Immunochromatography test strip and kit for detecting selurimab and application of immunochromatography test strip and kit

Abstract

The invention discloses an immunochromatography test strip and a kit for detecting a Slurimab and application of the immunochromatography test strip and the kit. According to the present invention, the fluorescence immunochromatography method has characteristics of simple operation, rapidness, quantitative result and the like, such that the high-sensitivity ([mu] g / ml), rapid and high-throughput detection and analysis of the Slurimab in the clinical samples such as the trace plasma (10 [mu] l) can be conveniently and rapidly achieved, and the Slurimab does not need to be purified; and the fluorescent immunochromatography test strip is convenient to prepare, about 200 test strips can be prepared from each prepared plate, and the test strip is suitable for large-scale detection and verification of the exposure concentration of the Slurimab of a clinical sample, so that the blank that no Slurimab drug exposure concentration detection is carried out clinically is filled up.

Description

Technical Field

[0001] The present invention belongs to the technical field of immune checkpoint inhibitors, and relates to an immunochromatographic test strip for detecting slulizumab, a kit and a detection system comprising the test strip, and also relates to an application of the test strip in the survival prognosis assessment of malignant tumors. Background Art

[0002] In recent years, immune checkpoint inhibitors (ICIs) have been shown to have significant therapeutic effects on a variety of tumors. Slulizumab is a monoclonal antibody among immune checkpoint inhibitors. It mainly blocks the binding of PD-1 on the surface of T cells to PD-L1 on the surface of tumor cells, thereby enhancing the anti-tumor immune function of T cells and preventing the proliferation and spread of cancer cells. The clinical application of slulizumab has brought patients better efficacy and medication compliance. However, the inter-individual efficacy differences in the use of slulizumab are still a huge obstacle to its clinical application.

[0003] For example, the results of the ASTRΜM-005 randomized clinical trial showed that 57.3% of patients treated with slulizumab combined with chemotherapy experienced disease progression or death, with a median progression-free survival of 5.7 months. In a phase 1 trial, slulizumab up to 10 mg / kg was well tolerated and showed similar pharmacokinetic characteristics to pembrolizumab and nivolumab. Bellesoeμr et al. demonstrated that patients with non-small cell lung cancer received 3 mg / kg Q2W nivolumab. After two cycles of administration, patients with Cmin ≤ 24.7 μg / ml tended to have a longer OS. Therefore, the factor that improves survival outcomes with slulizumab may be related to exposure concentration, and monitoring of exposure concentration has important application value for individualized dosing and efficacy prediction of slulizumab.

[0004] The detection methods of monoclonal antibody drugs generally include ELISA, LC-MS / MS, and chemiluminescence. The detection of slulizumab drug exposure concentration is almost blank in clinical application. The traditional LC-MS for monoclonal antibody drug concentration monitoring is costly, cumbersome, time-consuming, and difficult to develop methodology. For ELISA or chemiluminescence immunoassay methods, there are problems such as long detection time, large chemiluminescence equipment, and high detection cost.

[0005] Fluorescence immunochromatography has the characteristics of simple operation, rapidity and quantitative results. It can easily achieve high-sensitivity (μg / ml), rapid and high-throughput detection and analysis of slulizumab in clinical samples such as trace plasma (10μl) without the need to purify slulizumab. Each plate prepared can make approximately 200 test strips, which is suitable for large-scale clinical sample slulizumab exposure concentration detection and verification. The detection principle of the immunochromatographic test strip is: first, the PD-1 protein is coated on the test line (C line), and then the sample containing slulizumab is added to the detection well. Slulizumab can recognize the PD-1 protein and capture it; finally, the exposure level of slulizumab is quantitatively determined using an immunofluorescence analyzer.

