Kit for rapidly and efficiently diagnosing lung cancer meningeal metastasis

Abstract

The invention discloses a kit for rapidly and efficiently diagnosing lung cancer meningeal metastasis, and relates to the technical field of medical equipment, and the key points of the technical scheme are as follows: the kit is a disposable plastic shell, and four independent chromatography channels are arranged in parallel in the kit, namely a Compleent C7 channel, a Frizzled-9 channel, an Interleukin-1 receptor protein channel and an Alpha-1-anticmotrypsin channel. Four markers can be detected at the same time, when the kit is used for diagnosing lung cancer meningeal metastasis, the dosage of cerebrospinal fluid can be reduced, the diagnosis sensitivity is improved, the detection time is shortened, the sampling frequency is reduced, and by setting an objective interpretation threshold value and an automatic analysis module, subjective judgment errors are reduced, and diagnosis consistency and repeatability are improved.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and more specifically, to a rapid and efficient reagent kit for diagnosing dural metastases in lung cancer. Background Technology

[0002] Leydral metastasis in lung cancer is a common complication with a very poor prognosis in advanced lung cancer. Early identification and timely intervention are crucial for patient treatment and prolonging survival. However, current diagnostic techniques have some limitations. Commonly used diagnostic methods include cerebrospinal fluid cytology, imaging examinations, and liquid biopsy. Among these, cerebrospinal fluid cytology is currently considered the gold standard for diagnosing leydral metastasis in lung cancer. However, it still has the following drawbacks:

[0003] 1. Cerebrospinal fluid samples require large volumes, typically ≥10 mL;

[0004] 2. The detection sensitivity is low, with a first-positive rate of only 45–67%, and even with multiple sampling (≥3 times), the rate is <90%, delaying the diagnosis time;

[0005] 3. The testing cycle is long, typically taking 24–72 hours from sample collection to report issuance, which cannot meet the needs of rapid clinical decision-making;

[0006] 4. Repeated sampling is required, which not only increases patient discomfort but also raises the risk of complications such as infection and low intracranial pressure.

[0007] 5. It is highly dependent on the operator's experience, and the interpretation of cell morphology is highly subjective, making it easy to miss early or low-burden metastases. Summary of the Invention

[0008] The purpose of this invention is to provide a rapid and efficient reagent kit for diagnosing lung cancer dural metastases. Using this kit to diagnose lung cancer dural metastases can reduce the amount of cerebrospinal fluid used, improve diagnostic sensitivity, shorten detection time, reduce the number of samplings, and reduce subjective judgment errors by setting objective interpretation thresholds and an automated analysis module, thereby improving diagnostic consistency and repeatability.

[0009] The above-mentioned technical objective of this invention is achieved through the following technical solution: a rapid and efficient reagent kit for diagnosing dural metastases in lung cancer, wherein the reagent kit is a disposable plastic shell with four independent chromatography channels arranged in parallel inside, corresponding to:

[0010] ① Complement component C7 channel;

[0011] ② Frizzled-9 channel;

[0012] ③ Interleukin-1 receptor accessory protein channel;

[0013] ④ Alpha-1-antichymotrypsin channel.

[0014] The present invention is further configured such that the dimensions of the reagent kit are 120 mm long × 25 mm wide × 8 mm high.

[0015] The present invention is further configured such that the four independent chromatography channels are arranged sequentially from bottom to top:

[0016] a. Sample pad: pre-loaded with blocking protein + gold-labeled antibody microspheres for accelerated release;

[0017] b. Gold-labeled binding pad: 40 nm colloidal gold-labeled corresponding monoclonal antibody, lyophilized to a concentration of 15 μg / cm²;

[0018] c. NC membrane:

[0019] – T line: Coated with the corresponding capture antibody at a concentration of 300 pg / mL;

[0020] – Line C: Goat anti-mouse IgG 1 mg / mL;

[0021] d. Absorbent pad: ≥300 mm long, ensuring continuous chromatography for 15 min;

[0022] e. Quantitative windows: Four 5 mm × 2 mm reading windows are provided on the shell at the position corresponding to the T / C line, which can be used to obtain the concentration value by direct visual colorimetry or by inserting a colorimetric APP on a mobile phone.

