disease is judged; otherwise, it is a healthy control. (2) SVM model: when p is greater than 0.7, the hand-foot-and-mouth disease is judged; otherwise, it is a healthy control. (3) LDA model: when p is greater than 0.088, judging the hand-foot-and-mouth disease; otherwise, it is a healthy control.

The NNET-based decision results (fig. 3) show that the training and test groups have 98% and 77% accuracy, respectively. In 31 healthy controls in the training group, 31 controls are judged as healthy controls, and the true negative rate is 100%; in 28 healthy controls in the test group, 20 healthy controls are judged, the true negative rate is 71%, and the judgment of NNET by the marker combination has good specificity. In 49 children patients with hand-foot-and-mouth diseases in the training group, 47 children patients with hand-foot-and-mouth diseases are judged, 2 children patients are judged as healthy controls, and the true positive rate is 96%; in the test group, 40 of 50 children with hand-foot-and-mouth disease were judged as children with hand-foot-and-mouth disease, 10 were judged as healthy controls, and the true positive rate was 80%. Hand-foot-and-mouth disease condition evaluation is carried out on the test volunteers based on NNET modeling, and specific conditions are shown in Table 1. In conclusion, NNET has good sensitivity when determined by the marker combination.

The corresponding decision results (fig. 4) of the SVM show that the accuracy of the training set and the test set is 99% and 83%, respectively. In 31 healthy controls in the training group, 31 controls are judged as healthy controls, and the true negative rate is 100%; in 28 healthy controls in the test group, 22 healthy controls are judged, the true negative rate is 79%, and the SVM has good specificity through judgment of the marker combination. In the training group, of 49 children with hand-foot-and-mouth diseases, 48 children with hand-foot-and-mouth diseases are judged as children with hand-foot-and-mouth diseases, 1 child is judged as a healthy control, and the true positive rate is 98%; in the test group, of 50 children with hand-foot-and-mouth disease, 43 of the children with hand-foot-and-mouth disease were judged, 7 of the children with hand-foot-and-mouth disease were judged as healthy control, and the true positive rate was 86%. And (3) evaluating the hand-foot-and-mouth disease condition of the participating volunteers based on SVM modeling, wherein the specific conditions are shown in Table 2. In conclusion, the SVM has good sensitivity through the judgment of the marker combination.

The results of the LDA determinations (fig. 5) show that the accuracies of the training set and the test set were 93% and 81%, respectively. Among 31 healthy controls in the training group, 26 healthy controls are judged, and the true negative rate is 84%; in 28 healthy controls in the test group, 27 healthy controls are judged, the true negative rate is 96%, and the judgment of the LDA through the marker combination has good specificity. In 49 children patients with hand-foot-and-mouth diseases in the training group, 48 children patients with hand-foot-and-mouth diseases are judged, 1 child patient is judged as a healthy control, and the true positive rate is 98%; in the test group, of 50 children with hand-foot-and-mouth diseases, 36 children with hand-foot-and-mouth diseases are judged, 14 children with hand-foot-and-mouth diseases are judged as healthy controls, and the true positive rate is 72%. The hand-foot-and-mouth disease condition evaluation is carried out on the participating volunteers based on LDA modeling, and the specific conditions are shown in Table 3. In conclusion, LDA was judged to have good sensitivity by the present marker combinations.

TABLE 1 hand-foot-and-mouth disease assessment results based on NNET modeling with 9 marker combinations

Table 2 hand-foot-and-mouth disease assessment results corresponding to SVM (support vector machine) modeling by adopting 9 marker combinations

TABLE 3 hand-foot-and-mouth disease assessment results based on LDA modeling using 9 marker combinations

EXAMPLE 3 preparation and use of a kit for detecting hand-foot-and-mouth disease

(I) preparation of specific antibodies

First, the NCBI database was searched for the gene sequences corresponding to the nine marker factors, OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1 and Pentraxin 3, and primers were designed based on the known sequences. After the synthesized primer is obtained, the target gene is amplified by adopting a PCR method, and is transformed into escherichia coli after enzyme digestion and connection. Successful E.coli transformed by antibiotic selection and expanded culture will express recombinant proteins of the above nine marker factors. After the Escherichia coli culture solution and the thallus are purified by affinity chromatography or ion exchange chromatography, the recombinant proteins of the nine marker factors are obtained. These recombinant proteins will serve as immunogens for the antibody source required for the antibody chip.

