An immunological factor test paper and a method for preparing the same

Abstract

This invention discloses an immune factor detection test strip and its preparation method, belonging to the field of biomedical technology. The test strip consists of a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. By coating the conjugate pad with specific detection antibodies and the detection lines with specific trapping agents, multi-indicator joint detection is achieved. This test strip has advantages such as simple operation, rapid detection, and multi-indicator joint detection, and can provide strong support for the diagnosis of diseases such as immune-related adverse reactions.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an immune factor detection test strip and its preparation method. Background Technology

[0002] Immunotherapy, as an emerging cancer treatment method, has achieved certain successes in clinical practice in recent years, bringing new hope to cancer patients. Immunotherapy attacks tumor cells by activating the patient's own immune system, and compared to traditional chemotherapy and radiotherapy, it has high specificity and fewer side effects. However, with the widespread application of immunotherapy, it has been found that it is not without adverse reactions; some patients experience a series of immune-related adverse events (irAEs) after receiving immunotherapy. These adverse reactions involve multiple systems, such as the skin, gastrointestinal tract, endocrine system, liver, and lungs, seriously affecting the patient's quality of life and even potentially threatening their life.

[0003] Currently, clinical monitoring of adverse reactions to immunotherapy mainly relies on clinical examinations and laboratory tests. These tests are often time-consuming and costly. For certain adverse reactions, such as immune-associated pneumonia, imaging examinations, such as chest CT scans, are primarily used. However, these examinations usually only detect adverse reactions after symptoms have developed to a certain extent, making early prediction impossible. Furthermore, current methods for predicting adverse reactions to immunotherapy are mostly based on the detection of single biomarkers. However, the mechanisms underlying adverse reactions to immunotherapy are complex, and single biomarkers often fail to comprehensively reflect changes in the body's immune status during immunotherapy, resulting in low predictive accuracy. Additionally, some existing detection methods are complex to operate, requiring specialized equipment and technicians, making them unsuitable for primary healthcare institutions. For example, while flow cytometry-based methods can accurately detect immune cell surface markers, the equipment is expensive, the operation is cumbersome, and the technical requirements for operators are high, limiting their widespread clinical application.

[0004] In conclusion, there is an urgent need for a method and tool that can quickly, accurately, conveniently, and at low cost predict adverse reactions to immunotherapy, in order to help clinicians take effective intervention measures in advance, optimize immunotherapy regimens, and improve patients' treatment outcomes and quality of life. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an immune factor test strip and its preparation method, so as to achieve rapid, accurate, convenient and low-cost prediction of adverse reactions to immunotherapy.

[0006] The technical solution of this invention is as follows: An immune factor test strip includes a base plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper are sequentially arranged. Multiple detection lines and control lines are arranged on the nitrocellulose membrane. The conjugate pad is coated with detection antibodies, including IL-6 primary antibody, IFN-γ primary antibody, and / or mouse anti-human IgG antibody. The detection lines are coated with traps, including IL-6 secondary antibody, IFN-γ secondary antibody, and / or cardiac myosin.

[0007] Furthermore, the quality control line is coated with quality control antibodies.

[0008] Furthermore, the quality control cell body includes an anti-mouse IgG antibody.

[0009] Furthermore, in order to achieve simultaneous detection of different indicators, multiple detection lines need to be set on the nitrocellulose membrane, with each detection line coated with a specific antibody. For example, three detection lines can be set, with the three detection lines coated with IL-6 secondary antibody, IFN-γ secondary antibody and cardiac myosin, respectively.

[0010] Furthermore, the detection antibody on the binding pad is labeled with colloidal gold to form a colloidal gold-antibody complex.

[0011] Furthermore, the colloidal gold-antibody complex is resuspended in a buffer solution containing 0.05M~0.15M magnesium chloride and 5~15wt% peptidoglycan.

[0012] Furthermore, the buffer solution is 0.05M Tris-HCl with 0.05~0.15M magnesium chloride, 0.1~0.5wt% Tween 20, and 5~15wt% peptidoglycan added.

