Preparation and analysis method of a solution for detecting allergen activity and its application

Abstract

The present invention discloses a method for preparing a solution for detecting allergen activity, an analytical method, and its application, relating to the field of immunoassay technology. A solution for detecting serum and nasal secretion IgE levels is prepared, and the correlation between seasonal pollen concentrations, serum IgE and nasal secretion IgE levels, and SAR allergy symptoms and CSMS is analyzed. The present invention also discloses the application of the method in preparing reagents, kits, test strips, or chips to assist in screening patients for seasonal allergic rhinitis treatment, providing support for new treatment strategies and drug development.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassays, and in particular to a preparation method and an analysis method for a solution for detecting allergen activity and an application thereof. Background Art

[0002] Allergic rhinitis (AR) is an immunoglobulin E (IgE)-mediated type I allergic disease triggered by allergen exposure to the nasal mucosa in atopic individuals. Seasonal allergic rhinitis (SAR), also known as hay fever, is caused by allergens released by plant pollens and places a heavy burden on patients and healthcare resources. The development and progression of allergic reactions are primarily attributed to the persistent overproduction of IgE in response to inhaled allergens, particularly pollen. Inhaled allergens induce localized production of specific IgE (sIgE) in the nasal cavity. Studies have demonstrated that high-affinity IgE in the peripheral blood of sensitized individuals primarily originates from the nasal mucosa and enters the peripheral circulation via lymphatic vessels. Importantly, sIgE-positive nasal secretions can be detected in a subset of subjects with localized allergic rhinitis who are serum negative for allergens. These findings suggest that mucosal IgE synthesis can occur in the absence of systemic atopy and may be more responsive to allergens than serum sIgE.

[0003] Therefore, it is crucial to clarify the correlation between seasonal pollen concentrations, serum and nasal secretion IgE levels and SAR allergy symptoms and medication scores. This study aimed to explore the correlation between the fluctuation of local IgE levels under natural allergen exposure and the monitoring and evaluation of SAR symptoms. Summary of the Invention

[0004] In a first aspect, the present invention provides a method for preparing a solution for detecting allergen activity, the method comprising the following steps:

[0005] S1 Collection Preparation: Before starting to collect nasal secretions, ensure that the subject is in a resting state and comfortable environment;

[0006] S2 collection and processing: two cotton pads were placed in the left and right middle nasal passages of the subjects using forceps, adhered to the nasal mucosa, and left for 5-10 minutes;

[0007] S3 Drying: Remove the cotton pad from the nasal cavity and place it on a slotted tray, marking it. Place the tray in a dryer set at 35°C to 40°C for 2 to 2.5 hours.

[0008] After S4 centrifugation pretreatment, place the cotton pieces in 1.5 mL EP tubes. Add 500 μL–1000 μL of 0.9% saline solution to each tube. Place the EP tubes in a micromixer and mix thoroughly by inversion at 4°C–10°C for 10–20 minutes. Make a small hole at the bottom of the 0.5 mL EP tube, remove the cotton pieces, and place them in the 0.5 mL EP tube. Then, place the 0.5 mL EP tube containing the cotton pieces in the original 1.5 mL EP tube.

[0009] S5: Centrifugation treatment: Place the 1.5 mL EP tube treated in step S14 in a centrifuge at 4°C and a speed of 10,000-12,000 rpm for 5-10 minutes;

[0010] S6 cryopreservation treatment: After processing in the centrifuge, the nasal secretion solution collected in the 1.5 mL tube was stored in an ultra-low temperature freezer at -80℃~-40℃ until testing.

[0011] S7: Blood samples were collected and centrifuged at 3000-3500 rpm and 25-30°C for 10-15 minutes, and serum was collected for further analysis.

[0012] S8 After obtaining the nasal secretion solution of step S6 and the serum of step S7, the nasal secretion IgE level and the serum IgE level are detected using the ImmunoCAP 100 system, where the IgE value is the sIgE / tIgE value.

