NL2030266A - Combination kit and method for predicting childhood asthma attack - Google Patents

Abstract

The present disclosure provides a combination kit and a method for predicting a childhood asthma attack. Peripheral blood of children with asthma in acute and remission is collectedJ and. bioinformatics analysis of differentially 5 expressed proteins is carried out through a TMT TM—labeled quantitative proteomics technology, so as to obtain a target protein and build a library. The target protein is then subjected 11) a quantitative analysis through za parallel reaction monitoring (PRM)—targeted proteomics technology to 10 obtain a combination of plasma protein markers. Performance of each marker and a combination thereof in clinical application is determined through detecting peripheral serum of children with asthma using enzyme linked immunosorbent assay (ELISA) method. The combination kit is then used to 15 detect a combination of plasma markers, and a detection result is used to predict an asthma attack. The prediction result has a high diagnostic sensitivity and specificity, and is expected to be a laboratory diagnostic indicator of asthma.

Description

COMBINATION KIT AND METHOD FOR PREDICTING CHILDHOOD ASTHMA ATTACK

TECHNICAL FIELD This application relates to biomedical development, and more particularly to a combination kit and a method for predicting a childhood asthma attack.

BACKGROUND Asthma is a chronic airway disease, the symptoms of which includes reversible airflow obstruction, dyspnea, wheezing, coughing, airway inflammation and bronchial hyperresponsiveness. Although asthma has been known for hundreds of years, the diagnosis of asthma still lacks a gold standard.

Detection of airway hyperresponsiveness (AHR) is helpful for the diagnosis of symptomatic patients, but is limited for the airway of non-asthmatic and asymptomatic patients with allergic inflammation also shows hyperresponsive. It has been reported that 20%~30% of adult asthma patients are overdiagnosed.

Fiberoptic bronchoscopy was once considered to be the most important method for diagnosing asthma. Although it can accurately invade the airway for detection, it is expensive and cumbersome to operate. In addition, a slight improper operation may cause tissue perforation and bleeding. Furthermore, the fiberoptic bronchoscopy cannot be carried out in batches, and it cannot be used routinely in primary medical institutions.

Finding a serological indicator for the diagnosis of asthma has become a research hot spot for a long time, because the peripheral blood is easy to obtain and promote in clinical practice. However, no single serum marker can be used to accurately determine the condition of the patient. Although many serum markers have been reported in the literature for the diagnosis of asthma, their clinical translational application is rarely reported. Some teams identify asthma patients with different clinical phenotypes through data-driven approaches, but the risk factors obtained in different studies are inconsistent, that is, the same phenotype set is still heterogeneous. Several literatures identify the therapeutic effects of different phenotypes of asthma through data-driven approaches, and the results show that the therapeutic effects are similar. Serum markers with good sensitivity and specificity can identify childhood asthma with different phenotypes, and help individualize the choice of medication and predict the effect of treatment. Therefore, a comprehensive and systematic description of the occurrence and development of asthma, including clinical symptoms, genetically-related serum markers and lung function-related serum markers, will help to develop more effective treatment strategies. Proteomics was posed in the 1990s, and refers to using high-resolution protein separation technology and efficient protein identification technology to comprehensively, dynamically and quantitatively observe life phenomena and laws at the protein level, including protein expression levels, post-translational modification, protein-protein interaction. A proteome is a set of all expressed proteins in a cell, tissue, or organism, and the activities thereof. Tandem mass tag (TMT) is a commonly-used differential proteomics technology, which compares the differentially expressed proteins in different samples, and is widely used in disease marker screening and drug targets. Parallel reaction monitoring (PRM) is an ion monitoring technology based on high-resolution and high-precision mass spectrometry, which can selectively detect target proteins and target peptides, SO as to achieve absolute quantification of target proteins and peptides.

SUMMARY A first objective of the present disclosure is to provide a combination kit for predicting a childhood asthma attack. A second objective of the present disclosure 1s to provide a method for predicting a childhood asthma attack using the combination kit mentioned above.

The technical solutions of the present disclosure are described as follows.