[0006] At present, the detection of slulizumab drug exposure concentration has rarely been carried out clinically, and the relationship between drug exposure concentration and efficacy and efficacy prediction is also unclear. Therefore, it is difficult to carry out clinical monitoring of slulizumab drug concentration. Summary of the invention

[0007] The present invention aims at the above problems, explores the immunochromatographic detection of slulizumab, and provides an immunochromatographic test strip, a kit and a detection system for detecting slulizumab, which are used to predict the efficacy of slulizumab combined with chemotherapy for lung cancer.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] In the first aspect of the present invention, there is provided an immunochromatographic test strip for detecting slulizumab, comprising a bottom plate and a sample pad, a nitrocellulose membrane and a water-absorbing pad sequentially mounted on the bottom plate. The sample pad is provided with a sample addition hole; the nitrocellulose membrane is provided with a quality control line and a detection line, the quality control line is coated with GAM antibody, and the detection line is coated with recombinant PD-1 protein.

[0010] In a second aspect, the present invention provides an immunochromatography kit for detecting slulizumab, comprising an immunochromatography test strip and a sample processing solution.

[0011] Among them, the immunochromatographic test strip is as described above; the sample processing solution is composed of a sample diluent and a fluorescent solution, and the fluorescent solution is composed of quantum dot nanospheres coated with anti-PD-1 monoclonal antibodies and a fluorescent antibody diluent.

[0012] Preferably, the sample diluent is PBS; the mass-to-volume ratio between the quantum dot nanospheres of the anti-PD-1 monoclonal antibody and the fluorescent antibody diluent is 1:300.

[0013] The immunochromatographic kit also includes a slulizumab standard plasma sample as a control stock solution.

[0014] Principle of immunochromatography Figure 1It is an immunofluorescence competitive inhibition method. The slulizumab exposed in the sample will compete with the quantum dot nanosphere-slulizumab conjugate to be captured by the PD-1 protein fixed on the detection line (T line), and the quality control line (C line) will bind to the remaining fluorescent conjugate. The fluorescence intensity of the detection line is inversely proportional to the exposure concentration of slulizumab in the sample.

[0015] In a third aspect, the present invention provides a srulizumab exposure concentration detection system, comprising an immunochromatography kit for detecting srulizumab, a fluorescence intensity detection device, and a result analysis system.

[0016] Among them, the immunochromatographic kit for detecting slulizumab is as described above;

[0017] The fluorescence intensity detection system is used to detect the fluorescence intensity on the line;

[0018] The result analysis system includes an input display module, an analysis module and a storage module. The input display module is used to input the basic information of the patient and display the analysis results; the analysis module obtains the exposure concentration of slulizumab based on the standard curve and fluorescence intensity analysis in the storage module.

[0019] Preferably, the standard curve is obtained based on slulizumab standard plasma samples. Prepare slulizumab standard plasma samples of different concentrations, mix them with fluorescent liquid by pipetting, perform immunochromatography analysis, read the slulizumab fluorescence intensity T1 and the mean fluorescence intensity C of the quality control line, and fit the standard curve through the T / C value: y=A2+(A1-A2) / (1+(x / x0) p ), where A1 = 18.58 ± 0.04, A2 = 0.03 ± 0.04, x0 = 97.41 ± 0.26, p = 1.33 ± 0.05, in which y is the T / C value, x is the concentration of slulizumab, R 2 =0.99997.

[0020] In a fourth aspect, the present invention provides the use of the above-mentioned slulizumab exposure concentration detection system in the survival prognosis assessment of malignant tumors.

[0021] Preferably, since slulizumab is currently mainly used in combination with chemotherapy to treat lung cancer in clinical practice, the application is to evaluate the efficacy of slulizumab combined with chemotherapy on lung cancer, and then predict the survival period.

[0022] In the specific implementation of the present invention, among the collected patients, KM survival analysis (Log-rank) was performed according to the exposure concentration threshold determined by the exposure concentration-related efficacy, and the patients were divided into two groups: exposure concentration ≤ 51 μg / mL and exposure concentration > 51 μg / mL. The results showed that the PFS of patients with exposure concentration > 51 μg / mL was significantly longer than that of patients with exposure concentration ≤ 51 μg / mL. Therefore, the threshold of slulizumab exposure concentration is 51 μg / mL.