[0013] Furthermore, the present invention specifically provides a method for preparing the aforementioned immune factor test strip, the method comprising the following steps: (1) Mix the colloidal gold solution and the detection antibody for 40-60 minutes according to 20 μg~40 μg antibody / mL colloidal gold solution, then add BSA solution under stirring, incubate, centrifuge at low temperature, collect the colloidal gold-antibody complex, and resuspend the colloidal gold-antibody complex in buffer. (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it to obtain the conjugate pad; (4) Spray the captured material and the quality control antibody onto the nitrocellulose membrane to form the detection line and the quality control line, respectively; (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the PVC base plate to make an immune factor test strip.

[0014] The concentration of the captured substance coated in the detection line is at least 2 mg / mL.

[0015] The quality control line is coated with a quality control antibody at a concentration of at least 2 mg / mL.

[0016] Furthermore, this invention relates to the application of the aforementioned immune factor test strip in the preparation of products for detecting adverse reactions to immunotherapy. The indicators to be detected include IL-6, IFN-γ, and anti-myosin.

[0017] Compared with the prior art, the beneficial results of the present invention are as follows: The test strip of this invention can detect multiple immune factors simultaneously, and a single test can simultaneously obtain a variety of key immune-related information, providing a comprehensive and accurate basis for the diagnosis and monitoring of clinical diseases, and helping clinicians to more accurately judge the condition and formulate personalized treatment plans.

[0018] This invention, through the selection and processing of antibodies, enables the test strip to possess extremely high sensitivity and specificity for various immune factors. It can detect 5 pg / mL IL-6, 5 pg / mL IFN-γ, and 20 U / mL anti-cardiac antibodies, while effectively reducing cross-reactivity with other substances in the sample, thus lowering the probability of false positive and false negative results.

[0019] This invention's test strip is based on colloidal gold immunochromatography technology. It is simple and easy to operate, requiring no professional instruments or equipment. After adding the sample to be tested, the test results can be observed in about 15-20 minutes. It is suitable for primary healthcare units, on-site rapid testing, and patient self-testing, greatly improving testing efficiency and meeting the needs of rapid clinical diagnosis.

[0020] The preparation method of the test strip of this invention is relatively simple, does not require complicated instruments and equipment, and the cost of each raw material is low, making it suitable for large-scale industrial production.

[0021] When detecting multiple immune factors simultaneously, cross-influence can easily occur, and existing buffer solutions cannot provide an optimal detection environment for multiple immune factors, leading to poor detection results and affecting overall detection accuracy. This invention, by optimizing the buffer formulation, significantly reduces the adsorption of non-target substances in the sample onto the test strip detection area, lowers background signal, reduces cross-influence between substances, improves detection specificity and accuracy, and reduces false positive and false negative results. The improved buffer solution provides a suitable detection environment for multiple biomarkers, enhances compatibility with different immune factors or biomarkers, and enables the test strip to accurately detect multiple biomarkers simultaneously, improving the comprehensiveness and accuracy of predicting adverse reactions to immunotherapy. Detailed Implementation

[0022] To better understand the technical content of this invention, the invention will be further described below with reference to specific embodiments.

[0023] In the following embodiments, unless otherwise specified, the IL-6 primary antibody and the IFN-γ primary antibody are Anti-IL-6 antibody [B-E8] (ab11449) and IFN-γ antibody [NIB42] (ab180547), respectively.

[0024] The IL-6 secondary antibody and IFN-γ secondary antibody are Anti-IL-6 antibody [1.2-2B11-2G10] (ab9324) and Maichuan Bio's IFN-γ antibody (AB1023), respectively.

[0025] The peptidoglycan is lactobacillus peptidoglycan with a molecular weight of 56-63 kDa.

[0026] Example 1: Immune Factor Test Strip The immunoassay test strip includes a base plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper are sequentially arranged. The conjugate pad is coated with a colloidal gold-labeled detection antibody. The nitrocellulose membrane has multiple detection lines (T1-T3 lines) and a control line (C line).

