A diagnostic product and predictive system for blast-induced brain injury

Abstract

This invention provides a diagnostic product and a prediction system for blast shockwave-induced brain injury, belonging to the field of in vitro diagnostic technology. The diagnostic product for blast shockwave-induced brain injury provided by this invention includes a neurofilament light chain protein detection reagent and a ubiquitin C-terminal hydrolase L1 detection reagent. By using dual detection of neurofilament light chain protein and ubiquitin C-terminal hydrolase L1, this invention effectively improves the early detection rate of blast shockwave-induced brain injury. At the same time, the diagnostic product provided by this invention also has the advantages of high specificity and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically to a diagnostic product and a predictive system for blast shockwave-induced brain injury. Background Technology

[0002] Blast-wave brain injury (bTBI) is a unique type of traumatic brain injury (TBI). It is a leading cause of injury and death in wartime and industrial explosions. The brain, as a crucial component of the central nervous system, is a primary target organ of blast shock waves. Due to its protection by the skull, brain injuries are more insidious than those to hollow organs such as the lungs, tympanic membranes, and gastrointestinal tract under the same blast load, but the long-term harm is significant. bTBI is characterized by a high incidence, high rate of missed diagnosis, and high rate of chronicity. It not only causes acute physical injury but also has long-term adverse effects such as neuropsychiatric sequelae and loss of social function. Therefore, accurate detection of bTBI is of great importance for patients' quality of life, public health, and the development of blast protection equipment.

[0003] Currently, routine diagnosis of bTBI primarily relies on computed tomography (CT), magnetic resonance imaging (MRI), and the Glasgow Coma Scale (GCS). These methods are effective in diagnosing moderate to severe blast-induced brain injury. However, when patients have mild bTBI, even if the scores indicate damage, traditional imaging techniques such as CT or MRI often fail to detect significant abnormalities. In such cases, novel brain structural / functional imaging techniques such as diffusion tensor imaging (DTI), functional magnetic resonance imaging (fMRI), and magnetic resonance spectroscopy (MRS) are needed for further investigation. However, these devices are extremely expensive and complex to operate, limiting their rapid screening and widespread application in emergency departments. Due to the limitations and lag in imaging diagnosis, many bTBI patients may miss the optimal treatment window.

[0004] Body fluid diagnosis can reflect the occurrence and severity of brain injury in a timely and dynamic manner based on biomarkers in samples. Compared with imaging diagnosis, it has the advantages of timeliness, convenience, and high sensitivity. For example, Chinese invention patent CN118707112A discloses a combined detection kit for GFAP, NFL, and UCH-L1 for early detection of brain injury. However, this kit is mainly for conventional TBI. Due to its unique formation mechanism, the detection sensitivity and accuracy of bTBI by this kit are relatively low.

[0005] Therefore, there is a need to develop a new diagnostic product for brain injury caused by blast shockwaves. Summary of the Invention

[0006] The purpose of this invention is to address the technical deficiency of lacking effective and rapid diagnostic products for brain injury caused by blast shock waves by providing a highly specific rapid diagnostic product.

[0007] To achieve the above objectives, the first aspect of the present invention provides a diagnostic product for blast shock wave-induced brain injury, the diagnostic product comprising a biomarker detection reagent, the biomarker detection reagent being composed of a neurofilament light chain protein detection reagent and a ubiquitin carboxyl-terminal hydrolase L1 detection reagent.

[0008] Preferably, the neurofilament light chain protein detection reagent includes neurofilament light chain protein polyclonal antibody and / or neurofilament light chain protein monoclonal antibody.

[0009] Preferably, the ubiquitin C-terminal hydrolase L1 detection reagent includes ubiquitin C-terminal hydrolase L1 polyclonal antibody and / or ubiquitin C-terminal hydrolase L1 monoclonal antibody.

[0010] Preferably, the diagnostic product for blast shockwave-induced brain injury also includes quality control products and / or calibrators.