[0023] In a fifth aspect, the present invention provides a method for detecting the exposure concentration of slulizumab, which is performed using the above-mentioned slulizumab exposure concentration detection system, and comprises the following steps:

[0024] (1) Take a certain volume of fresh or thawed plasma sample (preferably 10 μL) and dilute it to 10 times (100 μL) with 1× PBS;

[0025] (2) Take a certain volume of treated sample (preferably 50 μL) and add an equal volume of fluorescent liquid to it. After blowing and mixing evenly, add the sample to the sample well of the immunochromatographic test strip and analyze it with a fluorescence meter electrically connected to the test strip. Wait for 15 minutes and then read the exposure concentration value of slulizumab.

[0026] In the sixth aspect of the present invention, a method for evaluating the efficacy of combined treatment of malignant tumors (lung cancer) with slulizumab and chemotherapy is provided by detecting the exposure concentration of slulizumab. According to the comparison between the exposure concentration value of slulizumab in the patient's plasma sample and the threshold value (51 μg / mL), the greater the exposure concentration is above the threshold value, the more obvious the PFS extension is, and the better the prognosis is.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention utilizes the characteristics of simple operation, rapidity and quantitative results of fluorescent immunochromatography, and can conveniently detect and analyze slulizumab in clinical samples such as trace plasma (10 μl) with high sensitivity (μg / ml), rapidity and high throughput, without the need to purify slulizumab. The fluorescent immunochromatography test strips are easy to prepare, and each plate can be used to make about 200 test strips, which is suitable for large-scale clinical sample slulizumab exposure concentration detection and verification.

[0029] At present, the detection of slulizumab drug exposure concentration has hardly been carried out clinically, and the relationship between drug exposure concentration and drug efficacy and efficacy prediction is unclear. Therefore, clinical monitoring of slulizumab drug concentration is also difficult to carry out. The present invention fills this gap. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It shows the working principle of immunochromatographic test strips;

[0031] Figure 2 The linearity verification experimental results of the kit of the present invention;

[0032] Figure 3 This is a bar graph comparing the patients’ PD and SD;

[0033] Figure 4 This is the ROC curve diagram for patients;

[0034] Figure 5 This is the KM survival analysis diagram for patients. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1 Preparation and performance analysis of slulizumab immunochromatography kit

[0037] 1. Kit Preparation

[0038] The slulizumab immunochromatographic kit includes a test strip and a sample processing solution. The test strip structure is shown in Figure 1 , including a base plate and a sample pad, a nitrocellulose membrane and absorbent paper mounted on the base plate in sequence, and a quality control line and a detection line are set on the nitrocellulose membrane; the sample processing liquid includes a sample diluent and a fluorescent liquid, and the fluorescent liquid includes quantum dot nanospheres coated with slulizumab and a fluorescent antibody diluent.

[0039] Quality control line: formed by coating GAM antibody on nitrocellulose membrane;

[0040] Detection line: formed by coating recombinant PD-1 protein on nitrocellulose membrane;

[0041] Fluorescent solution: The ratio of quantum dot nanospheres coated with anti-PD-1 monoclonal antibody to fluorescent antibody dilution is 1:300.

[0042] Production of test card: first take the plastic sheet and stick the nitrocellulose membrane and the absorbent pad in sequence to form a large plate; then put the large plate on a high-speed chopper and cut it into 4mm wide test strips; finally, assemble the test strips with the bottom cover of the card shell to obtain the test card.