[0013] Optionally, in step S2 of the embodiment, the cotton sheet is adhered to the nasal mucosa for a residence time of 5 minutes;

[0014] Optionally, in the embodiment, the temperature of the drying machine in step S3 is set to 35° C. and the processing time is 2 hours;

[0015] Optionally, in step S4 of the embodiment, the mixing treatment is performed by inverting and mixing at 4° C. for 10 minutes;

[0016] Optionally, in the 1.5 mL EP tubes described in step S4 of the embodiment, 500 μL of 0.9% saline solution is added to each tube;

[0017] Optionally, in step S5 of the embodiment, the centrifuge temperature is set to 4° C., the speed is 12000 rpm, and the processing time is 5 minutes;

[0018] Optionally, the ultra-low temperature refrigerator in step S6 of the embodiment is set to -40°C.

[0019] In a second aspect, an embodiment of the present invention provides a combined analysis method for allergen activity. After obtaining nasal secretion IgE and serum IgE level values, the correlation between seasonal pollen concentration, serum IgE and nasal secretion IgE levels and SAR allergy symptoms and CSMS is analyzed.

[0020] Optionally, the CSMC evaluation includes a symptom evaluation score and a daily medication score, with equal weights balancing the nasal and eye symptoms and the daily medication score, and is calculated as: CSMS=(TNSS+TOSS) / 6+DMS (0-6).

[0021] Optionally, the symptom evaluation score includes nasal and eye symptom scores, which are scored using a four-level quantitative scoring system based on severity, ranging from 0 to 3 points.

[0022] The total nasal symptom score is the sum of the four individual nasal symptoms: nasal itching, runny nose, sneezing, and nasal congestion;

[0023] The total eye symptom score is the sum of the two individual eye symptoms: itchy / red eyes and tearing.

[0024] Optionally, the daily medication score of 0 indicates no medication; 1 indicates use of only antihistamines; 2 indicates use of nasal corticosteroids (regardless of whether antihistamines are used); 3 indicates use of oral corticosteroids (regardless of whether other drugs are used in combination);

[0025] Optionally, the joint analysis method comprises the following steps:

[0026] Friedman repeated-measures rank analysis of variance was used to compare the differences in nasal secretion and serum IgE levels at the four follow-up points at T1;

[0027] T2 used generalized mixed linear models and time series analysis to investigate the effect of pollen concentration on symptom severity;

[0028] T3 Spearman analysis was used to analyze the correlation between each IgE level and pollen concentration and symptom severity;

[0029] T4 used causal mediation analysis to clarify the mediating role of changes in IgE levels in nasal secretions during the pollen season between changes in pollen concentration and CSMS differences, and evaluated the mediation effect using direct effects, indirect effects, and calculation of the proportion of the total effect.

[0030] T5 was repeated 1000 times to calculate the confidence interval using the Bootstrap method.

[0031] Optionally, the causal mediation analysis in step T4 is implemented by constructing an analysis model, specifically using a new variable M. If X affects Y by affecting variable M, then M is called a mediating variable. The relationship between the variables can be described by the following regression equation:

[0032] Y= cX+e1 (1)

[0033] Y= aX+e2 (2)

[0034] Y= c'X+bM+e3 (3)

[0035] Where X is the change in total pollen concentration under natural pollen exposure, Y is the change in the severity of seasonal allergic rhinitis symptoms, and M is the change in local IgE.

[0036] In a third aspect, embodiments of the present invention provide applications of methods for detecting and analyzing allergen activity, specifically applications in the preparation of a kit for assessing the severity of seasonal allergic rhinitis symptoms, wherein the test samples used in the test are nasal secretions and serum from patients with allergic rhinitis;

[0037] The ImmunoCAP 100 system was used to detect the IgE levels in nasal secretions and serum;

[0038] The analysis was a joint analysis of the correlation between seasonal pollen concentration, serum IgE and nasal secretion IgE levels and SAR allergic symptoms and CSMS.