The resulting recombinant protein was mixed with an adjuvant, and a mouse was immunized multiple times, and after detecting an antibody against the immunogen in the blood of the mouse, the spleen of the mouse was removed, and the B cells thereof were isolated and fused with mouse myeloma cells. And screening the hybridoma cells obtained by fusion, and selecting a hybridoma monoclonal cell strain which efficiently secretes the required antibody. These cell lines were subjected to amplification culture, culture supernatants were collected, and the obtained antibodies were purified by Protein A/G affinity chromatography columns to obtain monoclonal antibodies as antibody materials for antibody chips.

(II) preparing antibody chip

The monoclonal antibody prepared in this example was used as a raw material for a capture antibody of an antibody chip, and the capture antibody was immobilized on the surface of a slide coated with an amino group using a full-automatic spotting instrument (platinummer corporation, usa):

(1) Mixing the monoclonal antibody with a phosphate buffer solution of pH7.4 containing 1.4% -2% casein to form a capture antibody mixture;

(2) Immobilizing each antibody in an amount of 0.01ng to 2ng per well on the slide in the capture antibody mixture, each antibody being present in 2 to 4 replicates, one replicate per well;

(3) And (3) repeating the step (2) to form an antibody dot matrix on the slide, wherein 10-100 capture antibodies are arranged at each square centimeter point on the slide, in other embodiments, the arrangement of the capture antibodies can be adjusted according to the experimental design requirements, the array is arranged according to different antibody chips, and the copper drum controls the full-automatic sample spotting instrument to prepare the required product.

(III) preparing a reaction solution

Antibody mixture: the monoclonal antibody prepared in this example was used as a raw material for a detection antibody of an antibody chip, and a mixed solution containing a detection antibody labeled with biotin was prepared using a raw material for a detection antibody labeled with biotin.

Streptavidin: the streptavidin is labeled with a fluorescent dye, which is Cy3 or a fluorescent dye with a similar absorption wavelength.

And (3) standard substance: OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1 and Pentraxin 3 marker factor recombinant protein.

Washing liquid: 20X concentrated washing solution containing Tween 20, wherein 1X washing solution is 0.1mol/L phosphate buffer solution containing 0.1% Tween 20 and pH 7.2.

Diluting liquid: 2 bottles of 15mL 5X concentrated diluent D for diluting the sample, and 1 bottle of 15mL 5X concentrated diluent B for diluting the antibody and HRP-streptavidin; 1X concentrated dilution D is 15mM phosphate buffer, pH6.5, with the following solutes and their mass or molar or volume concentrations in dilution D: 2% -4% sucrose, 150mM NaCl;1X concentrated dilution B is 15mM phosphate buffer solution with pH7.4, and the mass concentration or molar concentration or volume concentration of solutes in the dilution B is as follows: 0.5% casein, 2% -4% sucrose, 150mM NaCl.

Streptavidin: 200 μ L of 300 Xconcentrated fluorescein-streptavidin solution, fluorescein was Cy3 or a fluorochrome with similar absorption wavelength.

Sample treatment liquid: 10mL of 2X cell lysate and 1X cell lysate comprising 1 mM Tris.HCl, 25mM NaCl,1% sodium deoxycholate, 1% Triton X-100, containing phosphatase and protease inhibitor.

(IV) use of kit for detecting hand-foot-and-mouth disease

1. Sample processing

And (3) putting the sample into a centrifuge tube, adding a sample treatment solution into the centrifuge tube, dissolving the sample, quickly centrifuging, and diluting the sample solution by using a diluent D.