[0023] The present invention is further configured such that the monoclonal antibody includes complementary component C7-mAb1, Frizzled-9-mAb1, Interleukin-1 receptor accessory protein-mAb1, and Alpha-1-antichymotrypsin-mAb1.

[0024] The present invention is further configured such that the capture antibody includes, but is not limited to, the complementary component C7-mAb2.

[0025] In summary, the present invention has the following beneficial effects:

[0026] Detection with minimal sample volume: This invention requires only about 50 μL of cerebrospinal fluid to simultaneously detect four lung cancer-related protein markers. The amount of cerebrospinal fluid required is only 0.5% of that required by traditional methods, successfully achieving "one drop" level diagnosis and completely solving the problem of requiring large amounts of sampling in traditional methods. It is especially suitable for patients with low intracranial pressure, children, weak patients, or patients who have difficulty with multiple sampling.

[0027] The sensitivity of lung cancer meningeal metastasis detection is significantly improved by combining multiple biomarkers: through parallel detection of 4 biomarkers + high-sensitivity immunochromatography, the first positive rate of this invention can be increased to ≥90%, which is close to or exceeds the sum of three cerebrospinal fluid cytology tests, significantly reducing the false negative rate and achieving the diagnostic efficacy of "one test ≈ three cytology tests", thus seizing the golden treatment window.

[0028] Shorten testing time: Test results can be obtained in about 15 minutes.

[0029] Diagnosis can be confirmed with a single sampling: Due to the high reliability of a single test, most patients only need one lumbar puncture to make a clear diagnosis, which significantly reduces complications such as infection, low intracranial pressure, bleeding, and pain caused by repeated punctures, and improves patient compliance and safety.

[0030] Objective quantitative interpretation, eliminating subjective errors: This invention establishes an objective positive threshold by converting "color depth to numerical concentration", eliminating the subjectivity and experience dependence of traditional cytology's "finding cells with the naked eye", achieving standardized, digital, and homogenized diagnosis, and significantly improving the consistency of detection of early or low-load metastases. Attached Figure Description

[0031] Figure 1 This is an example of protein differential analysis between the meningeal metastasis and control groups in this invention. (A) Violin bodies are color-coded according to the disease group and the control group. (B) Hierarchical clustering heatmaps show the quantitative changes in differentially expressed proteins. (C) An overview of quantitative proteomics comparisons. Bar charts indicate the number of proteins that are significantly upregulated (red) and downregulated (blue) in each pair of comparisons. (D) Volcano plots show the distribution of upregulated and downregulated proteins in the quantitative proteomics data.

[0032] Figure 2This is a preliminary exploration of machine learning and model performance evaluation for leptomeningeal metastasis diagnosis in this invention. (A) Flowchart of machine learning. (B) This study included two independent cohorts. The training cohort included 37 LM patients and 20 non-LM patients, all with NSCLC. The testing cohort included 24 LM patients and 14 non-LM patients, all with NSCLC. (C) Expression features were sorted according to their feature scores, and the top 30 features with the highest scores were selected. (D) Expression features were sorted according to their effect size scores and corresponding p-values. In the figure, the x-axis represents the score of each expression feature, and the y-axis represents the p-value transformed by −log10. Features with p-values ​​< 0.05 are shown in red, and features with p-values ​​≥ 0.05 are shown in gray. (E) The calculated accuracy values ​​were plotted against the number of features in the expression feature subset to generate an incremental feature selection (IFS) curve. (FH) Receiver operating characteristic (ROC) curves show the diagnostic accuracy (quantified by area under the curve AUC) of the 12, 8, and 4 protein feature combinations in the training and validation sets. (I) Heatmap of protein expression levels in four protein combinatorial models.

[0033] Figure 3 The PRM method was used to quantitatively analyze the differential expression of LM / NO-LM (LM group, N=12; NO-LM group, N=10). Detailed Implementation

[0034] The present invention will now be described in further detail.