[0039] Compared with the prior art, the present invention has at least the following beneficial effects:

[0040] The present invention has discovered a method for evaluating changes in the severity of allergic rhinitis symptoms using local IgE levels. By using the IgE levels detected by the method of the present invention and jointly analyzing the correlation between SAR allergic symptoms and CSMS, changes in seasonal allergic rhinitis symptoms under changing pollen concentrations can be quickly and timely predicted. This method has significant clinical and economic value in preparing reagents, kits, test strips or chips for evaluating the severity of seasonal allergic rhinitis symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Design diagrams for pollen monitoring and experimental data collection;

[0042] Figure 2 This is a graph showing the effect of pollen concentration (log2 transformed) on symptoms and medication scores based on the GLMM model;

[0043] Figure 3 A- Figure 3F is the comparison of humoral immunoglobulin E levels across seasons and the trend of total pollen concentration with seasons;

[0044] Figure 3 A is a graph showing seasonal changes in nasal secretion sIgE levels and total pollen concentrations;

[0045] Figure 3 B is a graph showing seasonal changes in tIgE levels and total pollen concentrations in nasal secretions;

[0046] Figure 3 C is a graph showing seasonal changes in nasal secretion sIgE / tIgE levels and total pollen concentration;

[0047] Figure 3 D is a graph showing seasonal changes in serum sIgE levels and total pollen concentration;

[0048] Figure 3 E is a graph showing seasonal changes in serum tIgE levels and total pollen concentration;

[0049] Figure 3 F is a graph showing seasonal changes in serum sIgE / tIgE levels and total pollen concentration;

[0050] Artemisia sIgE and tIgE were log2-logarithmized. Q < 0.05; Q < 0.01; Q < 0.001; Q < 0.0001;

[0051] Figure 4 A- Figure 4 F is the correlation analysis diagram between the difference in immunoglobulin E and the changes in symptom severity during the pollen season;

[0052] Figure 4 A is the correlation analysis diagram between ΔsIgE and ΔCSMS in nasal secretions during the pollen season;

[0053] Figure 4 B. Correlation analysis between ΔtIgE and ΔCSMS in nasal secretions during the pollen season;

[0054] Figure 4 C. Correlation analysis between the ΔsIgE / ΔtIgE ratio of nasal secretions and ΔCSMS during the pollen season;

[0055] Figure 4 D is the correlation analysis diagram between serum ΔsIgE and ΔCSMS during the pollen season;

[0056] Figure 4 E is the correlation analysis diagram between serum ΔtIgE and ΔCSMS during the pollen season;

[0057] Figure 4 F is the correlation analysis diagram between the ΔsIgE / ΔtIgE ratio and ΔCSMS during the pollen season;

[0058] Figure 5 Path diagram for mediation analysis. DETAILED DESCRIPTION

[0059] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0060] Example 1 Patient Data and Study Design

[0061] A total of 25 patients were included in this study. All patients had to meet the following inclusion criteria: 1) patients aged 18 to 60 years; 2) permanent residents in Beijing; 3) met the diagnostic criteria for AR in ARIA and had (a) two or more nasal symptoms, including nasal itching, runny nose, sneezing, and nasal congestion, in the past year; (b) ocular symptoms with or without concurrent onset, manifested as itchy eyes, red eyes, and tearing; (c) symptom onset was obvious seasonal, often occurring in autumn; and 4) serum UniCAP test results showed positive Artemisia sIgE (≥ 0.7 kU / L).

[0062] Patients were excluded if they had the following conditions: 1) concurrent non-allergic rhinitis, perennial allergic rhinitis, chronic sinusitis with or without nasal polyps, or other conditions that could interfere with the SAR symptom score; 2) sIgE-positive for other perennial allergens, such as dust mites and animal dander; 3) serum sIgE levels to ragweed or Humulus japonicus ≥ 3.5 kU / L or higher than sIgE to Artemisia; 4) concurrent complex medical conditions such as neoplastic or autoimmune diseases; and 5) women attempting pregnancy or who were pregnant. This study was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University (TRECKY2021-043). Written informed consent was obtained from all patients before participation.

