Novel serum protein marker combination for evaluating depth of anesthesia and application of novel serum protein marker combination

Abstract

The invention belongs to the technical field of in-vitro diagnosis and anesthesia monitoring. The invention discloses a serum protein marker combination for evaluating anesthesia depth and application of the serum protein marker combination. The composition is composed of orexin, S100 calcium binding protein B, C reactive protein and gamma-aminobutyric acid A type receptor alpha1 subunit, and can synergistically reflect the anesthesia sedation state from multiple dimensions. The invention also provides a detection kit containing the marker and application of the detection kit in evaluation of anesthesia depth. The combination has the advantages of being high in accuracy, objective, stable and easy to detect, can serve as an effective supplementary tool of an existing nerve electrophysiology monitoring technology, and provides a new reference means for intraoperative sedation depth monitoring and precise anesthesia management.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and anesthesia monitoring technology, specifically relating to a novel combination of serum protein biomarkers for assessing the depth of anesthesia and its applications. Background Technology

[0002] In the management of general anesthesia, accurate monitoring of sedation depth is a crucial step in ensuring surgical safety and improving patient outcomes. Insufficient sedation may lead to intraoperative awareness and drastic hemodynamic fluctuations, while excessive sedation is closely associated with adverse outcomes such as postoperative delirium, cognitive impairment, prolonged awakening time, and longer hospital stay.

[0003] Currently, the widely used tools for monitoring the depth of anesthesia in clinical practice mainly rely on neurophysiological devices based on electroencephalography (EEG), such as the bispectral index (BIS). However, this type of technology has the following drawbacks: First, it is susceptible to interference. Electromagnetic signals generated by devices such as electrosurgical units and electrocoagulation devices in the operating room can severely interfere with EEG signals, leading to distorted or interrupted monitoring values. Second, there are individual and drug variability. BIS values ​​vary for patients of different ages, with different pathophysiological states, and with different types of anesthetic drugs, and are not applicable to all populations. Third, the cost is high. Some more accurate EEG monitoring methods require the attachment of multiple electrodes, which is cumbersome to operate, and the equipment and consumables are expensive.

[0004] Therefore, there is an urgent clinical need for a highly accurate, objective, stable, and easily applicable detection method to compensate for the shortcomings of existing neurophysiological monitoring technologies and provide an effective supplementary tool for achieving individualized and precise anesthesia. Summary of the Invention

[0005] The purpose of this invention is to provide a novel combination of serum protein biomarkers, a detection kit, and their applications for assessing the depth of anesthesia, in order to solve or at least partially alleviate the problems of existing neurophysiological monitoring techniques being susceptible to interference, having individual variability, and being costly, and to provide a supplementary detection method that is highly accurate, objective, stable, and easy to promote.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a combination of serum protein biomarkers for assessing the depth of general anesthesia, the combination comprising orexin, S100 calcium-binding protein B, C-reactive protein and γ-aminobutyric acid type A receptor α1 subunit.

[0007] Secondly, the present invention provides a kit for detecting the serum protein biomarker combination described in the first aspect, the kit comprising reagents for the specific quantitative detection of the four proteins in a biological sample. Preferably, the kit comprises a solid-phase support having independent reaction regions thereon, each coated with a capture molecule targeting the four proteins.

[0008] Thirdly, the present invention provides a method for detecting a combination of serum protein biomarkers for assisting in the assessment of the depth of sedation under general anesthesia, comprising: detecting the concentrations of orexin, S100 calcium-binding protein B, C-reactive protein, and γ-aminobutyric acid type A receptor α1 subunit in a biological sample from a subject; and obtaining the concentration data of each of the four proteins.

[0009] Fourthly, the present invention provides the use of the serum protein biomarker combination or the kit in the preparation of in vitro diagnostic products for detecting or assisting in the assessment of the depth of sedation under general anesthesia. The assessment includes distinguishing between adequate and excessive sedation.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. For the first time, four key proteins characterizing arousal inhibition, glial stress, systemic inflammatory background, and the activity of core drug targets have been integrated into a single diagnostic synergy. This synergy simultaneously acquires information from multiple key pathways of anesthesia and sedation, and overcomes the deficiency of single biomarker specificity through mutual supplementation and validation of multiple indicators. Its combined diagnostic model demonstrates higher accuracy in distinguishing sedation depth.

[0011] 2. The peripheral blood sample-based detection method is minimally invasive, allows for repeated sampling, and facilitates dynamic monitoring throughout the perioperative period. This method is unaffected by physical interference such as intraoperative electrocautery, and the results are objective and stable.