In a first aspect, the present disclosure provides a combination kit for predicting a childhood asthma attack, the combination kit consisting of a sub kit for detecting human o-l-antichymotrypsin (AACT), a sub kit for detecting serum immunoglobulin A (IgA), a sub kit for detecting serum amyloid A (SAA) and a sub kit for detecting hemoglobin subunit beta (HBB).

In some embodiments, the combination kit comprises: an alpha 1 antichymotrypsin human enzyme linked immunosorbent assay (ELISA) kit (abl57706) for detecting the AACT; an IgA human SimpleStep ELISA kit {abl196263} for detecting the IgA; a human SAA ELISA kit (abl00635) for detecting the SAR; and an HBB human ELISA kit (abl57707) for detecting the HBB.

In a second aspect, the present disclosure provides a method for predicting a childhood asthma attack, comprising: predicting the childhood asthma attack using the combination kit mentioned above.

The beneficial effects of the present disclosure are described as follows.

A combination kit and a method for predicting a childhood asthma attack are provided herein. Peripheral blood of children with asthma in acute and remission is collected, and bioinformatics analysis of differentially expressed proteins is carried out through a TMT ™-labeled quantitative proteomics technology, so as to obtain a target protein and build a library. The target protein is then subjected to a quantitative analysis through a parallel reaction monitoring (PRM) -targeted proteomics technology to obtain a serum marker and a combination of plasma protein markers. Performance of each marker and a combination thereof in clinical application is determined through detecting peripheral serum of children with asthma using enzyme linked immunosorbent assay (ELISA) method. The combination kit is then used to detect a combination of plasma markers, and a detection result is used to predict the asthma attack. The prediction result has a high diagnostic sensitivity and specificity, and is expected to be a laboratory diagnostic indicator of asthma.

DETAILED DESCRIPTION OF EMBODIMENTS The objective, technical solutions and beneficial effects of the present disclosure are further described below with reference to the embodiments. It should be understood that the embodiments provided herein are illustrative, and not intended to limit the present disclosure. Material and method

1. Plasma sample The research process was approved by the ethics committee of Wuxi Children's Hospital. A test set included 4 children with asthma in clinical remission, 4 children with asthma in acute attack and 4 children in healthy control group (Table 1). A verification set included 16 children with asthma in clinical remission, 16 children with asthma in acute attack and 16 children in healthy control group (Table 1).

Table 1 Participant demographic and clinical information: TTT al 1E 5700 12100700) Zien es Boes |]

1.75 (0.74-14.43) 37,50 (26.25-48.20) aplasma was used for TMT and PRM proteomic analyses. 5 bAbbreviations: M, male; FF, female; DF-sIgk, D. farinae-specific IgE; DP-sIgE, D. pteronyssinus- specific IgE.

The diagnosis of asthma was based on the medical history, physical examination and pulmonary function test of the patient. For details, see the guidelines (Respiratory Group of Pediatric Branch of Chinese Medical Association.

Guidelines for the diagnosis and prevention of asthma in children. Chinese Journal of Pediatrics, 2016; 54(3):167-

181. DOI:10.3760/cma.j.issn.0578-1310.2016.03.003).

Exclusion criteria Patients with symptoms or signs of other lung diseases or systemic diseases and patients receiving oral hormone therapy were excluded. The three groups of selected subjects were the same in weight, height and body mass index (BMI). The children in the healthy control group had no history of respiratory diseases or allergies. All experiments were approved by the ethics committee of Wuxi Children's Hospital. 3 mL of venous blood of each subject was taken on an empty stomach in the early morning.

2. Protein extraction (1) 40 uL of blood was taken from each sample and was diluted 10 times with a binding buffer (kit: Binding Buffer).

(2) A lid of a column was removed, and the storage buffer was absorbed through paper.

(3) A tip at a bottom of the column was removed, and the column was put in a collection tube.

(4) The binding buffer was added into the column, and flowed through the column under the action of gravity.

(5) The column was put into a new collection tube.

(6) The diluted sample was added into the column, and flowed through the column under the action of gravity.