[0011] Furthermore, a second aspect of the present invention provides a system for predicting brain injury caused by explosive shockwaves, the system comprising: The data acquisition module is used to acquire and input the detection data of the content of neurofilament light chain protein and ubiquitin carboxyl-terminal hydrolase L1 in the plasma of the subject. The prediction calculation module calculates the probability of a subject suffering from blast shock wave brain injury based on a preset formula and generates a calculation result according to the detection data. An output module is used to output the calculation results.

[0012] Preferably, the preset formula includes a probability calculation formula for impact injury 6 hours later, as shown in formula (1): (1), Where P is the predicted probability of suffering from blast shockwave brain injury, and e is the base of the natural logarithm. NfL The results for the detection of neurofilament light chain protein are expressed in pg / mL. UCH-L1 The results are for the detection of ubiquitin carboxyl-terminal hydrolase L1, and the unit is pg / mL.

[0013] Preferably, the preset formula also includes a probability calculation formula for impact injury 3 days after the injury, as shown in formula (2): (2), Where P is the predicted probability of suffering from blast shockwave brain injury, and e is the base of the natural logarithm. NfL The results for the detection of neurofilament light chain protein are expressed in pg / mL. UCH-L1 The results are for the detection of ubiquitin carboxyl-terminal hydrolase L1, and the unit is pg / mL.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The diagnostic product for blast shock wave brain injury provided by this invention is a rapid and non-invasive detection solution. Compared with traditional CT and MRI detection, the diagnostic product provided by this invention can effectively accelerate the screening process of bTBI and has the advantage of fast detection speed. 2. The diagnostic product for blast shock wave brain injury provided by this invention has the advantages of high sensitivity and specificity, and can achieve early screening of mild bTBI. When used in conjunction with the blast shock wave brain injury prediction system provided by this invention, it can more accurately formulate personalized treatment plans. 3. This invention is the first to demonstrate that the combined use of neurofilament light chain protein and ubiquitin carboxyl-terminal hydrolase L1 can serve as a highly sensitive biomarker for bTBI. The detection results of the biomarker can be used to screen whether the injured person has suffered traumatic brain injury in the early stage, helping medical service providers to allocate medical resources more effectively, carry out intervention as early as possible, and improve treatment outcomes. This is of great significance in bTBI screening and management and can significantly improve the success rate of treatment for traumatic brain injury. Attached Figure Description

[0015] Figure 1 The results show the comparison of NfL content in mouse plasma at different time points after bTBI injury in Example 1 of this invention. Figure 2 This is a comparison of the UCH-L1 content in mouse plasma at different time points after bTBI injury in Example 1 of the present invention; Figure 3 This is the ROC curve of the single NfL index in Example 2 of the present invention, which distinguishes normal mice from those injured by bTBI 6 hours later. Figure 4 This is the ROC curve of the single UCH-L1 index distinguishing normal mice from bTBI-induced mice 6 hours after injury in Example 2 of the present invention; Figure 5 This is the ROC curve of NfL and UCH-L1 dual indicators in Example 2 of the present invention, which distinguishes normal mice from bTBI-induced mice 6 hours later. Figure 6 This is the ROC curve of the single NfL index in Example 2 of the present invention, which distinguishes normal mice from those injured by bTBI 3 days ago. Figure 7This is the ROC curve of the single UCH-L1 index in Example 2 of the present invention, which distinguishes normal mice from those injured by bTBI 3 days ago. Figure 8 This is the ROC curve of NfL and UCH-L1 dual indicators in Example 2 of the present invention, which distinguishes normal mice from bTBI-induced mice 3 days after injury. Detailed Implementation

[0016] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0017] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] As described in the background art, since bTBI has a unique formation mechanism compared with TBI caused by traffic accidents, falls from heights, and violent injuries, conventional body fluid diagnostic methods for traumatic brain injury have the drawbacks of low specificity and sensitivity for bTBI. In view of this, a specific embodiment of the present invention provides a diagnostic product for blast shock wave brain injury, which includes a biomarker detection reagent composed of a neurofilament light chain protein (NfL) detection reagent and a ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) detection reagent.