[0035] Example: A rapid and efficient reagent kit for diagnosing leptospirosis in lung cancer. The kit has a disposable plastic casing and its dimensions are 120 mm (length) × 25 mm (width) × 8 mm (height). Internally, it has four independent chromatography channels arranged side-by-side, corresponding to:

[0036] ① Complement component C7 channel;

[0037] ② Frizzled-9 channel;

[0038] ③ Interleukin-1 receptor accessory protein channel;

[0039] ④ Alpha-1-antichymotrypsin channel.

[0040] Four independent chromatography channels are arranged sequentially from bottom to top:

[0041] a. Sample pad: pre-loaded with blocking protein + gold-labeled antibody microspheres for accelerated release;

[0042] b. Gold-labeled binding pad: 40 nm colloidal gold-labeled corresponding monoclonal antibodies, all lyophilized at a concentration of 15 μg / cm²; the monoclonal antibodies include complementary component C7-mAb1, Frizzled-9-mAb1, Interleukin-1 receptor accessory protein-mAb1, and Alpha-1-antichymotrypsin-mAb1.

[0043] c. NC membrane:

[0044] – T line: Coated with the corresponding capture antibody at a concentration of 300 pg / mL; the capture antibody includes, but is not limited to, Complement component C7-mAb2.

[0045] – Line C: Goat anti-mouse IgG 1 mg / mL;

[0046] d. Absorbent pad: ≥300 mm long, ensuring continuous chromatography for 15 min;

[0047] e. Quantitative windows: Four 5 mm × 2 mm reading windows are provided on the shell at the position corresponding to the T / C line, which can be used to obtain the concentration value by direct visual colorimetry or by inserting a colorimetric APP on a mobile phone.

[0048] Application example: The combined detection of four biomarkers in the diagnosis of leptomeningeal metastases in lung cancer.

[0049] 1. Research cohort and sample source

[0050] Cerebrospinal fluid (CSF) samples were collected from 95 patients who were hospitalized in the Department of Oncology at the Second Affiliated Hospital of Nanchang University and were pathologically diagnosed with non-small cell lung cancer (NSCLC) between September 2023 and July 2025. The experimental group (LM group) consisted of 61 patients who met the following criteria: ① positive CSF cytology or ② typical linear / nodular enhancement of the pia mater on MRI and clinically presented with characteristic symptoms of LM such as headache, nausea, and vomiting. The control group (non-LM group) consisted of 34 patients who were negative CSF cytology and showed no signs of pia mater enhancement on MRI.

[0051] 2. Proteomics screening and machine learning feature ranking

[0052] 50 μL of each of 95 samples were collected and identified using a 10X Blood+DIA quantitative proteomics protocol, resulting in the quantification of 1350 differentially expressed proteins. Figure 1Using differential expression analysis between the LM group and the non-LM group (limma-voom, FDR < 0.05 and |log2FC| > 1.5) combined with support vector machine-recursive feature elimination (SVM-RFE) + incremental feature selection (IFS) strategy, the optimal diagnostic feature set of 4 proteins was finally determined: complementary component C7, Frizzled-9, interleukin-1 receptor accessory protein, and Alpha-1-antichymotrypsin. Its AUC in the training set (n=57) was 0.934, with an accuracy of 89.5%, significantly better than a single biomarker (C7 single protein AUC = 0.914, accuracy = 82.5%). Figure 2 (Table 1).

[0053] Table 1. Feature Selection for Machine Learning Models

[0054]

[0055] Note: MCC: Matthews correlation coefficient; AUC: area under the curve; ACC: accuracy.

[0056] 3. Independent cohort PRM target validation

[0057] To further confirm the high expression characteristics of the four-protein combination in the cerebrospinal fluid of LM patients, this invention used parallel response monitoring (PRM) technology to perform targeted validation on 22 CSF samples (12 LM-positive and 10 LM-negative) from an independent prospective cohort. Figure 3 The testing steps are as follows:

[0058] ① Collect and preprocess samples according to the method described in the embodiments;

[0059] ② Synthesize the corresponding isotope-labeled peptides for each protein (1–2 peptides / protein) and add them to the test sample as internal standards;

[0060] ③ Perform PRM scanning on an Orbitrap mass spectrometer to obtain the peak area of ​​each peptide;

[0061] ④ A peak area ratio of ≥1.2 in the LM group to the control group is considered a significant upregulation.