(7) The column was washed with 600 pL of the binding buffer.

(8) The column was washed with 600 pL of the binding buffer again. The elution components, that was, the sample after the removal of albumin/IgG, obtained in the previous three steps were collected and subjected to vacuum freeze drying.

(9) The lyophilized sample was added with 300 uL of

: sodium dodecyl sulphate (SDS) to split for redissolving.

(10) The solution was centrifuged at 12000xg for 10 min at room temperature to obtain a primary supernatant. The primary supernatant was centrifuged to obtain a secondary supernatant, that was, a total protein solution of the sample.

A protein concentration of the total protein solution of the sample was measured, and was aliquoted and stored at - 80°C.

3. Measurement of sample concentration The protein concentration was measured through the bicinchoninic acid (BCA) method (Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 1985, 150(1): 76-85.).

4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (1) 10 ug of each sample was respectively subjected to a separation using 12% SDS-PAGE.

(2) The separated gel was stained by the Coomassie brilliant blue staining method (Candiano G, Bruschi M, Musante L, et al. Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Flectrophoresis, 2004, 25(9): 1327-1333). The specific 2b operation was as follows. A. Fix for 2 h. B. Dye for 12 h. C. Wash with water until the background was clear.

(3) The dyed gel was scanned with a scanner (ImageScanner) with a full-color scanning mode and an optical density value of 300 dpi.

5. Trypsin digestion and labeling The protein was enzymatically digested according to the filter aided sample preparation (FASP)} (Wisniewski JR, Zougman A, Nagaraj N, et al. Universal sample preparation method for proteome analysis. Nature Methods, 2009, 6(5): 359-362.).

(1) After protein quantification, 100 ng of each sample was respectively added into a 10 K ultrafiltration tube, and added with 120 uL of reducing agent buffer (10 mM dithiothreitel (DTT), 8 M urea, 100 mM thermo scientific triethylamonium bicarbonat (TEAB), pH 8.0) for reacting at 60°C for 1 h.

(2) Indole-3-acetic acid (IAA) was added to a final concentration of 50 mM and reacted for 40 min at room temperature in the dark.

(3) A centrifuging was carried out at 12000 rpm at 4°C for 20 min. The solution at the bottom of the collection tube was discarded.

(4) 100 uL of a 300 mM TEAB buffer was added, and centrifuged at 12000 rpm for 20 min twice.

(5) The collection tube was replaced with a new collection tube. The ultrafiltration tube was added with 100 uL of the 300 mM TEAB buffer and 2 uL of 1 ug/ul trypsin sequencing grade solution followed by reacting at 37°C for 12 h.

(6) A centrifuging was carried out at 12000 rpm for 20 min to collect the peptides after enzymolysis. 50 uL of a 200 mM TEAB buffer was added into the ultrafiltration tube and was centrifuged at 12000 rpm for 20 min to collect a solution at a bottom of the ultrafiltration tube followed by lyophilizing.

(7) The lyophilized sample was added with 100 uL of the 200 mM TEAB buffer followed by vortex mixing. 40 uL of the sample was added into a 1.5 mL Ep tube for labeling reaction.

(8) A tandem mass tag (TMT) reagent was taken form a refrigerator and equilibrated to room temperature, and then added with 41 uL of anhydrous acetonitrile followed by vortex for 5 min and centrifuging.

(8) The sample was added with 41 uL of the TMT reagent followed by evenly vortex mixing, and was placed at room temperature for 1 h.

8 nL of 5% hydroxylamine was added to stop the reaction for 15 min, and then was lyophilized and stored at -80°C.

6. Reversed-phase chromatography separation Liquid chromatography: Agilent 1100 HPLC.

Chromatographic column: Agilent Zorbax Extend-C18 narrow-bore column, 2.1x150 mm, 5 um.

Detection wavelength: Ultraviolet (UV) 210 nm and 280 nm.

Mobile phase A: ACN-H:0 (2:98, v/v).

Mobile phase B: ACN-H,O (90:10, v/v).

Flow rate: 300 uL/min.