[0063] After signing informed consent, patients entered a four-month follow-up study. Four follow-up points were set up during this period: early pollen season (follow-up point 1, low pollen concentration); peak pollen season (follow-up point 2, high pollen concentration); pollen fall season (follow-up point 3, medium pollen concentration); and pollen season (follow-up point 4). All patients were monitored electronically during the autumn pollen season for daily nasal and eye symptoms and medication use. See the experimental procedures for details. Figure 1 Patients included in the study were allowed to use the first-line AR treatment drugs assigned by the investigators for symptomatic treatment during the trial, including oral loratadine tablets, budesonide nasal spray and / or oral methylprednisolone tablets.

[0064] TNSS is the total nasal symptom score, which includes four common nasal symptoms: nasal itching, nasal congestion, sneezing and runny nose. TNSS = 2.4 + 2.4 + 1.9 + 2.1 = 8.8

[0065] TOSS is the total score for eye symptoms (itching, tearing, and congestion), which includes two common eye symptoms: tearing and itchy / red eyes. TOSS = 1.3 + 1.9 = 3.2;

[0066] DMS, daily medication score, DMS=1.6;

[0067] CSMS, combined symptom-drug score, CSMS=(TNSS+TOSS) / 6+DMS=3.6.

[0068] Example 2 Clinical Evaluation Indicators

[0069] All patients were required to assess their symptoms and medication use daily via an electronic questionnaire. The questionnaire was distributed daily by researchers using a QR code and submitted to the research center via the backend between 6:30 PM and 8:30 AM the following day. The questionnaire used a symptom-drug combined score, equally weighting nasal and ocular symptoms and daily medication use. Symptoms included nasal itching, runny nose, sneezing, nasal congestion, itchy / red eyes, and tearing. Severity was assessed using a four-point scale, ranging from 0 to 3: 0 for no symptom; 1 for mild; 2 for moderate; and 3 for severe. Daily medication use was assessed as 0 for no medication; 1 for antihistamines only; 2 for nasal corticosteroids (regardless of whether antihistamines were used); and 3 for oral corticosteroids (regardless of whether other medications were used). The symptom-drug combined score was the sum of the mean symptom score and the medication use score. Higher scores indicate more severe symptoms.

[0070] Example 3 Sample Detection

[0071] Acquisition of local IgE and serum IgE:

[0072] Local IgE: Before collecting nasal secretions, ensure the subject is at rest and in a comfortable environment. Two cotton pads (Punot, China) (3 × 0.8 × 0.1 cm) were placed in the subject's left and right middle nasal meatus using forceps, adhering to the nasal mucosa for 5 minutes. The cotton pads were removed from the nasal cavity and placed in a slotted tray, which was labeled. A dryer was preheated to 35°C and the tray was placed in the dryer for 2 hours. After drying, the cotton pads were placed in 1.5 mL EP tubes. 500 μL of 0.9% saline solution was added to each tube. The tubes were then mixed by inversion in a micromixer at 4°C for 10 minutes. A small hole was made in the bottom of the 0.5 mL EP tube, and the cotton pads were removed and placed in the 0.5 mL EP tube, taking care to avoid spillage. The 0.5 mL EP tube containing the cotton pads was then placed in the original 1.5 mL EP tube. Centrifuge at 4°C, 12,000 rpm for 5 minutes; after centrifugation, the nasal secretion solution collected in a 1.5 mL tube was stored in a -80°C refrigerator until detection.

[0073] Serum IgE: After the patient's blood sample was centrifuged (3000 rpm, room temperature, 10 minutes), the serum was collected for further analysis.

[0074] IgE detection: Local IgE and serum IgE were detected using the ImmunoCAP 100 system (Pharmacia, Uppsala, Sweden).

[0075] Statistical Methods: Statistical analyses were performed using R software, version 4.1.2. Data are reported as means (with standard deviations) or medians (with interquartile ranges). Daily means were calculated for the six individual symptoms, the medication score, and the combined score. IgE levels were logarithmized due to skewed data distribution.

[0076] Friedman repeated measures rank analysis of variance was used to compare the differences in nasal secretion and serum IgE levels at the four follow-up points.

[0077] Generalized linear mixed models (GLMM) and time series analysis were used to investigate the effect of pollen concentration on symptom severity.