[0012] 3. The selected biomarkers are all known proteins, and their commercial detection reagents are readily available. The technology for developing standardized immunoassay kits is mature, and the barriers to industrialization and clinical application are low.

[0013] 4. The biomarker system and method provided by this invention can serve as an independent complementary tool to existing electroencephalogram (EEG) monitoring. When BIS signals are interfered with or insensitive to specific populations, they can provide anesthesiologists with objective biochemical references, helping to achieve more refined individualized sedation management. Attached Figure Description

[0014] Figure 1 This is a bar chart comparing serum orexin levels in patients in the appropriate sedation group and the excessively sedated group in Example 1 of the present invention. Figure 2 This is a bar chart comparing serum S100B protein levels in patients in the appropriate sedation group and the excessively sedated group in Example 1 of the present invention. Figure 3 This is a bar chart comparing serum CRP levels in patients in the appropriate sedation group and the excessively sedated group in Example 1 of the present invention. Figure 4This is a bar chart comparing the levels of GABA_A receptor α1 subunit in serum exosomes from patients in the appropriate sedation group and the excessively sedated group in Example 1 of the present invention. Figure 5 This is a receiver operating characteristic (ROC) curve diagram of four serum protein biomarkers used individually and in combination to differentiate between adequate and excessive sedation in Embodiment 1 of the present invention. Detailed Implementation

[0015] The present invention will be described in detail below through examples. These examples are merely illustrative and do not constitute a limitation on the scope of protection of the present invention. Unless otherwise stated, the methods, reagents, and materials used in the present invention are all conventional in the art or commercially available.

[0016] It should be noted that the embodiments of this invention are confirmatory studies, which use the bispectral index (BIS) of electroencephalography (EEG), a routine clinical monitoring tool, as a reference for grouping, aiming to explore serum protein biomarkers related to anesthesia and sedation. This invention aims to provide an objective and stable supplementary means of biochemical testing, especially to provide additional reference when BIS monitoring is limited or individual differences exist, rather than replacing existing neurophysiological monitoring. This invention provides objective biochemical reference data for clinicians by detecting the concentration of the aforementioned biomarker combination; the final clinical decision on sedation depth should be made by the physician by comprehensively considering the results of this test, other monitoring indicators, and clinical experience.

[0017] For the sake of brevity, orexin (also known as orexin A), S100 calcium-binding protein B (S100B), C-reactive protein (CRP), and γ-aminobutyric acid type A receptor α1 subunit (GABA_A Rα1) will be collectively referred to as the "four biomarkers" or their common abbreviations.

[0018] Example 1: Validation Study of Biomarker Combinations 1. Research Subjects and Sample Collection This was a prospective observational study, and all patients signed informed consent forms before the procedure.

[0019] Inclusion criteria: Age 18-75 years, American Society of Anesthesiologists (ASA) classification I-III, and scheduled for elective abdominal or orthopedic surgery under general anesthesia.

[0020] Exclusion criteria: severe neurological disease, severe liver or kidney dysfunction, systemic infection or active chronic inflammatory disease within one month prior to surgery, long-term use of glucocorticoids or immunosuppressants, or history of abuse of psychotropic drugs.

[0021] Anesthesia and monitoring protocol: All patients received standardized general anesthesia, such as target-controlled infusion of propofol combined with remifentanil, or maintenance with sevoflurane inhalation. Bispectral index (BIS), mean arterial pressure (MAP), and heart rate (HR) were monitored throughout the procedure.

[0022] Grouping and Sample Collection: Oversedation Group: During the maintenance of anesthesia, due to changes in surgical stimulation or routine adjustments by the anesthesiologist based on hemodynamics, some patients experienced a natural drop in BIS value that remained below 40 for more than 10 minutes. This state was defined as the "oversedation event period." The onset time of the event was recorded, and 5 mL of peripheral venous blood was collected from the patient when this state lasted for 10 minutes. Appropriate Sedation Group: Patients whose BIS value remained consistently between 45-60 throughout the entire maintenance of anesthesia, and whose hemodynamics were stable and did not require significant adjustments due to oversedation or undersedation, were selected. 5 mL of peripheral venous blood was collected 1 hour after the start of surgery.

[0023] Sample processing: After blood samples are left to stand at room temperature for 30 minutes, they are centrifuged at 3000 rpm for 10 minutes to carefully separate the serum. The serum is then aliquoted and stored in an ultra-low temperature freezer at -80℃ to avoid repeated freeze-thaw cycles.