Gradient elution conditions: 0-8 min, 98% A; 8-8.01 min, 984-954 A; 8.01-48 min, 95%-75% A; 48-60 min, 75-60% A; 60-

60.01 min, 60-10% A; 60.01-70 min, 10% A; 70-70.01 min, 10- 98% A; and 70.01-75 min, 98% A.

The samples during the 8-60 minutes were collected. The eluate was collected into No. 1-15 centrifuge tubes every one minute in turn. The samples were collected in this order until the end of gradient end. After the collection, the samples were vacuum freeze-dried and frozenly stored.

7. Chromatographic conditions and mass spectrometry conditions Chromatographic conditions The sample was loaded onto a pre-column Acclaim PepMapl00 100 umx2cm (RP-C18, Thermo Fisher) at a flow rate of 300 nL/min, and then separated through an analytical column Acclaim PepMap RSLC, 7bumxl5cm (RP-C18, Thermo Fisher).

Mobile phase A: H,O-FA (99.9:0.1, v/v).

Mobile phase B: ACN-H:0-FA (80:19.9:0.1, v/v/v).

Gradient elution conditions: 0-40 min, 5-30% B; 40-54 min, 30-50% B; 54-55 min, 50-1003 B; and 55-60 min, 100% B.

Mass spectrometry conditions A mass resolution of the primary MS was 70,000, and an automatic gain control value was le6. A scan mode of the mass spectrum scan was full scan. A charge-to-mass ratio m/z was 300-1600. The 10 highest peaks were scanned by MS/MS. MS/MS spectrum acquisition was completed through high-energy collision fragmentation in the data-dependent positive ion mode. A collision energy was 32. A resolution of MS/MS was

17500. An automatic gain control was Zeb. Maximum ion accumulation time was 80 ms. Dynamic elimination time was 30 s.

Data processing The experimental data was analyzed using Proteome Discoverer™ 2.2 (Thermo Company, USA), and the database used herein was a human database from UniProt. The false positive rate of peptide identification was controlled below 1%. The specific search parameter settings were shown in Table 2. Table 2 Mass spectrum search parameters 6 plex (Peptide Labeled) Parallel reaction monitoring (PRM) PRM is an ion monitoring technology based on high- resolution and high-precision mass spectrometry. The PRM combines the high selectivity of the quadrupole and the high-resolution and high-precision specificity of the Orbitrap/or ToF mass spectrometer, and can perform selective quantitative detection of target protein and target peptide. PRM targeted quantitative proteomics technology has become a very important mass spectrometry quantitative method, and mainly includes the following steps: protein extraction, protein quantification, protein enzymolysis, LC-PRM/MS analysis, PRM data Skyline analysis.

1. Instruments and reagents 1) UA buffer (8M Urea, 150 mM Tris-HCl, pHS8.0) 2) NH; HCO; {Sigama, A6141) 3) Acetonitrile (Merck, 1499230-935) 4) Q-exactive Plus (Thermo Scientific) 5) Easy-nLC1200 (Thermo Scientific) 6) Trap column {Reverse-Phase), 100 pmx20 mm (5 um, C18) 7) Thermo Scientific EASY column (Reverse-Phase), 75 umx120 mm (3 um, C18).

2. Sample preparation

About 200 ug of protein from each sample was respectively subjected to in-solution enzymolysis. The enzymolysis was as follows. An appropriate amount of a 50 mM NH HCO; buffer was added for diluting the protein by 10 times. DTT was added to a final concentration of 10 mM, and was kept at 37°C for 1 h followed by cooling to the room temperature.

An appropriate amount of 1 M IAA was added to a final concentration of 50 mM, and was kept in the dark at room temperature for 30 min. The sample was added with 2 ug of Trypsin, and was kept at 37°C for 16 h. The peptide after the enzymolysis was desalted and lyophilized, and then was redissolved with 0.1% FA. The peptide concentration was determined at optical density OD 280.

3. LC-PRM/MS analysis According to the results of TMT, three groups with significant differential expression and high abundance of single protein expression were selected.