[0027] Detection principle: Based on lateral chromatography immunoassay, the sample on the sample pad travels along the test strip. If the target immune factor is present in the sample, it binds to the colloidal gold-antibody complex on the binding pad and continues to the nitrocellulose membrane, where it binds to specific substances on the test line, forming a visible test line band, resulting in a positive result. If the target immune factor is absent, no obvious band will appear at the corresponding test line, resulting in a negative result. The sample continues to the control line, where it binds to the antibody, forming a control line band. If a control line band appears, the test is valid; if no control line band appears, the test is invalid.

[0028] Example 2: Preparation of Immunogenic Factor Test Strips (1) Heat 0.01wt% chloroauric acid solution to boiling, add 1wt% trisodium citrate solution at a volume ratio of 50:1, continue boiling for 15-20 minutes until the solution turns wine red to obtain colloidal solution.

[0029] Mix the colloidal gold solution and detection antibodies (IL-6 primary antibody, IFN-γ primary antibody, and mouse anti-human IgG antibody) at a concentration of 20 μg antibody / mL for 40-60 minutes. Then, while stirring, add 10 wt% BSA solution to bring the final BSA concentration to 1 wt%. After incubation for 1 hour, centrifuge at low temperature to collect the colloidal gold-antibody complex. Resuspend the colloidal gold-antibody complex in buffer (0.05 M Tris-HCl at pH 7.4 with 0.15 M magnesium chloride, 0.1 wt% Tween 20, and 5 wt% peptidoglycan).

[0030] (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it at 35±2℃ to obtain the conjugate pad.

[0031] (4) Spray the capture material (IL-6 secondary antibody, IFN-γ secondary antibody, myosin) and the quality control antibody onto the nitrocellulose membrane to form a detection line and a quality control line. The detection line is coated with the capture material at a concentration of 2 mg / mL, and the quality control line is coated with the quality control antibody (goat anti-mouse IgG antibody) at a concentration of 2 mg / mL.

[0032] (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the PVC base plate, with an overlap of about 1 mm between adjacent parts, to make an immune factor test strip.

[0033] This invention precisely controls the preparation method and antibody pairing of the test strip to accurately set the detection limits for IL-6 and IFN-γ at 5 pg / mL and for anticardiomyocyte antibodies at 20 U / mL. These limits are generally used to determine whether a patient has experienced immune-related adverse reactions; IL-6 and IFN-γ < 5 pg / mL and anticardiomyocyte antibodies < 20 U / mL are considered normal, while levels exceeding these limits are considered to indicate immune-related adverse reactions. The more pronounced the test bands, the higher the severity of the immune-related adverse reaction. Therefore, the presence and intensity of the bands can be used to preliminarily determine the severity of the patient's immune adverse reaction.

[0034] Detection and verification: 20 positive serum samples containing IL-6, IFN-γ, and anti-myocardial antibodies and 10 negative serum samples were taken and added to the sample wells for testing. After 15 minutes, it was observed that all positive samples showed positive results, and no obvious bands appeared on the test line for the negative samples, showing negative results. The detection accuracy was 100%.

[0035] Positive serum samples containing IL-6, IFN-γ, and anti-myocardial antibodies were separately added to the sample wells for testing. The results showed that each sample showed a band at its corresponding test line, while no bands appeared on other test lines, indicating that there was no cross-reaction and no interference between the analytes during testing.

[0036] Example 3: Preparation of Immunogenic Factor Test Strips (1) Heat 0.01wt% chloroauric acid solution to boiling, add 1wt% trisodium citrate solution at a volume ratio of 50:1, continue boiling for 15-20 minutes until the solution turns wine red to obtain colloidal solution.

[0037] Mix the colloidal gold solution and the detection antibody (IL-6 primary antibody) at a concentration of 20 μg antibody / mL for 40-60 minutes. Then, add 10 wt% BSA solution while stirring to bring the final BSA concentration to 1 wt%. After incubation for 1 hour, centrifuge at low temperature to collect the gold nanoparticle-antibody complex. Resuspend the gold nanoparticle-antibody complex in buffer (0.05 M Tris-HCl at pH 7.4 with 0.15 M magnesium chloride, 0.1 wt% Tween 20, and 5 wt% peptidoglycan).