[0062] result:

[0063] Complement component C7 peak area ratio = 1.36;

[0064] Interleukin-1 receptor accessory protein peak area ratio = 1.68;

[0065] Alpha-1-antichymotrypsin peak area ratio = 1.24;

[0066] Frizzled-9 was rejected due to insufficient signal strength.

[0067] The expression levels of the above three proteins in the LM group were significantly higher than those in the control group, confirming the high expression characteristics of the protein combination detected by the kit prepared in this invention in the cerebrospinal fluid of LM patients, and demonstrating its clinical diagnostic value.

[0068] 4. Head-to-head results of reagent kit testing and gold standard

[0069] The "four-channel colloidal gold chromatography kit" prepared in this invention was used to perform blinded retesting on the same 95 samples. Each sample consumed 50 μL, and the T-line color intensity was read within 15 minutes and quantified via a mobile app. The results are shown in Table 2.

[0070] Table 2. Head-to-head results of reagent kit testing and gold standard assay.

[0071]

[0072] 5. Statistics on the reduction in the rate of missed diagnoses

[0073] Of the 61 patients who were ultimately diagnosed with LM, this kit missed only 6 cases (9.8%), while the initial cytology missed 29 cases (47.5%). Among the 29 cytology-negative but clinically highly suspected LM samples in the "window period", this kit was positive in 23 cases, of which 19 were confirmed as LM by subsequent MRI or a second lumbar puncture. This suggests that the LM detection window can be advanced by about 7–14 days, reducing the need for repeated punctures and hospitalization.

[0074] 6. Conclusion

[0075] The above head-to-head studies confirm that the four-channel colloidal gold reagent kit prepared in this invention maintains a specificity of over 94%, while increasing the sensitivity of the first detection of lung cancer leptomeningeal metastases from 52.5% in cytology to 90.2%, with sample consumption only 1 / 20 of the traditional method, and the detection time shortened from 24–72 h to 15 min, significantly reducing patient suffering and medical costs, and has clinical feasibility as an alternative to CSF ​​cytology screening.

[0076] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A rapid and efficient reagent kit for diagnosing leptospirosis in lung cancer, characterized by: The kit is a disposable plastic casing with four independent chromatography channels arranged side-by-side inside, corresponding to: ① Complement component C7 channel; ② Frizzled-9 channel; ③ Interleukin-1 receptor accessory protein channel; ④ Alpha-1-antichymotrypsin channel.

2. The rapid and efficient diagnostic kit for lung cancer leptospirosis according to claim 1, characterized in that: The kit measures 120 mm long × 25 mm wide × 8 mm high.

3. The rapid and efficient diagnostic kit for lung cancer leptospirosis according to claim 1, characterized in that: The four independent chromatography channels are arranged sequentially from bottom to top: a. Sample pad: pre-loaded with blocking protein + gold-labeled antibody microspheres for accelerated release; b. Gold-labeled binding pad: 40 nm colloidal gold-labeled corresponding monoclonal antibody, lyophilized to a concentration of 15 μg / cm²; c. NC membrane: – T line: Coated with the corresponding capture antibody at a concentration of 300 pg / mL; – Line C: Goat anti-mouse IgG 1 mg / mL; d. Absorbent pad: ≥300 mm long, ensuring continuous chromatography for 15 min; e. Quantitative windows: Four 5 mm × 2 mm reading windows are provided on the shell at the position corresponding to the T / C line, which can be used to obtain the concentration value by direct visual colorimetry or by inserting a colorimetric APP on a mobile phone.

4. The rapid and efficient diagnostic kit for lung cancer leptospirosis according to claim 3, characterized in that: The monoclonal antibodies include Complement component C7-mAb1, Frizzled-9-mAb1, Interleukin-1 receptor accessory protein-mAb1, and Alpha-1-antichymotrypsin-mAb1.

5. The rapid and efficient diagnostic kit for lung cancer leptospirosis according to claim 3, characterized in that: The capture antibody includes, but is not limited to, Complement component C7-mAb2.

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