[0043] 2. Performance Analysis

[0044] In order to detect the analytical performance of the slulizumab immunochromatography kit prepared in Example 1, the following experiment was performed:

[0045] 2.1 Calibration curve

[0046] Take out the test card in the kit (Example 1), add 50 μL of slulizumab standard plasma sample and 50 μL of fluorescent liquid to 200 μL centrifuge tubes, mix by pipetting, and the concentrations are 0 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively; add 100 μL of the mixed solution to the buffer well of the test card and wait for 15 minutes; after 15 minutes, read the fluorescence intensity T1 of slulizumab and the mean fluorescence intensity C of the quality control line with a fluorescence meter, measure three times on average, and take the average value. The test results are shown in Table 1.

[0047] Table 1 Results of fluorescence immunochromatographic analysis of plasma samples with different concentrations of slulizumab standard

[0048]

[0049]

[0050] Fitting the standard curve by T / C value: y = A2 + (A1-A2) / (1 + (x / x0) p ), where A1 = 18.58 ± 0.04, A2 = 0.03 ± 0.04, x0 = 97.41 ± 0.26, p = 1.33 ± 0.05, in which y is the T / C value, x is the concentration of slulizumab, R 2 =0.99997( Figure 2 ).

[0051] 2.1 Precision

[0052] For the 50μg / mL and 100μg / mL slulizumab standard samples, 10 test strips were used to repeat the test 10 times, and then the corresponding concentrations were calculated according to the standard curve. The mean (M) and standard deviation (SD) of the two level measurement values ​​were calculated respectively; according to the formula CV = SD / M × 100%, CV-coefficient of variation; SD-standard deviation of 10 measurement results; M-average of 10 measurement results; calculate the coefficient of variation (CV), and then calculate the coefficient of variation (CV) of the results as shown in Table 2.

[0053] 2.2 Accuracy

[0054] The 50 μg / mL and 100 μg / mL slulizumab standard samples were tested three times using three test strips, and the test results were recorded as (X). According to the formula B = (X i -T) / T×100%; Where: B—relative deviation; X i—measured value; T—sample concentration; calculate the relative deviation B, and the results are shown in Table 2.

[0055] 2.3 Minimum detection limit

[0056] Using the zero concentration calibrator as the sample, the measurement was repeated 20 times, and the mean value (M) and standard deviation (SD) of the T / C values ​​corresponding to the 20 measurement results were calculated. According to the formula minimum detection limit = M + 2SD, the minimum detection limit concentration was calculated. The results are shown in Table 2.

[0057] Table 2 Summary of precision, accuracy and minimum detection limit

[0058]

[0059] Example 2 Clinical Sample Analysis

[0060] 1. Clinical Sample Design

[0061] Two groups of patients with PFS ≤ 6 months and PFS > 6 months were used for slulizumab combined with chemotherapy. Plasma samples at the trough concentration point after medication were selected, totaling 39 plasma samples. 10 μL of plasma was taken from each case, diluted to 100 μL with 1× PBS, and loaded. The grouping information is shown in Table 3 below:

[0062] Table 3 Summary of clinical plasma samples and slulizumab exposure concentrations

[0063] Patient number PFS Exposure concentration (μg / mL) Patient number PFS Exposure concentration (μg / mL) 1 >6M 57 21 ≤6M 31 2 >6M 162 22 ≤6M 66 3 >6M 48 23 ≤6M 45 4 >6M 71 24 >6M 54 5 ≤6M 26 25 >6M 35 6 ≤6M 35 26 >6M 153 7 >6M 210 27 >6M 244 8 ≤6M 49 28 >6M 100 9 >6M 49 29 >6M 59 10 >6M 132 30 >6M 150 11 >6M 42 31 >6M 122 12 >6M 173 32 >6M 84 13 ≤6M 15 33 ≤6M 41 14 >6M 76 34 ≤6M 70 15 >6M 61 35 ≤6M 88 16 >6M 109 36 >6M 92 17 >6M 53 37 >6M 41 18 >6M 53 38 ≤6M 15 19 >6M 54 39 ≤6M 8 20 >6M 60

[0064] 2. Sample processing and experimental operation steps

[0065] 2.1 Sample processing

[0066] (1) Small instruments: vortex machine, pipette.