1-3 peptides in each target protein having reliable identification information and good chromatographic separation behavior (chromatographic elution peaks were sharp and symmetrical) was/were selected for PRM quantitative analysis. The peptide information suitable for PRM analysis was imported into the software Xcalibur for setting the PRM method.

2 g of the peptide of each sample was respectively subjected to LC-PRM/MS analysis. After loading the sample, the Easy nLC 1200 chromatography system (Thermo Scientific, US) with nancliter flow rate was used for chromatographic separation. Regarding a buffer, A solution was a 0.1% aqueous formic acid solution, and B solution was a mixed solution of 0.1% formic acid, acetonitrile and water (in which a weight percentage of the acetonitrile was 95%). The column was balanced with 25% the A solution. The sample was injected into the Trap Column (100 m 20 mm, 5m, C18, Dr.

Maisch GmbH) and then passed through a chromatographic analysis column (75 m 150 mm, 3m, C18, Dr. Maisch GmbH) for gradient separation at a flow rate of 300 nl/min. The liquid phase separation gradient was as follows: 0-5 min, the linear gradient of the B solution was from 2% to 5%; 5-45 min, the linear gradient of the B solution was from 5% to 23%; 45-50 min, the linear gradient of the B solution was from 23 % to 40%; 50 min to 52 min, the linear gradient of the B solution was from 40% to 100%; 52-60 min, the B solution maintained at 100%. After separation of the peptide, a Q-Exactive Plus mass spectrometer (Thermo Scientific) was used for targeted PRM mass spectrometry analysis. Analysis time was 60 min. Detection mode was positive ion. A precursor ion scan range was 350-1500 m/z. A resolution of the primary mass spectrum was QAS 70, 000@m/z

200. AGC target was 3e6. Maximum IT of the primary mass spectrum was 200 ms. The secondary mass spectrometry analysis of the peptide was collected as follows. After each full MS scan, the Precursor m/z of the target peptide was selected according to the Inclusion list for the secondary mass spectrometry scan analysis (MS2). A resolution of MS2 was 17,500@ m/z 200. AGC target was 3e6. Maximum IT of the secondary mass spectrum was 100 ms. MS2 Activation Type was HCD. Isolation window was 2.0 Th. Normalized collision energy was 27. The obtained original RAW file of the mass spectrum was subjected to PRM data analysis using the software Skyline 4.1. 4, Enzyme linked immunosorbent assay (ELISA) verification According to the detection results of TMT and PRM, the ELISA kit was used for combined detection of the proteins with the same detection results to clarify the diagnostic performance. The number of the protein that the detection result of TMT was consistent with the detection result of PRM was 7; however, only the kits of AACT, IgA, SAA and HBB are commercially available. Therefore, a commercial alpha 1 antichymotrypsin human ELISA kit (abl57706), an IgA human SimpleStep ELISA kit (abl96263), a human SAA ELISA kit (ab100635) and an HBB human ELISA kit (abl57707) were purchased and operated according to the kit instructions.

The microplate reader used herein was iMark Microplate Reader S/N 10288. The wavelength was 450 nm. Optical density (OD) value was measured.

5. Statistical analysis The detection results were expressed in SD, and SPSS 19.0 (SPSS, Chicago, IL, USA) was used for data statistics and analysis, linear regression analysis and Pearson correlation analysis. Mann-Whitney U-test was used between the two groups of means, and P<0.05 indicated that the difference was statistically significant. The receiver operator characteristic (ROC) was calculated to evaluate the diagnostic performance of the candidate protein. Results

1. TMT identification of differentially expressed proteins in peripheral blood of children with asthma The plasma of 4 children with asthma in acute attack, 4 children with asthma in clinical remission and 4 children in healthy control group was identified using TMT, and 347 differentially expressed proteins were detected. With respect to the acute attack group and the control group, there were 125 differentially expressed proteins (FC>1.2, P<0.05), including 50 up-regulated proteins and 75 down- regulated proteins. With respect to the remission group and the control group, there were 142 differentially expressed proteins, including 72 up-regulated proteins and 70 down- regulated proteins. With respect to the acute attack group and the remission group there were 55 differentially expressed proteins, including 22 up-regulated proteins and 33 down-regulated proteins.