[0038] (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it at 35±2℃ to obtain the conjugate pad.

[0039] (4) Spray the capture antibody (IL-6 secondary antibody) and the quality control antibody onto the nitrocellulose membrane to form a detection line and a quality control line. The detection line is coated with a capture antibody at a concentration of 2 mg / mL, and the quality control line is coated with a quality control antibody (goat anti-mouse IgG antibody) at a concentration of 2 mg / mL.

[0040] (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the PVC base plate, with an overlap of about 1 mm between adjacent parts, to make an immune factor test strip.

[0041] Detection and verification: 10 positive serum samples containing IL-6 (>5 pg / mL) and 10 negative serum samples were taken and added to the sample wells for detection. After 15 minutes, it was observed that the positive samples all showed positive results, and the negative samples did not show obvious bands on the test line, showing negative results.

[0042] Example 3: Preparation of Immunogenic Factor Test Strips (1) Heat 0.01wt% chloroauric acid solution to boiling, add 1wt% trisodium citrate solution at a volume ratio of 50:1, continue boiling for 15-20 minutes until the solution turns wine red to obtain colloidal solution.

[0043] Mix the colloidal gold solution and the detection antibody (IFN-γ primary antibody) at a concentration of 20 μg antibody / mL for 40-60 minutes. Then, add 10 wt% BSA solution while stirring to bring the final BSA concentration to 1 wt%. After incubation for 1 hour, centrifuge at low temperature to collect the gold nanoparticle-antibody complex. Resuspend the gold nanoparticle-antibody complex in buffer (0.05 M Tris-HCl at pH 7.4 with 0.15 M magnesium chloride, 0.1 wt% Tween 20, and 5 wt% peptidoglycan).

[0044] (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it at 35±2℃ to obtain the conjugate pad.

[0045] (4) Spray the capture antibody (IFN-γ secondary antibody) and the quality control antibody onto the nitrocellulose membrane to form a detection line and a quality control line. The detection line is coated with a capture antibody at a concentration of 2 mg / mL, and the quality control line is coated with a quality control antibody (goat anti-mouse IgG antibody) at a concentration of 2 mg / mL.

[0046] (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the PVC base plate, with an overlap of about 1 mm between adjacent parts, to make an immune factor test strip.

[0047] Detection and verification: 10 positive serum samples containing IFN-γ (>5 pg / mL) and 10 negative serum samples were taken and added to the sample wells for detection. After 15 minutes, it was observed that the positive samples all showed positive results, and the negative samples did not show obvious bands on the test line, showing negative results.

[0048] Example 4: Preparation of Immunogenic Factor Test Strips In this example, a different antibody pairing was used. The second IL-6 antibody was chosen as Anti-IL-6 antibody [B-E4] (ab275971), and everything else remained the same.

[0049] (1) Heat 0.01wt% chloroauric acid solution to boiling, add 1wt% trisodium citrate solution at a volume ratio of 50:1, continue boiling for 15-20 minutes until the solution turns wine red to obtain colloidal solution.

[0050] Mix the colloidal gold solution and detection antibodies (IL-6 primary antibody, IFN-γ primary antibody, and mouse anti-human IgG antibody) at a concentration of 20 μg antibody / mL for 40-60 minutes. Then, add 10 wt% BSA solution while stirring to bring the final BSA concentration to 1 wt%. After incubation for 1 hour, centrifuge at low temperature to collect the gold nanoparticle-antibody complex. Resuspend the gold nanoparticle-antibody complex in buffer (0.05 M Tris-HCl at pH 7.4 with 0.15 M magnesium chloride, 0.1 wt% Tween 20, and 5 wt% peptidoglycan).

[0051] (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it at 35±2℃ to obtain the conjugate pad.

[0052] (4) Spray the capture material (IL-6 secondary antibody, IFN-γ secondary antibody, myosin) and the quality control antibody onto the nitrocellulose membrane to form a detection line and a quality control line. The detection line is coated with the capture material at a concentration of 2 mg / mL, and the quality control line is coated with the quality control antibody (goat anti-mouse IgG antibody) at a concentration of 2 mg / mL.