[0067] (2) Required reagents and consumables: sample diluent, PBS, centrifuge tubes.

[0068] (3) Experimental steps: Take the plasma sample out of the -80°C freezer, vortex and transfer 10 μL to a new centrifuge tube after thawing, and dilute to 100 μL with 1× PBS.

[0069] 2.2 Immunochromatographic test strip assay

[0070] (1) Instruments: fluorimeter, vortexer, sonicator, pipette.

[0071] (2) Required reagents and consumables: quantum dot nanospheres, fluorescent diluent, centrifuge tubes, and immunochromatographic test strips.

[0072] (3) Experimental steps:

[0073] First, take 50μl of the processed sample, then add 50μl of fluorescent liquid, and then pipette it evenly. Finally, add the liquid to the well to be tested. Wait for 15 minutes to read.

[0074] 3. Analysis of the efficacy of slulizumab in relation to exposure concentration

[0075] 3.1 Efficacy evaluation

[0076] The samples were divided into two groups: PFS ≤ 6 months and PFS > 6 months. The Mann-Whitney nonparametric test method was used to perform statistical tests on the association between exposure concentration and efficacy in the two groups. The test results showed that the association between exposure concentration and efficacy was significant (P value was 0.0002). The exposure concentration of slulizumab in patients with PFS > 6 months was significantly higher than that in patients with PFS ≤ 6 months. The bar chart analysis results are shown in Figure 3 shown.

[0077] 3.2 ROC analysis

[0078] The receiver operating characteristic curve (ROC curve) is also called the sensitivity curve. The area under the ROC curve (AMC) refers to the area between the ROC curve and the x-axis, (1, 0)-(1, 1). If the area under the ROC curve is greater than 0.5, it proves that the diagnostic test has a certain diagnostic value. The closer the area under the ROC curve is to 1 and the closer it is to the (0, 1) point, the better the authenticity of the diagnostic test.

[0079] The ROC curve analysis of the exposure concentration and the efficacy of slulizumab combined with chemotherapy was performed to determine that the above exposure concentration has good efficacy in predicting efficacy. Figure 4 As shown, the AUC of the exposure concentration-related efficacy was 0.83, the sensitivity was 82%, the specificity was 75%, and the maximum value of the Youden index was 0.565, which was the exposure concentration threshold, i.e. 51 μg / mL.

[0080] 3.3 Survival analysis

[0081] For malignant tumors, since it is impossible to judge the prognosis in a short period of time, it is not appropriate to use indicators such as cure rate and mortality rate. Instead, it is necessary to follow up the patients and analyze the survival or death of the patients after a certain period of time. The statistical method that combines the result of the event with the time it takes to achieve this result is called survival analysis. The KM method, namely the Kaplan-Meier survival estimate, is a non-parametric method to estimate the survival probability from the observed survival time. When the log rank test is used to test, when P<0.05, it can be considered that the difference in the overall survival curves of two or more groups is statistically significant.

[0082] Among the collected patients, KM survival analysis (Log-rank) was performed based on the exposure concentration threshold determined by the association between exposure concentration and efficacy, and the patients were divided into two groups: exposure concentration ≤ 51 μg / mL and exposure concentration > 51 μg / mL. Figure 5 As shown, the PFS of patients with exposure concentration >51 μg / mL was significantly longer than that of patients with exposure concentration ≤51 μg / mL.

[0083] 4. Summary of Results

[0084] 1) The analysis of exposure concentration-related efficacy showed that there was a significant difference in exposure concentration between benefited patients (PFS>6 months) and non-benefited patients (PFS≤6 months), indicating that higher exposure concentrations lead to better efficacy;

[0085] 2) Through ROC curve analysis, the AUC was 0.83, the sensitivity was 82%, and the specificity was 75%, indicating that the exposure concentration had good predictive efficacy in predicting patient efficacy. By detecting the exposure concentration of slulizumab in the patient's plasma at the trough concentration, it was used as a diagnostic marker to determine whether the patient benefited from the treatment and was applied to medication guidance;

[0086] 3) KM curve analysis was performed for the above exposure concentrations. Patients with exposure concentrations greater than 51 μg / mL had significantly prolonged PFS and better prognosis.