2. PRM identification of differentially expressed proteins in peripheral blood of children with asthma Based on the results of TMT identification, 11 proteins were selected according to the multiple of differential expression, P value and the abundance of single protein expression in plasma. The 11 proteins were pigment epithelium-derived {factor (PEDF')}, immunoglobulin heavy constant delta, immunoglobulin heavy constant gamma 4,

cofilin-1, alpha-l-antichymotrypsin, immunoglobulin heavy constant alpha 1, serum amyloid A-1 protein, ubiquitin-40S8 ribosomal protein S27a, hemoglobin subunit beta, hemoglobin subunit alpha (hemoglobin alpha chain). The LC-PRM/ MS was used to perform quantitative analysis. The result of PRM identification showed that 7 proteins that the expression levels thereof were consistent with the result of TMT identification, as shown in Table 3. Table 3 Result comparison of the PRM and TMT identifications of the 11 candidate proteins Fold changes Consisten Acute vs Remission vs Acute vs cy Gene Protein name Protein ID remission control control between name ™ PR PRM and

PRM TMT PRM TMT

T M TMT Pigment epithelium- SERPINF P36955 1.02 0.91 0.76 1.31 No derived factor 1 (PEDF) Immunoglobulin

AQAOAOM heavy constant 609 [GHD 1.10 1.00 7.47 2.27 2.28 Yes delta (IGHD Immunoglobulin heavy constant AOA286YF IGHG4 2.29 0.98 1.50 2.20 | 3.38 2.15 Yes gamma 4 J8 (IGHG4) Coin Enis GI i os 170 Alpha-~1-

SERPINA antichymotrypsin PO1011 ‚ 1.55 1.13 0.35 0.70 | 0.55 0.79 Yes

J (AACT) Immunoglobulin heavy constant ) ] PO1876 IGHA! 1.20 0.96 2.95 2.15 | 3.50 2.06 Yes alpha 1 (IgHAD) Fibronectin P0O2751 EN! 0.38 0.92 1.60 0.71 { 0.39 0.66 No (EN) Serum amyloid A-1 protein PODJI8 SAAI 4.51 1.41 0.03 021 | 0.14 0.29 Yes (SAAD

Ubiquitin-40S SC ribosomal protein 8974 P62979 RPS274 | 095 Li4 | 312 | 038 | 295 | 044 No EE] on [oee - EE Hemoglobin subunit beta P68871 HBB 1.14 1.14 0.12 0.27 | 0.14 0.31 Yes EEE en [om [ee [eee nn Hemoglobin subunit alpha P69905 HBA]I 1.83 1.07 0.17 0.32 | 031 0.34 Yes SE | oe [oe [ee [efen]

3. ELISA verification Commercially available kits for detecting the 7 proteins that the detection result of TMT was consistent with the detection result of PRM were an alpha 1 antichymotrypsin human ELISA kit (ablb57706), an IgA human SimpleStep ELISA kit (abl%6263), a human SAA ELISA kit (ab100635) and an HBB human ELISA kit (abl57707), which were used to detect AACT, IgA, SAA, HBB, respectively. The plasma of 16 children with asthma in acute attack, 16 children with asthma in clinical remission and 16 children in healthy control group was detected, and the result was shown in Tables 4-7. Table 4 ACCT detection result of the serum of the subjects in the three groups in zieh Standard Maximum Minimum antichymot Case Numbers Average value deviation value value rypsin Note: The difference among the three groups was statistically significant (F=4.231, P=0.021). Specifically, the difference between the control group and the remission group was not statistically significant; the average value of ACCT of the acute group was significantly increased, and the difference between the acute group and the control group and the difference between the acute group and the remission group were statistically significant. Table 5 SAA detection result of the serum of the subjects in the three groups Case Average Standard Maximum | Minimum