[0024] 2. Detection Method Orexin-A, S100B, and CRP: Validated commercial human ELISA kits were used, and all procedures were strictly performed according to the instructions. All samples were tested from the same batch.

[0025] GABA_A receptor α1 subunit: 200 μL of serum was collected, and exosomes were purified using an ultracentrifugation method combined with an exosome extraction kit. For example, Thermo Fisher Scientific's Total Exosome Isolation Reagent was used. Exosome protein concentration was determined using the BCA method. The GABA_A Rα1 content in the exosome lysate was detected using a commercially available ELISA kit specifically targeting the human GABA_A Rα1 subunit. The final result is expressed as ng / mg total exosome protein.

[0026] 3. Statistical Analysis SPSS 26.0 software was used. Normally distributed continuous data were expressed as mean ± standard deviation, and independent samples t-tests were used for comparisons between groups. Non-normally distributed data were expressed as median, and the Mann-Whitney U test was used. Chi-square tests were used for categorical data.

[0027] A joint diagnostic model was constructed using binary logistic regression. The regression equation was obtained with grouping (appropriate sedation = 0, excessive sedation = 1) as the dependent variable and the detection values ​​of four biomarkers as the independent variables.

[0028] Receiver operating characteristic (ROC) curves were plotted for the predicted probabilities of each individual biomarker and the combined model. The area under the curve (AUC), optimal cutoff value, sensitivity, and specificity were calculated. A p-value < 0.05 was considered statistically significant.

[0029] 4. Results 4.1 Comparison of basic data and single markers A total of 100 patients were ultimately included, with 50 in the appropriate sedation group and 50 in the excessive sedation group. There were no statistically significant differences between the two groups in baseline characteristics such as age, sex, BMI, ASA classification, type of surgery, and duration of surgery (P>0.05), making them comparable.

[0030] Table 1 Comparison of serum marker levels between the two groups of patients Note: *P<0.05, **P<0.01, ***P<0.001 vs. appropriate sedation group.

[0031] As shown in Table 1 and Figure 1-4 As shown, compared with the appropriate sedation group, patients in the excessively sedated group had significantly lower serum orexin levels (P<0.001), significantly higher serum S100B and CRP levels (P<0.01), and significantly higher serum exosomal GABA_A Rα1 subunit levels (P<0.01).

[0032] 4.2 Diagnostic efficacy analysis like Figure 5 As shown in the ROC curve analysis, among the single biomarkers, orexin had the highest AUC, at 0.83 (95% CI: 0.75–0.90). The AUCs for S100B, CRP, and GABA_A Rα1 were 0.76 (95% CI: 0.67–0.84), 0.74 (95% CI: 0.65–0.83), and 0.75 (95% CI: 0.66–0.84), respectively. The combined diagnostic model of the four biomarkers constructed using logistic regression showed an AUC of 0.92 (95% CI: 0.87–0.97). At the optimal cutoff value, the sensitivity was 88.0%, and the specificity was 86.0%.

[0033] Conclusion: Under natural clinical conditions, the four serum protein biomarkers provided by this invention showed significant differences in distinguishing between patients who were appropriately sedated and those who were excessively sedated. Furthermore, their combined use has significant synergistic diagnostic value, and their accuracy is significantly better than that of any single biomarker.

[0034] Example 2: Construction of a multiplex detection ELISA kit This embodiment provides an ELISA kit that can simultaneously detect four biomarkers.

[0035] 1. Kit Components Microplate: 96-well plate, each well is physically divided into four independent reaction microcells.

[0036] Coating antibodies: The four micropools were pre-coated with monoclonal antibodies against human orexin, human S100B, human CRP and human GABA_AR α1 subunit, respectively.

[0037] Standards: Four lyophilized powders, which, after reconstitution, form a series of concentration gradients, corresponding to recombinant human orexin, S100B, CRP, and GABA_A Rα1 protein, respectively.

[0038] Detection antibodies: ready-to-use mixture containing horseradish peroxidase (HRP)-labeled detection antibodies against the four proteins mentioned above.

[0039] Auxiliary reagents: sample diluent, 20× concentrated wash buffer, TMB chromogenic substrate solution A and B, stop solution (2MH2SO4), sealing film, product instructions.

[0040] 2. Brief Description of the Testing Process Add the appropriately diluted serum sample or standard to each micropool.

[0041] Incubation and washing: Incubate at 37°C for 60 minutes, then wash thoroughly.

[0042] Add enzyme-labeled antibody: Add HRP-labeled detection antibody mixture, incubate at 37°C for 45 minutes, and wash.