[0019] In clinical practice, to improve the sensitivity and specificity of body fluid diagnosis, multiple biomarkers are often used in combination to diagnose a particular disease. However, since the formation of bTBI is related to the impact of a high-energy shock wave on the skull surface and the formation of a stress wave within the skull, unlike TBI caused by blunt impact, bTBI usually presents as diffuse damage. In diffuse damage scenarios, the response of existing biomarkers for conventional TBI, such as glial fibrillary acidic protein (GFAP), may be weakened. In a specific embodiment of this invention, NfL and UCH-L1 are used together for the first time as diagnostic biomarkers for blast shock wave brain injury, which has higher diagnostic sensitivity and specificity than using NfL alone; using UCH-L1 alone; or using NfL, UCH-L1, and GFAP in a triple combination.

[0020] In the above embodiments, the neurofilament light chain protein detection reagent includes neurofilament light chain protein polyclonal antibody and / or neurofilament light chain protein monoclonal antibody.

[0021] In the above embodiments, the ubiquitin carboxy-terminal hydrolase L1 detection reagent includes ubiquitin carboxy-terminal hydrolase L1 polyclonal antibody and / or ubiquitin carboxy-terminal hydrolase L1 monoclonal antibody.

[0022] The core principle of the diagnostic product for blast shock wave brain injury provided in the specific embodiments of the present invention is the antigen-antibody binding principle. Specifically, the specific antibody in the diagnostic product provided in the above embodiments can specifically capture the corresponding neurofilament light chain protein and ubiquitin carboxyl-terminal hydrolase L1 to form an antigen-antibody complex.

[0023] In some specific implementations, the aforementioned antigen-antibody complex can be detected using methods such as chemiluminescence, immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), or colloidal gold.

[0024] In the above embodiments, the chemiluminescent luminescent marker can be selected from any one or more of horseradish peroxidase, alkaline phosphatase, acridine ester, isoluminol, and terpyridine ruthenium.

[0025] In the above embodiments, immunoturbidimetry is preferably performed using latex particle-enhanced immunoturbidimetry.

[0026] In the above embodiments, enzyme-linked immunosorbent assay (ELISA) is preferably sandwich ELISA, the enzyme label can be horseradish peroxidase or alkaline phosphatase, and the chromogenic substrate can be tetramethylbenzidine or p-nitrophenyl phosphate.

[0027] In the above embodiments, colloidal gold is preferably colloidal gold immunochromatography.

[0028] In the above embodiments, the diagnostic product for blast shock wave-induced brain injury also includes quality control products and / or calibrators.

[0029] More specifically, in the above embodiments, the concentration of the quality control sample is preferably two.

[0030] More specifically, in the above embodiments, the concentration of the calibrator is preferably 2 to 8.

[0031] More specifically, in the above embodiments, the matrix of the quality control products and calibrators is preferably human plasma.

[0032] A specific embodiment of the present invention also provides a prediction system for blast shockwave-induced brain injury, the prediction system comprising: The data acquisition module is used to acquire and input the detection data of the content of neurofilament light chain protein and ubiquitin carboxyl-terminal hydrolase L1 in the plasma of the subject. The prediction calculation module calculates the probability of a subject suffering from blast shock wave brain injury based on a preset formula and generates a calculation result according to the detection data. An output module is used to output the calculation results.

[0033] In the above embodiments, the preset formula includes a probability calculation formula for impact injury 6 hours later, and the probability calculation formula for impact injury 6 hours later is shown in formula (1): (1), Where P is the predicted probability of suffering from blast shockwave brain injury, and e is the base of the natural logarithm. NfL The results for the detection of neurofilament light chain protein are expressed in pg / mL. UCH-L1 The results are for the detection of ubiquitin carboxyl-terminal hydrolase L1, and the unit is pg / mL.

[0034] In the above embodiments, the preset formula also includes a probability calculation formula for impact injury 3 days after the injury, as shown in formula (2): (2), Where P is the predicted probability of suffering from blast shockwave brain injury, and e is the base of the natural logarithm. NfL The results for the detection of neurofilament light chain protein are expressed in pg / mL. UCH-L1 The results are for the detection of ubiquitin carboxyl-terminal hydrolase L1, and the unit is pg / mL.