[0078] The correlations between various IgE levels and pollen concentrations and symptom severity were compared using Spearman analysis.

[0079] In addition, causal mediation analysis was performed to clarify the mediating role of changes in IgE levels in nasal secretions during the pollen season between changes in pollen concentration and CSMS differences (V2 - V1 and V3 - V2). The mediation effect was evaluated using direct effect (DE), indirect effect (IE) and calculation of the total effect ratio [IE / (IE+DE)].

[0080] Confidence intervals (CI) were calculated by bootstrap method with 1000 repetitions.

[0081] Example 4 Sample test results

[0082] We further verified the temporal changes in the relationship between pollen concentration and symptom scores using a generalized linear mixed model and found that the combined symptom and medication scores of SAR patients increased with increasing pollen concentration. This trend was also confirmed in single symptom scores and daily medication scores.

[0083] like Figure 2 As shown, pollen concentration showed a significant effect on all response variables analyzed (Q < 0.0001). Significance was assessed using a two-sided Wald test. Solid circles indicate statistically significant parameter estimates (Q < 0.0001), with 95% confidence intervals shown. The ACF was used to calculate the temporal effect of pollen concentration on the day of symptoms ± 7 days. The highest correlation with pollen exposure was observed for the total nasal symptom score and the combined symptom and medication score on the day of symptoms, with the remaining indicators presented with a one-day lag.

[0084] We analyzed the dynamic changes of IgE levels in body fluids during natural pollen exposure by comparing IgE levels in peripheral blood and nasal secretions at each follow-up point.

[0085] In nasal secretions, Artemisia sIgE peaked during the peak pollen season (V2) and was significantly higher than that during the early pollen season (V1) ( Figure 3 A, Q < 0.05). A downward trend in sIgE levels was observed during the pollen fall period (V3) and the non-pollen season (V4), but the difference from the peak period was not statistically significant. No statistical difference was observed in the tIgE levels of nasal secretions at different time periods ( Figure 3 B). Comparison of nasal secretion sIgE / tIgE observed an expression pattern consistent with sIgE, with more significant cross-sectional differences ( Figure 3C, V1 to V2, Q < 0.001; V2 to V3, Q < 0.05). To further explore the relationship between dynamic changes in nasal IgE and pollen concentration, a Spearman correlation test was performed. Only the sIgE / tIgE ratio in nasal secretions (Spearman correlation coefficient r = 0.415, Q < 0.01) was positively correlated with total pollen concentration.

[0086] In serum, Artemisia sIgE was significantly higher in the pollen fall period (V3) than in the pollen early period (V1) and pollen peak period (V2) ( Figure 3 D, V2 vs. V3, Q < 0.05; V1 vs. V3, Q < 0.0001). Meanwhile, in the non-pollen season (V4), sIgE levels did not return to the levels of the early pollen season (Q < 0.0001). Serum tIgE also peaked during the pollen fall period ( Figure 3 -E, Q <0.05). The expression patterns of serum sIgE / tIgE and sIgE were different, and showed significant differences compared with the previous follow-up point in the non-pollen season ( Figure 3 F, V1 vs. V4, Q < 0.05; V2 vs. V4, Q < 0.001). No clear monotonic trend was observed between serum IgE levels and pollen concentration. Considering the specific effect of Artemisia pollen, we repeated the above analysis comparing Artemisia pollen concentration with IgE levels, and the conclusions obtained were consistent with those for total pollen concentration.

[0087] Thus, Artemisia sIgE and sIgE / tIgE levels in nasal secretions from SAR patients reached their highest levels during the peak of the pollen season. Nasal secretion sIgE / tIgE responses showed greater timeliness and a significant positive correlation with changes in pollen concentration. However, serum IgE levels peaked at different times than pollen concentrations, lagging behind changes in pollen concentration.

[0088] We further analyzed whether changes in nasal IgE levels could serve as a quantifiable biomarker to track changes in SAR symptom severity during the pollen season.