[0053] (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the PVC base plate, with an overlap of about 1 mm between adjacent parts, to make an immune factor test strip.

[0054] The test results showed that positive serum samples with high IL-6 levels (10 pg / mL) were detectable, but positive serum samples with IL-6 levels (5 pg / mL) were not detected. This indicates that the optimal antibody pairing is the antibody described in Example 1.

[0055] Example 5: Preparation of Immunogenic Factor Test Strips (1) Heat 0.01wt% chloroauric acid solution to boiling, add 1wt% trisodium citrate solution at a volume ratio of 50:1, continue boiling for 15-20 minutes until the solution turns wine red to obtain colloidal solution.

[0056] Mix the colloidal gold solution and detection antibodies (IL-6 primary antibody, IFN-γ primary antibody, and mouse anti-human IgG antibody) at a concentration of 40 μg antibody / mL for 40-60 minutes. Then, add 10 wt% BSA solution while stirring to bring the final BSA concentration to 1 wt%. After incubation for 1 hour, centrifuge at low temperature to collect the gold nanoparticle-antibody complex. Resuspend the gold nanoparticle-antibody complex in buffer (0.05 M Tris-HCl at pH 7.4 with 0.05 M magnesium chloride, 0.1 wt% Tween 20, and 5 wt% peptidoglycan).

[0057] (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it at 35±2℃ to obtain the conjugate pad.

[0058] (4) Spray the capture material (IL-6 secondary antibody, IFN-γ secondary antibody, myosin) and the quality control antibody onto the nitrocellulose membrane to form a detection line and a quality control line. The detection line is coated with the capture material at a concentration of 2 mg / mL, and the quality control line is coated with the quality control antibody (goat anti-mouse IgG antibody) at a concentration of 2 mg / mL.

[0059] (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the PVC base plate, with an overlap of about 1 mm between adjacent parts, to make an immune factor test strip.

[0060] Example 6: Preparation of Immunogenic Factor Test Strips In this example, the peptidoglycan in the buffer solution used to resuspend the gold nanoparticle-antibody complex is replaced with polyethylene glycol.

[0061] (1) Heat 0.01wt% chloroauric acid solution to boiling, add 1wt% trisodium citrate solution at a volume ratio of 50:1, continue boiling for 15-20 minutes until the solution turns wine red to obtain colloidal solution.

[0062] Mix the colloidal gold solution and detection antibodies (IL-6 primary antibody, IFN-γ primary antibody, and mouse anti-human IgG antibody) at a concentration of 20 μg antibody / mL for 40-60 minutes. Then, add 10 wt% BSA solution while stirring to bring the final BSA concentration to 1 wt%. After incubation for 1 hour, centrifuge at low temperature to collect the gold nanoparticle-antibody complex. Resuspend the gold nanoparticle-antibody complex in buffer (0.05 M Tris-HCl at pH 7.4 with 0.15 M magnesium chloride, 0.1 wt% Tween 20, and 5 wt% polyethylene glycol 4000).

[0063] (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it at 35±2℃ to obtain the conjugate pad.

[0064] (4) Spray the capture material (IL-6 secondary antibody, IFN-γ secondary antibody, myosin) and the quality control antibody onto the nitrocellulose membrane to form a detection line and a quality control line. The detection line is coated with the capture material at a concentration of 2 mg / mL, and the quality control line is coated with the quality control antibody (goat anti-mouse IgG antibody) at a concentration of 2 mg / mL.

[0065] (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the PVC base plate, with an overlap of about 1 mm between adjacent parts, to make an immune factor test strip.

[0066] The test results showed that the tested items may interfere with each other, and 2 of the 30 positive samples were false negatives.

[0067] Example 7: Preparation of Immunogenic Factor Test Strips In this example, peptidoglycan in the buffer solution used to resuspend the gold nanoparticle-antibody complex is replaced with trehalose.