[0035] The technical solutions of the present invention are further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. In some cases, terms with conventional meanings are limited herein for clarification or ease of reference, and such limitations should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters. Example 1

[0036] NfL and UCH-L1 as diagnostic markers for brain injury caused by blast shock waves A mouse model of intracranial shock injury was established using a BST-I type bio-shock tube. The specific procedure was as follows: Mice were anesthetized and placed in a fixation device, which was then placed inside a shock tube capable of generating an explosive shock wave. The mice's heads were facing the direction of the incoming shock wave. An injury was induced using a driving pressure of 4.5 MPa. Peripheral blood was collected from the injured animals at 6 h, 3 d, and 7 d post-injury. Plasma was separated, and the levels of NfL and UCH-L1 in the plasma were measured. The differences in the levels of these two biomarkers in mouse plasma at different time points after bTBI injury compared to normal mice were analyzed. Relevant test results are as follows: Figure 1 and Figure 2 As shown, where, Figure 1 The results show the comparison of NfL content in mouse plasma at different time points after bTBI injury, where N represents the control group; Figure 2 This section compares the levels of UCH-L1 in mouse plasma at different time points after bTBI injury, where N represents the control group. Figure 1 and Figure 2 The results showed that, 6 hours after injury, bTBI mice exhibited a trend of increased plasma NfL and significantly increased UCH-L1 compared to normal mice; both biomarkers were significantly elevated 3 days after injury. This indicates that NfL and UCH-L1 have the potential to serve as diagnostic biomarkers for blast shockwave-induced brain injury. Example 2

[0037] Based on whether bTBI injury was induced and the different detection times of biomarkers after injury, the participants were divided into a healthy group, a 6-hour injury group, and a 3-day injury group. The ROC curve was plotted with the diagnosis of the enrolled subjects (the healthy mice were the negative group, and the mice in the 6-hour and 3-day injury groups were the positive groups, respectively) as the classification variable, and the detection results of the two biomarkers and their combinations as variables.

[0038] Figure 3 The ROC curve for distinguishing normal mice from bTBI-induced mice 6 hours after injury using the single NfL index is shown, with an AUC of 0.7214.

[0039] Figure 4 The ROC curve for distinguishing normal mice from bTBI-induced mice 6 hours after injury using the single UCH-L1 index is shown, with an AUC of 0.9529.

[0040] Figure 5 The ROC curves for distinguishing normal mice from bTBI-induced mice 6 hours after injury using NfL and UCH-L1 dual indicators were plotted, with an AUC of 0.9708.

[0041] Figure 6 The ROC curve for distinguishing normal mice from bTBI-induced mice 3 days after injury using the single NfL index has an AUC of 0.8167.

[0042] Figure 7The ROC curve for distinguishing normal mice from bTBI-induced mice 3 days after injury using the single UCH-L1 index is shown, with an AUC of 0.8526.

[0043] Figure 8 The ROC curves of NfL and UCH-L1 in differentiating normal mice from bTBI-induced mice 3 days after injury were plotted, with an AUC of 0.9694.

[0044] Depend on Figures 3-8 The results showed that, whether in the early stage of bTBI (6h) or the late stage of bTBI (3d), the combination of NfL and UCH-L1 showed better diagnostic efficacy than NfL and UCH-L1 single biomarkers, which was more pronounced in the late stage of bTBI.

[0045] Table 1 shows the AUC, cutoff value, sensitivity, and specificity of single-indicator and dual-indicator methods 6 hours after bTBI injury.

[0046] Table 1 Table 2 shows the AUC, cutoff value, sensitivity, and specificity of single-indicator and dual-indicator methods 3 days after bTBI injury.