SAA Numbers value deviation value value amste | 16 | ems | 17m | soe | se | Remissio 16 5.315 0.689 4.150 6.749 n : ] 07 Note: The difference among the three groups was statistically significant (F=8.069, P=0.001). Specifically, the difference between the control group and the remission group was not statistically significant; the average value of SAA the acute group was significantly increased, and the difference between the acute group and the control group and the difference between the acute group and the remission group were statistically significant. Table 6 HBB detection result of the serum of the subjects in the three groups Case Average Standard Maximum Minimum Hemoglobin Numbers value deviation value value Note: The difference among the three groups was statistically significant (F=7.757, P=0.001). The difference between the acute group and the remission group was not statistically significant. The average value of hemoglobin of the acute group and the remission group were significantly lower than that of the control group.

Table 7 IgA detection result of the serum of the subjects in the three groups Case Average Standard Maximum Minimum IgA Numbers value deviation value value Remissi 16 33.756 8.350 18.734 47.533 on Note: The difference among the three groups was statistically significant (F=9.282, P<0.001).

Compared with the control group, the expression level of AACT and SAA in the serum of children with asthma (including the acute group and remission group) increased (P<0.05), and the expression level of HBB and IgA decreased (P<0.01). The AUCs of AACT, IgA, SAA and HBB were 0.688 [95% confidence interval (CI), 0.481-0.834], 0.707 (95% CI, 0.522-0.892),

0.623 (95% CI, 0.409-0.837) and 0.525 (95% CI, 0.342-0.704). With respect to the area under the receiver operating characteristic (ROC) curve of the combined diagnosis, the area under the ROC curve of the combined diagnosis of AACT+IgA+SAA was 0.801; the area under the ROC curve of the combined diagnosis of IgA+SAA was 0.789 and the area under the ROC curve of the combined diagnosis of AACT+IgA+SAA+HBB was 0.840. The area under the ROC curve of the combined diagnosis of AACT+IgA+SAA+HBB was the largest. IgA and HBB had the best diagnostic sensitivity (87.50%), and IgA+SAA and AACT+IgA+SAA had the highest diagnostic specificity (83.75%). Details were shown in Table 8.

Table 8 Diagnostic performance of AACT, IgA, SAA and HBB detection levels Variable AUC 95% CI Sensitivity | Specificity Z

0.688 0.481-0.894 68.75 75.00 0.4375 1.782 0.0747

0.623 0.109 0.409-0.837 75.00 0.3750 1.129 0.2588 AACT+IgA+SAA 0.801 0.08012 0.622-0.920 62.50 93.75 0.5625 3.652 0.0003 IgA+SAA 0.789 0.0818 0.609-0.913 56.25 0.5000 3.532 0.0004 HBB 0.525 0.107 0.342-0.704 31.25 0.1875 0.8123 AACT+IgA+SAA+HBB 0.840 0.0689 0.667-0.945 75.00 81.25 0.5625 4.931 <0.0001 apbbreviations: AUC, area under the curve; SE, Standard Error; 954 CI, 953 confidence interval.

The embodiments of the present disclosure are preferred embodiments, and not intended to limit the present disclosure.

Modifications, replacements and improvements made by those skilled in the art without departing from the spirit of this disclosure should fall within the scope of the present disclosure defined by the appended claims.

Claims (3)

CONCLUSIONS A combination kit for predicting an asthma attack in children, characterized in that the combination kit consists of a subkit for detecting human α-l-antichymotrypsin (AACT), a subkit for detecting serum immunoglobulin A (IgA), a subkit for detecting serum amyloid A (SAA), and a subkit for detecting hemoglobin subunit beta (HBB). A combination kit according to claim 1, comprising: an alpha-l-antichymotrypsin human enzyme-linked immunosorbent assay (ELISA) kit (ab157706) for detecting the AACT; an IgA human SimpleStep ELISA kit (ab196263) for detecting the IgA; a human SAA ELISA kit (abl00635) for detecting the SAA; and an HBB human ELISA kit (abl157707) for detecting the HBB. A method for predicting an asthma attack in children, comprising: predicting an asthma attack in children using the combination kit according to any one of claims 1-2. -0-0-0-