[0068] (1) Heat 0.01wt% chloroauric acid solution to boiling, add 1wt% trisodium citrate solution at a volume ratio of 50:1, continue boiling for 15-20 minutes until the solution turns wine red to obtain colloidal solution.

[0069] Mix the colloidal gold solution and detection antibodies (IL-6 primary antibody, IFN-γ primary antibody, and mouse anti-human IgG antibody) at a concentration of 20 μg antibody / mL for 40-60 minutes. Then, add 10 wt% BSA solution while stirring to bring the final BSA concentration to 1 wt%. After incubation for 1 hour, centrifuge at low temperature to collect the gold nanoparticle-antibody complex. Resuspend the gold nanoparticle-antibody complex in buffer (0.05 M Tris-HCl at pH 7.4 with 0.15 M magnesium chloride, 0.1 wt% Tween 20, and 5 wt% trehalose).

[0070] (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it at 35±2℃ to obtain the conjugate pad.

[0071] (4) Spray the capture material (IL-6 secondary antibody, IFN-γ secondary antibody, myosin) and the quality control antibody onto the nitrocellulose membrane to form a detection line and a quality control line. The detection line is coated with the capture material at a concentration of 2 mg / mL, and the quality control line is coated with the quality control antibody (goat anti-mouse IgG antibody) at a concentration of 2 mg / mL.

[0072] (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the PVC base plate, with an overlap of about 1 mm between adjacent parts, to make an immune factor test strip.

[0073] The test results showed that the tested substances may interfere with each other, with 3 false negatives and 1 invalid sample out of 30 positive samples.

[0074] The above description is only a part of the embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. An immune factor test strip, the immune factor test strip comprising a base plate, wherein a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper are sequentially disposed on the base plate, and the nitrocellulose membrane is provided with multiple detection lines and control lines, characterized in that, The detection pad is coated with detection antibodies, which include IL-6 primary antibody, IFN-γ primary antibody and / or mouse anti-human IgG antibody. The detection line is coated with traps, which include IL-6 secondary antibody, IFN-γ secondary antibody and / or cardiac myosin. The detection antibody on the binding pad is labeled with colloidal gold to form a colloidal gold-antibody complex. The colloidal gold-antibody complex is resuspended in a buffer solution, which is 0.05M Tris-HCl with 0.05~0.15M magnesium chloride, 0.1~0.5wt% Tween 20, and 5~15wt% peptidoglycan added.

2. The immune factor detection test strip according to claim 1, characterized in that, The quality control line is coated with quality control antibodies.

3. The immune factor detection test strip according to claim 2, characterized in that, The quality control antibody includes anti-mouse IgG antibody.

4. The immune factor detection test strip according to claim 1, characterized in that, The nitrocellulose membrane has three detection lines, which are respectively coated with IL-6 secondary antibody, IFN-γ secondary antibody and cardiac myosin.

5. The method for preparing the immune factor detection test strip according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Heat 0.01wt% chloroauric acid solution to boiling, add 1wt% trisodium citrate solution at a volume ratio of 50:1, continue boiling for 15-20 minutes until the solution turns wine red to obtain colloidal gold solution. (2) Mix the colloidal gold solution and the detection antibody for 40-60 minutes according to 20-40 μg antibody / mL colloidal gold solution, then add BSA solution while stirring, incubate, centrifuge at low temperature, collect the colloidal gold-antibody complex, and resuspend the colloidal gold-antibody complex in buffer. (3) Spray the colloidal gold-antibody complex solution evenly onto glass wool and dry it to obtain the conjugate pad; (4) Spray the captured material and the quality control antibody onto the nitrocellulose membrane to form the detection line and the quality control line, respectively; (5) The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper are sequentially overlapped on the base plate to make an immune factor test strip.

6. The preparation method according to claim 5, characterized in that, The concentration of the capture material coated in the detection line is at least 2 mg / mL, and the concentration of the control antibody coated in the control line is at least 2 mg / mL.

7. The use of the immune factor test strip according to any one of claims 1 to 4 in the preparation of products for detecting adverse reactions to immunotherapy.