[0047] Table 2 As shown in Tables 1 and 2, the combination of NfL and UCH-L1 as dual indicators can achieve a sensitivity of 89.5%, a specificity of 100%, and an area under the ROC curve (AUC) of 97.1% 6 hours after injury. The combination of NfL and UCH-L1 as dual indicators can achieve a sensitivity of 100%, a specificity of 88.9%, and an AUC of 96.9% 3 days after injury. Both the sensitivity and specificity are significantly higher than those of single indicators. Example 3

[0048] Prediction of brain injury caused by blast shockwave A multiple linear regression model was used to establish the relationship between the biomarker combination and bTBI in the samples detected in Example 1. Multiple regression analysis was performed using SPSS statistical software to obtain estimated values ​​of the regression coefficients. Based on the estimated values ​​of the regression coefficients, the influence of different biomarker concentrations on bTBI was explained, and the regression coefficients composed of the two biomarkers were finally obtained, as shown in formulas (1) and (2), respectively.

[0049] The probability calculation formula for impact injury 6 hours later is shown in formula (1), which is: (1), Where P is the predicted probability of suffering from blast shockwave brain injury, and e is the base of the natural logarithm. NfL The results for the detection of neurofilament light chain protein are expressed in pg / mL. UCH-L1 The results are for the detection of ubiquitin carboxyl-terminal hydrolase L1, and the unit is pg / mL.

[0050] The probability calculation formula for impact injury 3 days later is shown in formula (2), which is: (2), Where P is the predicted probability of suffering from blast shockwave brain injury, and e is the base of the natural logarithm. NfL The results for the detection of neurofilament light chain protein are expressed in pg / mL. UCH-L1 The results are for the detection of ubiquitin carboxyl-terminal hydrolase L1, and the unit is pg / mL.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A diagnostic product for brain injury caused by explosive shockwaves, characterized in that, The diagnostic product for blast shockwave-induced brain injury includes biomarker detection reagents, which consist of neurofilament light chain protein detection reagents and ubiquitin carboxyl-terminal hydrolase L1 detection reagents.

2. The diagnostic product for blast shockwave-induced brain injury as described in claim 1, characterized in that, The neurofilament light chain protein detection reagent includes neurofilament light chain protein polyclonal antibodies and / or neurofilament light chain protein monoclonal antibodies.

3. The diagnostic product for blast shockwave-induced brain injury as described in claim 1, characterized in that, The ubiquitin carboxyl-terminal hydrolase L1 detection reagent includes ubiquitin carboxyl-terminal hydrolase L1 polyclonal antibody and / or ubiquitin carboxyl-terminal hydrolase L1 monoclonal antibody.

4. The diagnostic product for blast shockwave-induced brain injury as described in claim 1, characterized in that, The diagnostic products for blast shockwave-induced brain injury also include quality control products and / or calibrators.

5. A system for predicting brain injury caused by explosive shock waves, characterized in that, The blast shockwave brain injury prediction system includes: The data acquisition module is used to acquire and input the detection data of the content of neurofilament light chain protein and ubiquitin carboxyl-terminal hydrolase L1 in the plasma of the subject. The prediction calculation module calculates the probability of a subject suffering from blast shock wave brain injury based on a preset formula and generates a calculation result according to the detection data. An output module is used to output the calculation results.

6. The blast shockwave-induced brain injury prediction system as described in claim 5, characterized in that, The preset formula includes a probability calculation formula for impact injury 6 hours later, as shown in formula (1): （1）， Where P is the predicted probability of suffering from blast shockwave brain injury, and e is the base of the natural logarithm. NfL The results for the detection of neurofilament light chain protein are expressed in pg / mL. UCH-L1 The results are for the detection of ubiquitin carboxyl-terminal hydrolase L1, and the unit is pg / mL.

7. The blast shockwave-induced brain injury prediction system as described in claim 5, characterized in that, The preset formula also includes a probability calculation formula for impact injury 3 days after the injury, as shown in formula (2): （2）， Where P is the predicted probability of suffering from blast shockwave brain injury, and e is the base of the natural logarithm. NfL The results for the detection of neurofilament light chain protein are expressed in pg / mL. UCH-L1 The results are for the detection of ubiquitin carboxyl-terminal hydrolase L1, and the unit is pg / mL.

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