[0087] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. An immunochromatographic test strip for detecting slulizumab, characterized in that: It includes a base plate and a sample pad, a nitrocellulose membrane and a water-absorbing pad sequentially mounted on the base plate. The sample pad is provided with a sample addition hole. The nitrocellulose membrane is provided with a quality control line and a detection line, the quality control line is coated with GAM antibody, and the detection line is coated with recombinant PD-1 protein.

2. An immunochromatographic kit for detecting slulizumab, characterized in that: Including immunochromatographic test strips and sample processing fluid, Wherein, the immunochromatographic test strip is as described in claim 1; The sample processing solution is composed of a sample diluent and a fluorescent solution, and the fluorescent solution is composed of quantum dot nanospheres coated with anti-PD-1 monoclonal antibodies and a fluorescent antibody diluent.

3. The immunochromatographic kit for detecting slulizumab according to claim 2, characterized in that: in, The sample diluent is PBS; The mass-to-volume ratio between the quantum dot nanospheres of the anti-PD-1 monoclonal antibody and the fluorescent antibody dilution is 1:

300.

4. The immunochromatographic kit for detecting slulizumab according to claim 2, characterized in that: in, The immunochromatography kit also includes a slulizumab standard plasma sample as a control substance mother solution.

5. A slulizumab exposure concentration detection system, characterized in that: It includes an immunochromatographic kit for detecting slulizumab, a fluorescence intensity detection device, and a result analysis system. The immunochromatographic kit for detecting slulizumab is as described in any one of claims 2 to 4; The fluorescence intensity detection system is used to detect the fluorescence intensity on the line; The result analysis system includes an input display module, an analysis module and a storage module. The input display module is used to input the basic information of the patient and display the analysis results; the analysis module obtains the exposure concentration of slulizumab based on the standard curve in the storage module and the fluorescence intensity analysis.

6. The slulizumab exposure concentration detection system according to claim 5, characterized in that: in, The standard curve was obtained based on slulizumab standard plasma samples as follows: Prepare plasma samples of slulizumab standard with different concentrations, mix them with the fluorescent solution by pipetting, and then perform immunochromatography analysis. Read the fluorescence intensity T1 of slulizumab and the mean fluorescence intensity C of the quality control line, and fit the standard curve by T / C value: y=A2+(A1-A2) / (1+(x / x0) p ), where y is the T / C value, x is the concentration of slulizumab, A1=18.58±0.04, A2=0.03±0.04, x0=97.41±0.26, p=1.33±0.05, R 2 =0.99997.

7. Use of the slulizumab exposure concentration detection system according to claim 5 in the survival prognosis assessment of malignant tumors.

8. The use according to claim 7, characterized in that: The malignant tumor is lung cancer, Survival prognosis evaluation is a predictive assessment of the efficacy of slulizumab combined with chemotherapy in lung cancer.

9. The use according to claim 7, characterized in that: The threshold exposure concentration of slulizumab was 51 μg / mL.

10. A method for detecting the exposure concentration of slulizumab, characterized in that: The method is carried out using the slulizumab exposure concentration detection system according to claim 5 or 6, comprising the following steps: (1) Take a certain volume of fresh or thawed plasma sample and dilute it to 10 times with 1× PBS; (2) Take a certain volume of the treated sample and add an equal volume of fluorescent liquid to it. After blowing and mixing evenly, add the sample to the sample well of the immunochromatographic test strip and analyze it with a fluorescence meter electrically connected to the test strip. Wait for 15 minutes and then read the exposure concentration value of slulizumab.

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