2. Complete drying of antibody chips

And taking the antibody chip out of the box, after the box is equilibrated at room temperature for 20-30 minutes, opening the packaging bag, uncovering the sealing strip, and then placing the antibody chip in a vacuum drier or drying the antibody chip at room temperature for 1-2 hours.

3. Incubation of sample solutions

Adding 50-100 μ L of diluted sample solution into each well on the antibody chip; the sample solution in each well was removed and washed 5 times with washing solution, each time with shaking table shaking at room temperature for 5 minutes, 150 μ L of 1X washing solution (prepared by diluting 20X washing solution with deionized water, the same applies below) per well, and the washing solution was removed completely for each washing.

4. Incubation of detection antibody mixtures

Centrifuging the detection antibody mixture tube, adding 1.4mL of diluent B, mixing uniformly, centrifuging quickly again, adding 80 mu L of antibody mixed solution into each hole of the antibody chip, and incubating for 2 hours on a shaking table at room temperature; and (3) pumping out the detection antibody in each hole, washing for 5 times by using washing liquid, shaking by a shaking table at room temperature for 5 minutes each time, and pumping out the washing liquid for each washing, wherein 150 mu L of 1X washing liquid is used for each hole.

Incubation of Cy3-streptavidin

Centrifuging a Cy 3-streptavidin tubule, adding 1.4mL of diluent B, mixing uniformly, quickly centrifuging again, adding 80 mu L of Cy 3-streptavidin into each hole of the antibody chip, wrapping the antibody chip with aluminum foil paper, incubating in a dark place, and incubating for 1 hour on a shaking table at room temperature; and (3) extracting Cy 3-streptavidin from each hole, washing for 5 times by using washing liquid, shaking by a shaking table at room temperature for 5 minutes each time, and extracting the washing liquid completely from 150 mu L of 1X washing liquid in each hole for each time of washing.

6. Fluorescence detection

6.1 the frame of the antibody chip is removed, taking care not to touch the antibody-printed side of the antibody chip by hand;

6.2 placing the antibody chip in a glass slide cleaning tube, adding about 30mL of cleaning solution to cover the glass slide completely, shaking for 15 minutes on a shaking table at room temperature, and removing the cleaning solution;

6.3 placing the antibody chip in a slide glass washing tube/drying tube, and centrifuging at 1000rpm for 3 minutes, thereby removing the residual washing solution of the antibody chip;

6.4 scanning the signal with a laser scanner, e.g.axon GenePix, using Cy3 or green channel (excitation wavelength 532 nm).

7. Data extraction of the chip and data analysis by analysis software.

Finally, it should be noted that the above-mentioned embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the modifications and equivalents of the specific embodiments of the present invention can be made by those skilled in the art after reading the present specification, but these modifications and variations do not depart from the scope of the claims of the present application.

Claims (4)

1. An antibody chip for detecting hand-foot-and-mouth disease is characterized in that: the kit comprises a solid phase carrier and capture antibodies coated on the surface of the solid phase carrier, wherein the capture antibodies are specific antibodies respectively aiming at OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1 and Pentraxin 3, and the solid phase carrier is a glass slide coated with amino on the surface.

2. A kit for detecting hand-foot-and-mouth disease is characterized in that: an antibody chip for detecting hand-foot-and-mouth disease according to claim 1.

3. The kit for detecting hand-foot-and-mouth disease according to claim 2, further comprising the following components:

an antibody mixture containing biotin-labeled detection antibodies specific to OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1 and Pentraxin 3, respectively;

streptavidin: the streptavidin is labeled with a fluorescent dye.

4. The kit for detecting hand-foot-and-mouth disease according to claim 3, characterized in that: also included are marker standards including OPN, APRIL, TRAIL R1, MIP-3b, TWEAK, BLC, ICOS, ICAM-1, and Pentraxin 3.

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