[0089] The change in each patient's CSMS score corresponded to the change in their IgE level during the pollen season (expressed as Δ = V2 - V1, V3 - V2). Spearman correlation analysis compared the correlation between ΔCSMS and body fluid ΔIgE levels, and the Spearman r coefficient was annotated. Bonferroni correction was performed for multiple hypothesis testing, and the corrected P value was defined as Q. Spearman correlation analysis showed that Δ nasal secretion sIgE and sIgE / tIgE were significantly positively correlated with ΔCSMS ( Figure 4 A and Figure 4C (r = 0.424, Q < 0.05; r = 0.444, Q < 0.01). In serum, only Δserum sIgE / tIgE showed a significant negative correlation with ΔCSMS ( Figure 4 -F) (r = 0.385, Q < 0.05). Seasonal changes in other IgE variables were not significantly correlated with ΔCSMS.

[0090] Based on the above analysis results, only changes in nasal secretion sIgE / tIgE showed a significant correlation with both pollen concentration and SAR symptoms. To better understand the interrelationships among the three, we conducted a mediation analysis to analyze the mediating effect of nasal secretion ΔsIgE / tIgE between changes in pollen concentration and changes in SAR symptoms during the pollen season. The analytical model is as follows:

[0091] A new variable M is used. If X affects Y by affecting variable M, then M is called a mediating variable. The following regression equation can be used to describe the relationship between the variables.

[0092] Y= cX+e1 (1)

[0093] Y= aX+e2 (2)

[0094] Y= c'X+bM+e3 (3)

[0095] Where X is the change in total pollen concentration under natural pollen exposure, Y is the change in the severity of seasonal allergic rhinitis symptoms, and M is the change in local IgE.

[0096] like Figure 5 Results showed that Δ nasal secretion sIgE / tIgE significantly mediated the effect of pollen on CSMS in SAR patients (mediating effect β = 0.177, 95% CI, P < 0.01), accounting for 27.3% of the total effect. The direct effect between Δ pollen and Δ CSMS was β = 0.471, 95% CI, P < 0.0001, and the total effect β = 0.648, 95% CI, P < 0.0001.

[0097] Example 5 Verification and Analysis Model

[0098] Based on the screening data set, it was found that the changes in sIgE / tIgE in nasal secretions showed a significant correlation trend with the changes in pollen concentration and SAR symptoms, and a mediation effect analysis was performed. The analysis model was the same as that of Example (1)-(3): The placebo group in another clinical study, which was composed of SAR patients, was followed up regularly during the pollen season, and the severity of SAR symptoms was recorded and nasal secretion IgE was tested at different time points, which was consistent with the data type of the screening set.

[0099] The mediation effect analysis was performed on the validation data set, and the regression equation calculated based on the validation data set was as follows:

[0100] Y= 0.007627X-1.691545 (4)

[0101] M= -0.000111X-0.10440 (5)

[0102] Y= 0.006682X-8.49262M-1.780250 (6)

[0103] The results were as follows: Δnasal secretion sIgE / tIgE significantly mediated the effect of pollen on symptom scores in SAR patients (mediation effect β = 0.062, 95% CI, P < 0.01), accounting for 12.4% of the total effect. The direct effect between Δpollen and Δsymptom score β = 0.436, 95% CI, P < 0.0001; the total effect β = 0.498, 95% CI, P < 0.0001.