[0043] Developing and stopping: Add TMB developing solution, develop color in the dark, and then add stop solution.

[0044] Reading and calculation: The absorbance value of each microcell at 450 nm was read using an ELISA reader, and the concentration of the four proteins in the sample was calculated according to their respective standard curves.

[0045] This kit enables high-throughput, simultaneous, and quantitative detection of the core biomarker combination of this invention. It is easy to operate and suitable for use in clinical laboratories.

[0046] Example 3: Clinical validation test of the reagent kit To verify the clinical performance of the kit of the present invention, the reserved independent validation set samples were tested using the quadruple ELISA kit prepared according to the principle described in Example 2, and the results were compared with the clinical BIS monitoring results.

[0047] 1. Test Samples and Methods Sample set: In this study design, we randomly reserved 40 out of a total of 100 cases as an independent validation set, which was not used in the construction of the Logistic regression combined diagnostic model in Example 1. In this validation set, 20 cases were judged as "appropriately sedated" and 20 cases as "excessively sedated" based on clinical BIS records.

[0048] Reagent kit: Using the prototype quadruple ELISA kit described in this invention, all validation set samples were tested under the same detection conditions to obtain the concentration values ​​of orexin, S100B, CRP, and GABA_A Rα1.

[0049] Diagnostic Model: The detected values ​​were substituted into a Logistic Regression Joint Diagnostic Model established from the training set (60 samples) to calculate the predicted probability P for each validation set sample. The higher the P value, the greater the probability of "over-sedation". The optimal cutoff value (P=0.55) determined by this model on the training set was used for grouping and judgment.

[0050] 2. Test Results 2.1 Examples of Partial Sample Detection Data and Judgment Results Table 2. Detection results and judgment examples for part of the validation set samples. Table 2 presents detailed test data, model-calculated P-values, and kit-based judgment results for 8 samples in the validation set (4 with appropriate sedation and 4 with excessive sedation), and compares them with clinical BIS status.

[0051] 2.2 Overall Validation Performance Summary Based on the detection and judgment results of all 40 independent validation set samples, the overall performance of the combined diagnostic model of the kit is summarized in Table 3.

[0052] Table 3 Diagnostic performance of the reagent kit combination model on the independent validation set. 3. Conclusion As shown in Tables 2 and 3, in the independent validation set reserved for this study, the quadruple diagnostic kit provided by this invention achieved an overall diagnostic accuracy of 85.0% by detecting four serum protein biomarkers and applying a combined diagnostic model, with a balance between sensitivity and specificity. A few samples observed in the study that were inconsistent with BIS determination (such as V-03) suggest that changes in serum protein biomarkers may have different temporal dynamics or physiological associations with EEG signals, which warrants further investigation in subsequent studies. The above results demonstrate the good clinical diagnostic potential of this kit, providing preliminary and objective data support for its further clinical translation and application. Future research needs to validate its universality and clinical efficacy in larger-scale, multi-center prospective studies.

[0053] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination of serum protein biomarkers for assessing the depth of general anesthesia, characterized in that, The biomarker combination consists of orexin, S100 calcium-binding protein B, C-reactive protein, and the α1 subunit of the γ-aminobutyric acid type A receptor.

2. A kit for detecting the combination of serum protein biomarkers according to claim 1, characterized in that, The kit contains reagents for the specific quantitative detection of the four proteins in biological samples.

3. The reagent kit according to claim 2, characterized in that, It includes a solid support, on which independent reaction regions are provided, each coated with a capture molecule targeting the four proteins.

4. The reagent kit according to claim 3, characterized in that, The solid support is a microplate, with each well physically divided into four independent microcells, each coated with an anti-human orexin antibody, an anti-human S100 calcium-binding protein B antibody, an anti-human C-reactive protein antibody, and an anti-human γ-aminobutyric acid type A receptor α1 subunit antibody.

5. A method for detecting a combination of serum protein biomarkers to aid in assessing the depth of sedation during general anesthesia, characterized in that, include: The concentrations of orexin, S100 calcium-binding protein B, C-reactive protein, and γ-aminobutyric acid type A receptor α1 subunit were detected in biological samples from subjects; and the concentration data of each of the four proteins were obtained.

6. The use of the biomarker combination of claim 1 or the kit of any one of claims 2 to 4 in the preparation of in vitro diagnostic products for detecting or assisting in the assessment of the depth of sedation under general anesthesia.

7. The application according to claim 6, characterized in that, The assessment includes distinguishing between appropriate sedation and excessive sedation.

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