[0104] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A combined analysis method for allergen activity, characterized in that: (1) preparing a solution for detecting allergen activity, obtaining nasal secretion IgE and serum IgE level values, wherein the nasal secretion IgE and serum IgE level values ​​are sIgE / tIgE values; (2) After obtaining the nasal secretion IgE and serum IgE level values ​​described in (1), analyzing the correlation between seasonal pollen concentration, serum IgE and nasal secretion IgE levels and SAR allergy symptoms and CSMS, specifically comprising the following steps: Friedman repeated-measures rank analysis of variance was used to compare the differences in nasal secretion and serum IgE levels at the four follow-up points at T1; T2 used generalized mixed linear models and time series analysis to investigate the effect of pollen concentration on symptom severity; T3 Spearman analysis was used to analyze the correlation between each IgE level and pollen concentration and symptom severity; T4 used causal mediation analysis to clarify the mediating role of changes in IgE levels in nasal secretions during the pollen season between changes in pollen concentration and CSMS differences, and evaluated the mediation effect using direct effects, indirect effects, and calculation of the proportion of the total effect. T5 calculated the confidence interval by repeating the bootstrap method 1000 times; The CSMC evaluation includes symptom evaluation score and daily medication score, with equal weights balancing nose and eye symptoms and daily medication scores. The calculation method is: CSMS = (TNSS + TOSS) / 6 + DMS (0-6); The causal mediation analysis in step T4 is carried out by constructing an analysis model, specifically by using a new variable M and the following regression equation to describe the relationship between variables: Y= cX+e1 (1) Y= aX+e2 (2) Y= c'X+bM+e3 (3) Where X is the change in total pollen concentration under natural pollen exposure, Y is the change in the severity of seasonal allergic rhinitis symptoms, and M is the change in local IgE.

2. The analysis method according to claim 1, characterized in that The step (1) comprises the following steps: S1 Collection Preparation: Before starting to collect nasal secretions, ensure that the subject is in a resting state and comfortable environment; S2 collection and processing: two cotton pads were placed in the left and right middle nasal passages of the subjects using forceps, adhered to the nasal mucosa, and left for 5-10 minutes; S3 Drying: Remove the cotton pad from the nasal cavity and place it on a slotted tray, marking it. Place the tray in a dryer set at 35°C to 40°C for 2 to 2.5 hours. After S4 centrifugation pretreatment, place the cotton pieces in 1.5 mL EP tubes. Add 500 μL–1000 μL of 0.9% saline solution to each tube. Place the EP tubes in a micromixer and mix thoroughly by inversion at 4°C–10°C for 10–20 minutes. Make a small hole at the bottom of the 0.5 mL EP tube, remove the cotton pieces, and place them in the 0.5 mL EP tube. Then, place the 0.5 mL EP tube containing the cotton pieces in the original 1.5 mL EP tube. S5: Centrifugation treatment: Place the 1.5 mL EP tube treated in step S14 in a centrifuge at 4°C and a speed of 10,000-12,000 rpm for 5-10 minutes; S6 cryopreservation treatment: After processing in a centrifuge, the nasal secretion solution collected in a 1.5 mL tube was stored in an ultra-low temperature freezer at -80°C to -40°C until testing; S7: Collect blood samples and centrifuge them at 3000-3500 rpm, 25-30°C for 10-15 minutes. Serum is then collected for further analysis. S8 After obtaining the nasal secretion solution of step S6 and the serum of step S7, the nasal secretion IgE level and the serum IgE level are detected using the ImmunoCAP 100 system, where the IgE value is the sIgE / tIgE value.

3. The analysis method according to claim 1, characterized in that The symptom evaluation score includes nasal and eye symptom scores, which are graded and quantified into four levels according to severity, ranging from 0 to 3 points. The total nasal symptom score is the sum of the four individual nasal symptoms: nasal itching, runny nose, sneezing, and nasal congestion; The total eye symptom score was the sum of the two individual eye symptoms: itchy / red eyes and tearing.

4. The analysis method according to claim 1, characterized in that The daily medication score of 0 indicates no medication; 1 indicates use of only antihistamines; 2 indicates use of nasal corticosteroids; and 3 indicates use of oral corticosteroids.

5. Use of the combined analysis method for allergen activity according to any one of claims 1 to 4, characterized in that: The invention is used in the preparation of a kit for evaluating the severity of seasonal allergic rhinitis symptoms, wherein the test samples used in the test are nasal secretions and serum of patients with allergic rhinitis.

6. The use according to claim 5, characterized in that The ImmunoCAP 100 system was used to detect the nasal secretion IgE level and the serum IgE level.

7. The use according to claim 5, characterized in that The analysis was a joint analysis of the correlation between seasonal pollen concentration, serum IgE and nasal secretion IgE levels and SAR allergic symptoms and CSMS.

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